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Sample records for b-box protein involved

  1. CENP-B box, a nucleotide motif involved in centromere formation, occurs in a New World monkey.

    PubMed

    Suntronpong, Aorarat; Kugou, Kazuto; Masumoto, Hiroshi; Srikulnath, Kornsorn; Ohshima, Kazuhiko; Hirai, Hirohisa; Koga, Akihiko

    2016-03-01

    Centromere protein B (CENP-B) is one of the major proteins involved in centromere formation, binding to centromeric repetitive DNA by recognizing a 17 bp motif called the CENP-B box. Hominids (humans and great apes) carry large numbers of CENP-B boxes in alpha satellite DNA (AS, the major centromeric repetitive DNA of simian primates). Only negative results have been reported regarding the presence of the CENP-B box in other primate taxa. Consequently, it is widely believed that the CENP-B box is confined, within primates, to the hominids. We report here that the common marmoset, a New World monkey, contains an abundance of CENP-B boxes in its AS. First, in a long contig sequence we constructed and analysed, we identified the motif in 17 of the 38 alpha satellite repeat units. We then sequenced terminal regions of additional clones and found the motif in many of them. Immunostaining of marmoset cells demonstrated that CENP-B binds to DNA in the centromeric regions of chromosomes. Therefore, functional CENP-B boxes are not confined to hominids. Our results indicate that the efficiency of identification of the CENP-B box may depend largely on the sequencing methods used, and that the CENP-B box in centromeric repetitive DNA may be more common than researchers previously thought. PMID:27029836

  2. Formation of functional CENP-B boxes at diverse locations in repeat units of centromeric DNA in New World monkeys.

    PubMed

    Kugou, Kazuto; Hirai, Hirohisa; Masumoto, Hiroshi; Koga, Akihiko

    2016-01-01

    Centromere protein B, which is involved in centromere formation, binds to centromeric repetitive DNA by recognizing a nucleotide motif called the CENP-B box. Humans have large numbers of CENP-B boxes in the centromeric repetitive DNA of their autosomes and X chromosome. The current understanding is that these CENP-B boxes are located at identical positions in the repeat units of centromeric DNA. Great apes also have CENP-B boxes in locations that are identical to humans. The purpose of the present study was to examine the location of CENP-B box in New World monkeys. We recently identified CENP-B box in one species of New World monkeys (marmosets). In this study, we found functional CENP-B boxes in CENP-A-assembled repeat units of centromeric DNA in 2 additional New World monkeys (squirrel monkeys and tamarins) by immunostaining and ChIP-qPCR analyses. The locations of the 3 CENP-B boxes in the repeat units differed from one another. The repeat unit size of centromeric DNA of New World monkeys (340-350 bp) is approximately twice that of humans and great apes (171 bp). This might be, associated with higher-order repeat structures of centromeric DNA, a factor for the observed variation in the CENP-B box location in New World monkeys. PMID:27292628

  3. Formation of functional CENP-B boxes at diverse locations in repeat units of centromeric DNA in New World monkeys

    PubMed Central

    Kugou, Kazuto; Hirai, Hirohisa; Masumoto, Hiroshi; Koga, Akihiko

    2016-01-01

    Centromere protein B, which is involved in centromere formation, binds to centromeric repetitive DNA by recognizing a nucleotide motif called the CENP-B box. Humans have large numbers of CENP-B boxes in the centromeric repetitive DNA of their autosomes and X chromosome. The current understanding is that these CENP-B boxes are located at identical positions in the repeat units of centromeric DNA. Great apes also have CENP-B boxes in locations that are identical to humans. The purpose of the present study was to examine the location of CENP-B box in New World monkeys. We recently identified CENP-B box in one species of New World monkeys (marmosets). In this study, we found functional CENP-B boxes in CENP-A-assembled repeat units of centromeric DNA in 2 additional New World monkeys (squirrel monkeys and tamarins) by immunostaining and ChIP-qPCR analyses. The locations of the 3 CENP-B boxes in the repeat units differed from one another. The repeat unit size of centromeric DNA of New World monkeys (340–350 bp) is approximately twice that of humans and great apes (171 bp). This might be, associated with higher-order repeat structures of centromeric DNA, a factor for the observed variation in the CENP-B box location in New World monkeys. PMID:27292628

  4. The Arabidopsis B-BOX Protein BBX25 Interacts with HY5, Negatively Regulating BBX22 Expression to Suppress Seedling Photomorphogenesis[C][W

    PubMed Central

    Gangappa, Sreeramaiah N.; Crocco, Carlos D.; Johansson, Henrik; Datta, Sourav; Hettiarachchi, Chamari; Holm, Magnus; Botto, Javier F.

    2013-01-01

    ELONGATED HYPOCOTYL5 (HY5) is a basic domain/leucine zipper (bZIP) transcription factor, central for the regulation of seedling photomorphogenesis. Here, we identified a B-BOX (BBX)–containing protein, BBX25/SALT TOLERANCE HOMOLOG, as an interacting partner of HY5, which has been previously found to physically interact with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). BBX25 physically interacts with HY5 both in vitro and in vivo. By physiological and genetic approaches, we showed that BBX25 is a negative regulator of seedling photomorphogenesis. BBX25 and its homolog BBX24 regulate deetiolation processes and hypocotyl shade avoidance response in an additive manner. Moreover, genetic relationships of bbx25 and bbx24 with hy5 and cop1 revealed that BBX25 and BBX24 additively enhance COP1 and suppress HY5 functions. BBX25 accumulates in a light-dependent manner and undergoes COP1-mediated degradation in dark and light conditions. Furthermore, a protoplast cotransfection assay showed that BBX24 and BBX25 repress BBX22 expression by interfering with HY5 transcriptional activity. As HY5 binds to the BBX22 promoter and promotes its expression, our results identify a direct mechanism through which the expression of BBX22 is regulated. We suggest that BBX25 and BBX24 function as transcriptional corepressors, probably by forming inactive heterodimers with HY5, downregulating BBX22 expression for the fine-tuning of light-mediated seedling development. PMID:23624715

  5. The B-box dominates SAP-1-SRF interactions in the structure of the ternary complex.

    PubMed

    Hassler, M; Richmond, T J

    2001-06-15

    The serum response element (SRE) is found in several immediate-early gene promoters. This DNA sequence is necessary and sufficient for rapid transcriptional induction of the human c-fos proto-oncogene in response to stimuli external to the cell. Full activation of the SRE requires the cooperative binding of a ternary complex factor (TCF) and serum response factor (SRF) to their specific DNA sites. The X-ray structure of the human SAP-1-SRF-SRE DNA ternary complex was determined (Protein Data Bank code 1hbx). It shows SAP-1 TCF bound to SRF through interactions between the SAP-1 B-box and SRF MADS domain in addition to contacts between their respective DNA-binding motifs. The SAP-1 B-box is part of a flexible linker of which 21 amino acids become ordered upon ternary complex formation. Comparison with a similar region from the yeast MATalpha2-MCM1-DNA complex suggests a common binding motif through which MADS-box proteins may interact with additional factors such as Fli-1. PMID:11406578

  6. Modulation of retroviral restriction and proteasome inhibitor-resistant turnover by changes in the TRIM5alpha B-box 2 domain.

    PubMed

    Diaz-Griffero, Felipe; Kar, Alak; Perron, Michel; Xiang, Shi-Hua; Javanbakht, Hassan; Li, Xing; Sodroski, Joseph

    2007-10-01

    An intact B-box 2 domain is essential for the antiretroviral activity of TRIM5alpha. We modeled the structure of the B-box 2 domain of TRIM5alpha based on the existing three-dimensional structure of the B-box 2 domain of human TRIM29. Using this model, we altered the residues predicted to be exposed on the surface of this globular structure. Most of the alanine substitutions in these residues exerted little effect on the antiretroviral activity of human TRIM5alphahu or rhesus monkey TRIM5alpharh. However, alteration of arginine 119 of TRIM5alphahu or the corresponding arginine 121 of TRIM5alpharh diminished the abilities of the proteins to restrict retroviral infection without affecting trimerization or recognition of the viral capsid. The abilities of these functionally defective TRIM5alpha proteins to accelerate the uncoating of the targeted retroviral capsid were abolished. Removal of the positively charged side chain from B-box 2 arginines 119/120/121 resulted in diminished proteasome-independent turnover of TRIM5alpha and the related restriction factor TRIMCyp. However, testing of an array of mutants revealed that the rapid turnover and retroviral restriction functions of this B-box 2 region are separable. PMID:17626085

  7. Protein phosphorylation is involved in bacterial chemotaxis.

    PubMed Central

    Hess, J F; Oosawa, K; Matsumura, P; Simon, M I

    1987-01-01

    The nature of the biochemical signal that is involved in the excitation response in bacterial chemotaxis is not known. However, ATP is required for chemotaxis. We have purified all of the proteins involved in signal transduction and show that the product of the cheA gene is rapidly autophosphorylated, while some mutant CheA proteins cannot be phosphorylated. The presence of stoichiometric levels of two other purified components in the chemotaxis system, the CheY and CheZ proteins, induces dephosphorylation. We suggest that the phosphorylation of CheA by ATP plays a central role in signal transduction in chemotaxis. Images PMID:3313398

  8. Autophagy and proteins involved in vesicular trafficking.

    PubMed

    Amaya, Celina; Fader, Claudio Marcelo; Colombo, María Isabel

    2015-11-14

    Autophagy is an intracellular degradation system that, as a basic mechanism it delivers cytoplasmic components to the lysosomes in order to maintain adequate energy levels and cellular homeostasis. This complex cellular process is activated by low cellular nutrient levels and other stress situations such as low ATP levels, the accumulation of damaged proteins or organelles, or pathogen invasion. Autophagy as a multistep process involves vesicular transport events leading to tethering and fusion of autophagic vesicles with several intracellular compartments. This review summarizes our current understanding of the autophagic pathway with emphasis in the trafficking machinery (i.e. Rabs GTPases and SNAP receptors (SNAREs)) involved in specific steps of the pathway. PMID:26450776

  9. Mechanism of B-box 2 domain-mediated higher-order assembly of the retroviral restriction factor TRIM5α.

    PubMed

    Wagner, Jonathan M; Roganowicz, Marcin D; Skorupka, Katarzyna; Alam, Steven L; Christensen, Devin; Doss, Ginna; Wan, Yueping; Frank, Gabriel A; Ganser-Pornillos, Barbie K; Sundquist, Wesley I; Pornillos, Owen

    2016-01-01

    Restriction factors and pattern recognition receptors are important components of intrinsic cellular defenses against viral infection. Mammalian TRIM5α proteins are restriction factors and receptors that target the capsid cores of retroviruses and activate ubiquitin-dependent antiviral responses upon capsid recognition. Here, we report crystallographic and functional studies of the TRIM5α B-box 2 domain, which mediates higher-order assembly of TRIM5 proteins. The B-box can form both dimers and trimers, and the trimers can link multiple TRIM5α proteins into a hexagonal net that matches the lattice arrangement of capsid subunits and enables avid capsid binding. Two modes of conformational flexibility allow TRIM5α to accommodate the variable curvature of retroviral capsids. B-box mediated interactions also modulate TRIM5α's E3 ubiquitin ligase activity, by stereochemically restricting how the N-terminal RING domain can dimerize. Overall, these studies define important molecular details of cellular recognition of retroviruses, and how recognition links to downstream processes to disable the virus. PMID:27253059

  10. Mechanism of B-box 2 domain-mediated higher-order assembly of the retroviral restriction factor TRIM5α

    PubMed Central

    Wagner, Jonathan M; Roganowicz, Marcin D; Skorupka, Katarzyna; Alam, Steven L; Christensen, Devin; Doss, Ginna; Wan, Yueping; Frank, Gabriel A; Ganser-Pornillos, Barbie K; Sundquist, Wesley I; Pornillos, Owen

    2016-01-01

    Restriction factors and pattern recognition receptors are important components of intrinsic cellular defenses against viral infection. Mammalian TRIM5α proteins are restriction factors and receptors that target the capsid cores of retroviruses and activate ubiquitin-dependent antiviral responses upon capsid recognition. Here, we report crystallographic and functional studies of the TRIM5α B-box 2 domain, which mediates higher-order assembly of TRIM5 proteins. The B-box can form both dimers and trimers, and the trimers can link multiple TRIM5α proteins into a hexagonal net that matches the lattice arrangement of capsid subunits and enables avid capsid binding. Two modes of conformational flexibility allow TRIM5α to accommodate the variable curvature of retroviral capsids. B-box mediated interactions also modulate TRIM5α’s E3 ubiquitin ligase activity, by stereochemically restricting how the N-terminal RING domain can dimerize. Overall, these studies define important molecular details of cellular recognition of retroviruses, and how recognition links to downstream processes to disable the virus. DOI: http://dx.doi.org/10.7554/eLife.16309.001 PMID:27253059

  11. Transcriptional regulation of the human H ferritin-encoding gene (FERH) in G418-treated cells: role of the B-box-binding factor.

    PubMed

    Bevilacqua, M A; Faniello, M C; Russo, T; Cimino, F; Costanzo, F

    1994-04-20

    We have analysed the molecular basis underlying the increase in ferritin heavy-chain mRNA (FERH) levels in cells exposed to the antibiotic Geneticin (G418). Transient transfection experiments demonstrate that this increase is paralleled by an enhanced transcription driven by the promoter (pFERH) for the human FERH gene, in which the most proximal promoter element (B-box) appears to play a key role. This region is conserved in human and rat, and binds an unknown factor. The DNA-protein complex composed of B-box-binding factor and its cis-element becomes more abundant in the G418-treated cells, as compared with the untreated ones. PMID:8163204

  12. Involvement of the eye in protein malnutrition*

    PubMed Central

    McLaren, D. S.

    1958-01-01

    An extensive review of the literature on protein malnutrition, with special reference to the frequency of involvement of the eyes, has been made by the author. Consideration of accounts from all parts of the world and in many different languages, including early as well as more recent descriptions of the syndrome, indicates that this important complication has not received sufficient attention hitherto. The evidence available suggests that it is nearly always an accompanying deficiency of vitamin A that is responsible. Less commonly reported—and producing less severe effects—is deficiency of the B-complex vitamins, and there is no clear evidence to date that protein deficiency itself damages the eyes in these cases. The ways in which protein lack might interfere with various aspects of vitamin-A metabolism are discussed, but it is pointed out that their actual significance in human disease is not yet known. A low dietary intake of vitamin A is regarded by the author as being the prime factor in the causation of eye complications, and attention is drawn to the necessity to correct this as part of any prophylactic or therapeutic programme aimed primarily at combating protein malnutrition. PMID:13585077

  13. Yeast ABC proteins involved in multidrug resistance.

    PubMed

    Piecuch, Agata; Obłąk, Ewa

    2014-03-01

    Pleiotropic drug resistance is a complex phenomenon that involves many proteins that together create a network. One of the common mechanisms of multidrug resistance in eukaryotic cells is the active efflux of a broad range of xenobiotics through ATP-binding cassette (ABC) transporters. Saccharomyces cerevisiae is often used as a model to study such activity because of the functional and structural similarities of its ABC transporters to mammalian ones. Numerous ABC transporters are found in humans and some are associated with the resistance of tumors to chemotherapeutics. Efflux pump modulators that change the activity of ABC proteins are the most promising candidate drugs to overcome such resistance. These modulators can be chemically synthesized or isolated from natural sources (e.g., plant alkaloids) and might also be used in the treatment of fungal infections. There are several generations of synthetic modulators that differ in specificity, toxicity and effectiveness, and are often used for other clinical effects. PMID:24297686

  14. Van der Waals interactions involving proteins.

    PubMed Central

    Roth, C M; Neal, B L; Lenhoff, A M

    1996-01-01

    Van der Waals (dispersion) forces contribute to interactions of proteins with other molecules or with surfaces, but because of the structural complexity of protein molecules, the magnitude of these effects is usually estimated based on idealized models of the molecular geometry, e.g., spheres or spheroids. The calculations reported here seek to account for both the geometric irregularity of protein molecules and the material properties of the interacting media. Whereas the latter are found to fall in the generally accepted range, the molecular shape is shown to cause the magnitudes of the interactions to differ significantly from those calculated using idealized models, with important consequences. First, the roughness of the molecular surface leads to much lower average interaction energies for both protein-protein and protein-surface cases relative to calculations in which the protein molecule is approximated as a sphere. These results indicate that a form of steric stabilization may be an important effect in protein solutions. Underlying this behavior is appreciable orientational dependence, one reflection of which is that molecules of complementary shape are found to exhibit very strong attractive dispersion interactions. Although this has been widely discussed previously in the context of molecular recognition processes, the broader implications of these phenomena may also be important at larger molecular separations, e.g., in the dynamics of aggregation, precipitation, and crystal growth. Images FIGURE 3 PMID:8789115

  15. Van der Waals Interactions Involving Proteins

    NASA Technical Reports Server (NTRS)

    Roth, Charles M.; Neal, Brian L.; Lenhoff, Abraham M.

    1996-01-01

    Van der Waals (dispersion) forces contribute to interactions of proteins with other molecules or with surfaces, but because of the structural complexity of protein molecules, the magnitude of these effects is usually estimated based on idealized models of the molecular geometry, e.g., spheres or spheroids. The calculations reported here seek to account for both the geometric irregularity of protein molecules and the material properties of the interacting media. Whereas the latter are found to fall in the generally accepted range, the molecular shape is shown to cause the magnitudes of the interactions to differ significantly from those calculated using idealized models. with important consequences. First, the roughness of the molecular surface leads to much lower average interaction energies for both protein-protein and protein-surface cases relative to calculations in which the protein molecule is approximated as a sphere. These results indicate that a form of steric stabilization may be an important effect in protein solutions. Underlying this behavior is appreciable orientational dependence, one reflection of which is that molecules of complementary shape are found to exhibit very strong attractive dispersion interactions. Although this has been widely discussed previously in the context of molecular recognition processes, the broader implications of these phenomena may also be important at larger molecular separations, e.g., in the dynamics of aggregation, precipitation, and crystal growth.

  16. Methods for Mapping of Interaction Networks Involving Membrane Proteins

    SciTech Connect

    Hooker, Brian S.; Bigelow, Diana J.; Lin, Chiann Tso

    2007-11-23

    Numerous approaches have been taken to study protein interactions, such as tagged protein complex isolation followed by mass spectrometry, yeast two-hybrid methods, fluorescence resonance energy transfer, surface plasmon resonance, site-directed mutagenesis, and crystallography. Membrane protein interactions pose significant challenges due to the need to solubilize membranes without disrupting protein-protein interactions. Traditionally, analysis of isolated protein complexes by high-resolution 2D gel electrophoresis has been the main method used to obtain an overall picture of proteome constituents and interactions. However, this method is time consuming, labor intensive, detects only abundant proteins and is not suitable for the coverage required to elucidate large interaction networks. In this review, we discuss the application of various methods to elucidate interactions involving membrane proteins. These techniques include methods for the direct isolation of single complexes or interactors as well as methods for characterization of entire subcellular and cellular interactomes.

  17. Identification of Inhibitors of Biological Interactions Involving Intrinsically Disordered Proteins

    PubMed Central

    Marasco, Daniela; Scognamiglio, Pasqualina Liana

    2015-01-01

    Protein–protein interactions involving disordered partners have unique features and represent prominent targets in drug discovery processes. Intrinsically Disordered Proteins (IDPs) are involved in cellular regulation, signaling and control: they bind to multiple partners and these high-specificity/low-affinity interactions play crucial roles in many human diseases. Disordered regions, terminal tails and flexible linkers are particularly abundant in DNA-binding proteins and play crucial roles in the affinity and specificity of DNA recognizing processes. Protein complexes involving IDPs are short-lived and typically involve short amino acid stretches bearing few “hot spots”, thus the identification of molecules able to modulate them can produce important lead compounds: in this scenario peptides and/or peptidomimetics, deriving from structure-based, combinatorial or protein dissection approaches, can play a key role as hit compounds. Here, we propose a panoramic review of the structural features of IDPs and how they regulate molecular recognition mechanisms focusing attention on recently reported drug-design strategies in the field of IDPs. PMID:25849651

  18. Oligouridylate Binding Protein 1b Plays an Integral Role in Plant Heat Stress Tolerance.

    PubMed

    Nguyen, Cam Chau; Nakaminami, Kentaro; Matsui, Akihiro; Kobayashi, Shuhei; Kurihara, Yukio; Toyooka, Kiminori; Tanaka, Maho; Seki, Motoaki

    2016-01-01

    Stress granules (SGs), which are formed in the plant cytoplasm under stress conditions, are transient dynamic sites (particles) for mRNA storage. SGs are actively involved in protecting mRNAs from degradation. Oligouridylate binding protein 1b (UBP1b) is a component of SGs. The formation of microscopically visible cytoplasmic foci, referred to as UBP1b SG, was induced by heat treatment in UBP1b-overexpressing Arabidopsis plants (UBP1b-ox). A detailed understanding of the function of UBP1b, however, is still not clear. UBP1b-ox plants displayed increased heat tolerance, relative to control plants, while ubp1b mutants were more sensitive to heat stress than control plants. Microarray analysis identified 117 genes whose expression was heat-inducible and higher in the UBP1b-ox plants. RNA decay analysis was performed using cordycepin, a transcriptional inhibitor. In order to determine if those genes serve as targets of UBP1b, the rate of RNA degradation of a DnaJ heat shock protein and a stress-associated protein (AtSAP3) in UBP1b-ox plants was slower than in control plants; indicating that the mRNAs of these genes were protected within the UBP1b SG granule. Collectively, these data demonstrate that UBP1b plays an integral role in heat stress tolerance in plants. PMID:27379136

  19. Oligouridylate Binding Protein 1b Plays an Integral Role in Plant Heat Stress Tolerance

    PubMed Central

    Nguyen, Cam Chau; Nakaminami, Kentaro; Matsui, Akihiro; Kobayashi, Shuhei; Kurihara, Yukio; Toyooka, Kiminori; Tanaka, Maho; Seki, Motoaki

    2016-01-01

    Stress granules (SGs), which are formed in the plant cytoplasm under stress conditions, are transient dynamic sites (particles) for mRNA storage. SGs are actively involved in protecting mRNAs from degradation. Oligouridylate binding protein 1b (UBP1b) is a component of SGs. The formation of microscopically visible cytoplasmic foci, referred to as UBP1b SG, was induced by heat treatment in UBP1b-overexpressing Arabidopsis plants (UBP1b-ox). A detailed understanding of the function of UBP1b, however, is still not clear. UBP1b-ox plants displayed increased heat tolerance, relative to control plants, while ubp1b mutants were more sensitive to heat stress than control plants. Microarray analysis identified 117 genes whose expression was heat-inducible and higher in the UBP1b-ox plants. RNA decay analysis was performed using cordycepin, a transcriptional inhibitor. In order to determine if those genes serve as targets of UBP1b, the rate of RNA degradation of a DnaJ heat shock protein and a stress-associated protein (AtSAP3) in UBP1b-ox plants was slower than in control plants; indicating that the mRNAs of these genes were protected within the UBP1b SG granule. Collectively, these data demonstrate that UBP1b plays an integral role in heat stress tolerance in plants. PMID:27379136

  20. Viral and host proteins involved in picornavirus life cycle.

    PubMed

    Lin, Jing-Yi; Chen, Tzu-Chun; Weng, Kuo-Feng; Chang, Shih-Cheng; Chen, Li-Lien; Shih, Shin-Ru

    2009-01-01

    Picornaviruses cause several diseases, not only in humans but also in various animal hosts. For instance, human enteroviruses can cause hand-foot-and-mouth disease, herpangina, myocarditis, acute flaccid paralysis, acute hemorrhagic conjunctivitis, severe neurological complications, including brainstem encephalitis, meningitis and poliomyelitis, and even death. The interaction between the virus and the host is important for viral replication, virulence and pathogenicity. This article reviews studies of the functions of viral and host factors that are involved in the life cycle of picornavirus. The interactions of viral capsid proteins with host cell receptors is discussed first, and the mechanisms by which the viral and host cell factors are involved in viral replication, viral translation and the switch from translation to RNA replication are then addressed. Understanding how cellular proteins interact with viral RNA or viral proteins, as well as the roles of each in viral infection, will provide insights for the design of novel antiviral agents based on these interactions. PMID:19925687

  1. Dialysis-related amyloidosis: visceral involvement and protein constituents.

    PubMed

    Campistol, J M; Argilés, A

    1996-01-01

    beta 2-M amyloidosis mainly concerns dialysis patients and typically presents with osteoarticular symptoms. In order to precise the incidence and gravity of visceral involvement, subcutaneous abdominal fat aspirates, skin and rectal biopsies, as well as echocardiograms were performed in 26 patients with severe beta 2-M amyloidosis. Visceral amyloidosis was confirmed in 58% and the numbers were even higher when including heart abnormalities suggestive of amyloidosis (81%). Clinical manifestations of visceral involvement were usually not severe and include odynophagia, gastrointestinal haemorrhage, intestinal obstruction, kidney stones, myocardial dysfunction and subcutaneous tumours. The removal and synthesis rates of beta 2-M were assessed during dialysis. Serum 131I-beta 2-M levels decreased by 5-10% with cuprophane and by 40-45% with polysulfone and polyacrylonitrile membranes. These reduction rates were higher than those found with unlabelled beta 2-M suggesting an increased synthesis or release during dialysis. The protein constituents of amyloid deposits were studied. Two different preparative methods to extract the proteins from amyloid deposits were used. TCA precipitation showed the presence of several proteins which were not observed with PBS homogenizing and resuspending in guanidine. The protein constituents of amyloid fibrils were studied by both, two dimensional gel electrophoresis (2D-gel) as well as protein sequencing after gel filtration. Similarly, the technical approach used for protein analysis greatly influenced the results. It was observed that 2D-gel displayed the presence of proteins which were missed by the gel filtration technique. Some of the proteins contained in amyloid deposits in addition to beta 2-M, were identified as globin chains, kappa and lambda light chains of immunoglobulins, and alpha 2 macroglobulin. A putative participation of these other protein constituents on the pathogenesis of beta 2-microglobulin amyloidosis is

  2. Identifying Unstable Regions of Proteins Involved in Misfolding Diseases

    NASA Astrophysics Data System (ADS)

    Guest, Will; Cashman, Neil; Plotkin, Steven

    2009-05-01

    Protein misfolding is a necessary step in the pathogenesis of many diseases, including Creutzfeldt-Jakob disease (CJD) and familial amyotrophic lateral sclerosis (fALS). Identifying unstable structural elements in their causative proteins elucidates the early events of misfolding and presents targets for inhibition of the disease process. An algorithm was developed to calculate the Gibbs free energy of unfolding for all sequence-contiguous regions of a protein using three methods to parameterize energy changes: a modified G=o model, changes in solvent-accessible surface area, and all-atoms molecular dynamics. The entropic effects of disulfide bonds and post-translational modifications are treated analytically. It incorporates a novel method for finding local dielectric constants inside a protein to accurately handle charge effects. We have predicted the unstable parts of prion protein and superoxide dismutase 1, the proteins involved in CJD and fALS respectively, and have used these regions as epitopes to prepare antibodies that are specific to the misfolded conformation and show promise as therapeutic agents.

  3. First identification of proteins involved in motility of Mycoplasma gallisepticum.

    PubMed

    Indikova, Ivana; Vronka, Martin; Szostak, Michael P

    2014-01-01

    Mycoplasma gallisepticum, the most pathogenic mycoplasma in poultry, is able to glide over solid surfaces. Although this gliding motility was first observed in 1968, no specific protein has yet been shown to be involved in gliding. We examined M. gallisepticum strains and clonal variants for motility and found that the cytadherence proteins GapA and CrmA were required for gliding. Loss of GapA or CrmA resulted in the loss of motility and hemadsorption and led to drastic changes in the characteristic flask-shape of the cells. To identify further genes involved in motility, a transposon mutant library of M. gallisepticum was generated and screened for motility-deficient mutants, using a screening assay based on colony morphology. Motility-deficient mutants had transposon insertions in gapA and the neighbouring downstream gene crmA. In addition, insertions were seen in gene mgc2, immediately upstream of gapA, in two motility-deficient mutants. In contrast to the GapA/CrmA mutants, the mgc2 motility mutants still possessed the ability to hemadsorb. Complementation of these mutants with a mgc2-hexahistidine fusion gene restored the motile phenotype. This is the first report assigning specific M. gallisepticum proteins to involvement in gliding motility. PMID:25323771

  4. Proteomic detection of proteins involved in perchlorate and chlorate metabolism.

    PubMed

    Bansal, Reema; Deobald, Lee A; Crawford, Ronald L; Paszczynski, Andrzej J

    2009-09-01

    Mass spectrometry and a time-course cell lysis method were used to study proteins involved in perchlorate and chlorate metabolism in pure bacterial cultures and environmental samples. The bacterial cultures used included Dechlorosoma sp. KJ, Dechloromonas hortensis, Pseudomonas chloritidismutans ASK-1, and Pseudomonas stutzeri. The environmental samples included an anaerobic sludge enrichment culture from a sewage treatment plant, a sample of a biomass-covered activated carbon matrix from a bioreactor used for treating perchlorate-contaminated drinking water, and a waste water effluent sample from a paper mill. The approach focused on detection of perchlorate (and chlorate) reductase and chlorite dismutase proteins, which are the two central enzymes in the perchlorate (or chlorate) reduction pathways. In addition, acetate-metabolizing enzymes in pure bacterial samples and housekeeping proteins from perchlorate (or chlorate)-reducing microorganisms in environmental samples were also identified. PMID:19199051

  5. Molecular signaling involving intrinsically disordered proteins in prostate cancer

    PubMed Central

    Russo, Anna; Manna, Sara La; Novellino, Ettore; Malfitano, Anna Maria; Marasco, Daniela

    2016-01-01

    Investigations on cellular protein interaction networks (PINs) reveal that proteins that constitute hubs in a PIN are notably enriched in Intrinsically Disordered Proteins (IDPs) compared to proteins that constitute edges, highlighting the role of IDPs in signaling pathways. Most IDPs rapidly undergo disorder-to-order transitions upon binding to their biological targets to perform their function. Conformational dynamics enables IDPs to be versatile and to interact with a broad range of interactors under normal physiological conditions where their expression is tightly modulated. IDPs are involved in many cellular processes such as cellular signaling, transcriptional regulation, and splicing; thus, their high-specificity/low-affinity interactions play crucial roles in many human diseases including cancer. Prostate cancer (PCa) is one of the leading causes of cancer-related mortality in men worldwide. Therefore, identifying molecular mechanisms of the oncogenic signaling pathways that are involved in prostate carcinogenesis is crucial. In this review, we focus on the aspects of cellular pathways leading to PCa in which IDPs exert a primary role. PMID:27212129

  6. Molecular signaling involving intrinsically disordered proteins in prostate cancer.

    PubMed

    Russo, Anna; Manna, Sara La; Novellino, Ettore; Malfitano, Anna Maria; Marasco, Daniela

    2016-01-01

    Investigations on cellular protein interaction networks (PINs) reveal that proteins that constitute hubs in a PIN are notably enriched in Intrinsically Disordered Proteins (IDPs) compared to proteins that constitute edges, highlighting the role of IDPs in signaling pathways. Most IDPs rapidly undergo disorder-to-order transitions upon binding to their biological targets to perform their function. Conformational dynamics enables IDPs to be versatile and to interact with a broad range of interactors under normal physiological conditions where their expression is tightly modulated. IDPs are involved in many cellular processes such as cellular signaling, transcriptional regulation, and splicing; thus, their high-specificity/low-affinity interactions play crucial roles in many human diseases including cancer. Prostate cancer (PCa) is one of the leading causes of cancer-related mortality in men worldwide. Therefore, identifying molecular mechanisms of the oncogenic signaling pathways that are involved in prostate carcinogenesis is crucial. In this review, we focus on the aspects of cellular pathways leading to PCa in which IDPs exert a primary role. PMID:27212129

  7. Analysis of proteins involved in biodegradation of crop biomass

    NASA Technical Reports Server (NTRS)

    Crawford, Kamau; Trotman, Audrey

    1998-01-01

    The biodegradation of crop biomass for re-use in crop production is part of the bioregenerative life support concept proposed by the National Aeronautics and Space Administration (NASA) for long duration, manned space exploration. The current research was conducted in the laboratory to evaluate the use of electrophoretic analysis as a means of rapidly assaying for constitutive and induced proteins associated with the bacterial degradation of crop residue. The proteins involved in crop biomass biodegradation are either constitutive or induced. As a result, effluent and cultures were examined to investigate the potential of using electrophoretic techniques as a means of monitoring the biodegradation process. Protein concentration for optimum banding patterns was determined using the Bio-Rad Protein Assay kit. Four bacterial soil isolates were obtained from the G.W. Carver research Farm at Tuskegee University and used in the decomposition of components of plant biomass. The culture, WDSt3A was inoculated into 500 mL of either Tryptic Soy Broth or Nutrient Broth. Incubation, with shaking of each flask was for 96 hours at 30 C. The cultures consistently gave unique banding patterns under denaturing protein electrophoresis conditions, The associated extracellular enzymes also yielded characteristic banding patterns over a 14-day period, when native electrophoresis techniques were used to examine effluent from batch culture bioreactors. The current study evaluated sample preparation and staining protocols to determine the ease of use, reproducibility and reliability, as well as the potential for automation.

  8. Involvement of heat shock proteins in gluten-sensitive enteropathy

    PubMed Central

    Sziksz, Erna; Pap, Domonkos; Veres, Gábor; Fekete, Andrea; Tulassay, Tivadar; Vannay, Ádám

    2014-01-01

    Gluten-sensitive enteropathy, also known as coeliac disease (CD), is an autoimmune disorder occurring in genetically susceptible individuals that damages the small intestine and interferes with the absorption of other nutrients. As it is triggered by dietary gluten and related prolamins present in wheat, rye and barley, the accepted treatment for CD is a strict gluten-free diet. However, a complete exclusion of gluten-containing cereals from the diet is often difficult, and new therapeutic strategies are urgently needed. A class of proteins that have already emerged as drug targets for other autoimmune diseases are the heat shock proteins (HSPs), which are highly conserved stress-induced chaperones that protect cells against harmful extracellular factors. HSPs are expressed in several tissues, including the gastrointestinal tract, and their levels are significantly increased under stress circumstances. HSPs exert immunomodulatory effects, and also play a crucial role in the maintenance of epithelial cell structure and function, as they are responsible for adequate protein folding, influence the degradation of proteins and cell repair processes after damage, and modulate cell signalling, cell proliferation and apoptosis. The present review discusses the involvement of HSPs in the pathophysiology of CD. Furthermore, HSPs may represent a useful therapeutic target for the treatment of CD due to the cytoprotective, immunomodulatory, and anti-apoptotic effects in the intestinal mucosal barrier. PMID:24914370

  9. Interleukin 2 signaling involves the phosphorylation of Stat proteins.

    PubMed

    Frank, D A; Robertson, M J; Bonni, A; Ritz, J; Greenberg, M E

    1995-08-15

    One of the most important cytokines involved in immune response regulation is interleukin 2 (IL-2), a potent activator of the proliferation and function of T lymphocytes and natural killer cells. The mechanisms by which the effects of IL-2 are propagated within cells are not understood. While the binding of IL-2 to its receptor was recently shown to lead to the activation of two kinases, Jak-1 and Jak-3, subsequent steps in the signaling pathway to the nucleus that lead to the activation of specific genes had not been characterized. Since many cytokines that activate Jak kinases also lead to the tyrosine phosphorylation and activation of members of the Stat family of transcription factors, the ability of IL-2 to trigger Stat phosphorylation was examined. Exposure of activated human T lymphocytes or of a natural killer cell line (NKL) to IL-2 leads to the phosphorylation of Stat1 alpha, Stat1 beta, and Stat3, as well as of two Stat-related proteins, p94 and p95. p94 and p95 share homology with Stat1 at the phosphorylation site and in the Src homology 2 (SH2) domain, but otherwise are immunologically distinct from Stat1. These Stat proteins were found to translocate to the nucleus and to bind to a specific DNA sequence. These findings suggest a mechanism by which IL-2 binding to its receptor may activate specific genes involved in immune cell function. PMID:7544001

  10. Structural Insights into Protein-Protein Interactions Involved in Bacterial Cell Wall Biogenesis

    PubMed Central

    Laddomada, Federica; Miyachiro, Mayara M.; Dessen, Andréa

    2016-01-01

    The bacterial cell wall is essential for survival, and proteins that participate in its biosynthesis have been the targets of antibiotic development efforts for decades. The biosynthesis of its main component, the peptidoglycan, involves the coordinated action of proteins that are involved in multi-member complexes which are essential for cell division (the “divisome”) and/or cell wall elongation (the “elongasome”), in the case of rod-shaped cells. Our knowledge regarding these interactions has greatly benefitted from the visualization of different aspects of the bacterial cell wall and its cytoskeleton by cryoelectron microscopy and tomography, as well as genetic and biochemical screens that have complemented information from high resolution crystal structures of protein complexes involved in divisome or elongasome formation. This review summarizes structural and functional aspects of protein complexes involved in the cytoplasmic and membrane-related steps of peptidoglycan biosynthesis, with a particular focus on protein-protein interactions whereby disruption could lead to the development of novel antibacterial strategies. PMID:27136593

  11. Adenanthin targets proteins involved in the regulation of disulphide bonds.

    PubMed

    Muchowicz, Angelika; Firczuk, Małgorzata; Chlebowska, Justyna; Nowis, Dominika; Stachura, Joanna; Barankiewicz, Joanna; Trzeciecka, Anna; Kłossowski, Szymon; Ostaszewski, Ryszard; Zagożdżon, Radosław; Pu, Jian-Xin; Sun, Han-Dong; Golab, Jakub

    2014-05-15

    Adenanthin has been recently shown to inhibit the enzymatic activities of peroxiredoxins (Prdx) I and II through its functional α,β-unsaturated ketone group serving as a Michael acceptor. A similar group is found in SK053, a compound recently developed by our group to target the thioredoxin-thioredoxin reductase (Trx-TrxR) system. This work provides evidence that next to Prdx I and II adenanthin targets additional proteins including thioredoxin-thioredoxin reductase system as well as protein disulfide isomerase (PDI) that contain a characteristic structural motif, referred to as a thioredoxin fold. Adenanthin inhibits the activity of Trx-TR system and PDI in vitro in the insulin reduction assay and decreases the activity of Trx in cultured cells. Moreover, we identified Trx-1 as an adenanthin binding protein in cells incubated with biotinylated adenanthin as an affinity probe. The results of our studies indicate that adenanthin is a mechanism-selective, rather than an enzyme-specific inhibitor of enzymes containing readily accessible, nucleophilic cysteines. This observation might be of importance in considering potential therapeutic applications of adenanthin to include a range of diseases, where aberrant activity of Prdx, Trx-TrxR and PDI is involved in their pathogenesis. PMID:24630929

  12. Life under tension: Computational studies of proteins involved in mechanotransduction

    NASA Astrophysics Data System (ADS)

    Sotomayor, Marcos Manuel

    cadherins. Simulations also revealed how calcium ions control cadherin's shape and the availability of key residues involved in cell-cell adhesion, suggesting a conceptual framework for interpreting mutations in cadherin calcium binding motifs causing hereditary deafness. Overall, simulations provided a unique nanoscopic view of the dynamics and function of some of the proteins involved in mechanotransduction.

  13. A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum.

    PubMed Central

    Dierks, T; Volkmer, J; Schlenstedt, G; Jung, C; Sandholzer, U; Zachmann, K; Schlotterhose, P; Neifer, K; Schmidt, B; Zimmermann, R

    1996-01-01

    Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates. Images PMID:9003769

  14. Protein kinase C in pain: Involvement of multiple isoforms

    PubMed Central

    Velázquez, Kandy T.; Mohammad, Husam; Sweitzer, Sarah M.

    2007-01-01

    Pain is the primary reason that people seek medical care. At present chronic unremitting pain is the third greatest health problem after heart disease and cancer. Chronic pain is an economic burden in lost wages, lost productivity, medical expenses, legal fees and compensation. Chronic pain is defined as a pain of greater than two months duration and can be of an inflammatory or neuropathic origin that can arise following nerve injury or in the absence of any apparent injury. Chronic pain is characterized by an altered pain perception that includes allodynia (a response to a normally non-noxious stimuli), and hyperalgesia (an exaggerated response to a normally noxious stimuli). This type of pain is often insensitive to the traditional pain drugs or surgical intervention and thus the study of the cellular and molecular mechanisms that contribute to chronic pain are of the up-most importance for the development of a new generation of analgesic agents. Protein kinase C isozymes are under investigation as potential therapeutics for the treatment of chronic pain conditions. The anatomical localization of protein kinase C isozymes in both peripheral and central nervous system sites that process pain have made them the topic of basic science research for close to two decades. This review will outline the research to date on protein kinase C involvement in pain and analgesia. In addition, this review will try to synthesize these works to begin to develop a comprehensive mechanistic understanding of how protein kinase C may function as the master regulator of peripheral and central sensitization that underlies many chronic pain conditions. PMID:17548207

  15. Ex vivo identification of protein-protein interactions involving the dopamine transporter.

    PubMed

    Hadlock, Gregory C; Nelson, Chad C; Baucum, Anthony J; Hanson, Glen R; Fleckenstein, Annette E

    2011-03-30

    The dopamine (DA) transporter (DAT) is a key regulator of dopaminergic signaling as it mediates the reuptake of extrasynaptic DA and thereby terminates dopaminergic signaling. Emerging evidence indicates that DAT function is influenced through interactions with other proteins. The current report describes a method to identify such interactions following DAT immunoprecipitation from a rat striatal synaptosomal preparation. This subcellular fraction was selected since DAT function is often determined ex vivo by measuring DA uptake in this preparation and few reports investigating DAT-protein interactions have utilized this preparation. Following SDS-PAGE and colloidal Coomassie staining, selected protein bands from a DAT-immunoprecipitate were excised, digested with trypsin, extracted, and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). From the analysis of the tryptic peptides, several proteins were identified including DAT, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) β, CaMKII δ, protein kinase C (PKC) β, and PKC γ. Co-immunoprecipitation of PKC, CaMKII, and protein interacting with C kinase-1 with DAT was confirmed by Western blotting. Thus, the present study highlights a method to immunoprecipitate DAT and to identify co-immunoprecipitating proteins using LC/MS/MS and Western blotting. This method can be utilized to evaluate DAT protein-protein interactions but also to assess interactions involving other synaptic proteins. Ex vivo identification of protein-protein interactions will provide new insight into the function and regulation of a variety of synaptic, membrane-associated proteins, including DAT. PMID:21291912

  16. Structural Reconstruction of Protein-Protein Complexes Involved in Intracellular Signaling.

    PubMed

    Kirsch, Klára; Sok, Péter; Reményi, Attila

    2016-01-01

    Signaling complexes within the cell convert extracellular cues into physiological outcomes. Their assembly involves signaling enzymes, allosteric regulators and scaffold proteins that often contain long stretches of disordered protein regions, display multi-domain architectures, and binding affinity between individual components is low. These features are indispensable for their central roles as dynamic information processing hubs, on the other hand they also make reconstruction of structurally homogeneous complex samples highly challenging. In this present chapter we discuss protein machinery which influences extracellular signal reception, intracellular pathway activity, and cytoskeletal or transcriptional activity. PMID:27165334

  17. Protein Acetylation Is Involved in Salmonella enterica Serovar Typhimurium Virulence.

    PubMed

    Sang, Yu; Ren, Jie; Ni, Jinjing; Tao, Jing; Lu, Jie; Yao, Yu-Feng

    2016-06-01

    Salmonella causes a range of diseases in different hosts, including enterocolitis and systemic infection. Lysine acetylation regulates many eukaryotic cellular processes, but its function in bacteria is largely unexplored. The acetyltransferase Pat and NAD(+)-dependent deacetylase CobB are involved in the reversible protein acetylation in Salmonella Typhimurium. Here, we used cell and animal models to evaluate the virulence of pat and cobB deletion mutants in S. Typhimurium and found that pat is critical for bacterial intestinal colonization and systemic infection. Next, to understand the underlying mechanism, genome-wide transcriptome was analyzed. RNA sequencing data showed that the expression of Salmonella pathogenicity island 1 (SPI-1) is partially dependent on pat In addition, we found that HilD, a key transcriptional regulator of SPI-1, is a substrate of Pat. The acetylation of HilD by Pat maintained HilD stability and was essential for the transcriptional activation of HilA. Taken together, these results suggest that a protein acetylation system regulates SPI-1 expression by controlling HilD in a posttranslational manner to mediate S. Typhimurium virulence. PMID:26810370

  18. Arabinogalactan proteins are involved in root hair development in barley

    PubMed Central

    Marzec, Marek; Szarejko, Iwona; Melzer, Michael

    2015-01-01

    The arabinogalactan proteins (AGPs) are involved in a range of plant processes, including cell differentiation and expansion. Here, barley root hair mutants and their wild-type parent cultivars were used, as a model system, to reveal the role of AGPs in root hair development. The treatment of roots with different concentrations of βGlcY (a reagent which binds to all classes of AGPs) inhibited or totally suppressed the development of root hairs in all of the cultivars. Three groups of AGP (recognized by the monoclonal antibodies LM2, LM14, and MAC207) were diversely localized in trichoblasts and atrichoblasts of root hair-producing plants. The relevant epitopes were present in wild-type trichoblast cell walls and cytoplasm, whereas in wild-type atrichoblasts and in all epidermal cells of a root hairless mutant, they were only present in the cytoplasm. In all of cultivars the higher expression of LM2, LM14, and MAC207 was observed in trichoblasts at an early stage of development. Additionally, the LM2 epitope was detected on the surface of primordia and root hair tubes in plants able to generate root hairs. The major conclusion was that the AGPs recognized by LM2, LM14, and MAC207 are involved in the differentiation of barley root epidermal cells, thereby implying a requirement for these AGPs for root hair development in barley. PMID:25465033

  19. Arabinogalactan proteins are involved in root hair development in barley.

    PubMed

    Marzec, Marek; Szarejko, Iwona; Melzer, Michael

    2015-03-01

    The arabinogalactan proteins (AGPs) are involved in a range of plant processes, including cell differentiation and expansion. Here, barley root hair mutants and their wild-type parent cultivars were used, as a model system, to reveal the role of AGPs in root hair development. The treatment of roots with different concentrations of βGlcY (a reagent which binds to all classes of AGPs) inhibited or totally suppressed the development of root hairs in all of the cultivars. Three groups of AGP (recognized by the monoclonal antibodies LM2, LM14, and MAC207) were diversely localized in trichoblasts and atrichoblasts of root hair-producing plants. The relevant epitopes were present in wild-type trichoblast cell walls and cytoplasm, whereas in wild-type atrichoblasts and in all epidermal cells of a root hairless mutant, they were only present in the cytoplasm. In all of cultivars the higher expression of LM2, LM14, and MAC207 was observed in trichoblasts at an early stage of development. Additionally, the LM2 epitope was detected on the surface of primordia and root hair tubes in plants able to generate root hairs. The major conclusion was that the AGPs recognized by LM2, LM14, and MAC207 are involved in the differentiation of barley root epidermal cells, thereby implying a requirement for these AGPs for root hair development in barley. PMID:25465033

  20. Interacting Protein Kinases Involved in the Regulation of Flagellar Length

    PubMed Central

    Erdmann, Maja; Scholz, Anne; Melzer, Inga M.; Schmetz, Christel; Wiese, Martin

    2006-01-01

    A striking difference of the life stages of the protozoan parasite Leishmania is a long flagellum in the insect stage promastigotes and a rudimentary organelle in the mammalian amastigotes. LmxMKK, a mitogen-activated protein (MAP) kinase kinase from Leishmania mexicana, is required for growth of a full-length flagellum. We identified LmxMPK3, a MAP kinase homologue, with a similar expression pattern as LmxMKK being not detectable in amastigotes, up-regulated during the differentiation to promastigotes, constantly expressed in promastigotes, and shut down during the differentiation to amastigotes. LmxMPK3 null mutants resemble the LmxMKK knockouts with flagella reduced to one-fifth of the wild-type length, stumpy cell bodies, and vesicles and membrane fragments in the flagellar pocket. A constitutively activated recombinant LmxMKK activates LmxMPK3 in vitro. Moreover, LmxMKK is likely to be directly involved in the phosphorylation of LmxMPK3 in vivo. Finally, LmxMPK3 is able to phosphorylate LmxMKK, indicating a possible feedback regulation. This is the first time that two interacting components of a signaling cascade have been described in the genus Leishmania. Moreover, we set the stage for the analysis of reversible phosphorylation in flagellar morphogenesis. PMID:16467378

  1. Interplay between circadian rhythm, time of the day and osmotic stress constraints in the regulation of the expression of a Solanum Double B-box gene

    PubMed Central

    Kiełbowicz-Matuk, Agnieszka; Rey, Pascal; Rorat, Tadeusz

    2014-01-01

    Background and Aims Double B-box zinc finger (DBB) proteins are recently identified plant transcription regulators that participate in the response to sodium chloride-induced stress in arabidopsis plants. Little is known regarding their subcellular localization and expression patterns, particularly in relation to other osmotic constraints and the day/night cycle. This study investigated natural variations in the amount of a Solanum DBB protein, SsBBX24, during plant development, and also under various environmental constraints leading to cell dehydration in relation to the circadian clock and the time of day. Methods SsBBX24 transcript and protein abundance in various organs of phytotron-grown Solanum tuberosum and S. sogarandinum plants were investigated at different time points of the day and under various osmotic constraints. The intracellular location of SsBBX24 was determined by western blot analysis of subcellular fractions. Key Results Western blot analysis of SsBBX24 protein revealed that it was located in the nucleus at the beginning of the light period and in the cytosol at the end, suggesting movement (‘trafficking’) during the light phase. SsBBX24 gene expression exhibited circadian cycling under control conditions, with the highest and lowest abundances of both transcript and protein occurring 8 and 18 h after dawn, respectively. Exposing Solanum plants to low temperature, salinity and polyethylene glycol (PEG), but not to drought, disturbed the circadian regulation of SsBBX24 gene expression at the protein level. SsBBX24 transcript and protein accumulated in Solanum plants in response to salt and PEG treatments, but not in response to low temperature or water deficit. Most interestingly, the time of the day modulated the magnitude of SsBBX24 expression in response to high salt concentration. Conclusions The interplay between circadian rhythm and osmotic constraints in the regulation of the expression of a Solanum DBB transcriptional regulator is

  2. Identification of a plastid protein involved in vesicle fusion and/or membrane protein translocation.

    PubMed Central

    Hugueney, P; Bouvier, F; Badillo, A; d'Harlingue, A; Kuntz, M; Camara, B

    1995-01-01

    Structural evidence has accumulated suggesting that fusion and/or translocation factors are involved in plastid membrane biogenesis. To test this hypothesis, we have developed an in vitro system in which the extent of fusion and/or translocation is monitored by the conversion of the xanthophyll epoxide (antheraxanthin) into the red ketocarotenoid (capsanthin). Only chromoplast membrane vesicles from red pepper fruits (Capsicum annuum) contain the required enzyme. Vesicles prepared from the mutant yellow cultivar are devoid of this enzyme and accumulate antheraxanthin. The fusion and/or translocation activity is characterized by complementation due to the synthesis of capsanthin and the parallel decrease of antheraxanthin when the two types of vesicles are incubated together in the presence of plastid stroma. We show that the extent of conversion is dependent upon an ATP-requiring protein that is sensitive to N-ethylmaleimide. Further purification and immunological analysis have revealed that the active factor, designated plastid fusion and/or translocation factor (Pftf), resides in a protein of 72 kDa. cDNA cloning revealed that mature Pftf has significant homology to yeast and animal (NSF) or bacterial (Ftsh) proteins involved in vesicle fusion or membrane protein translocation. Images Fig. 1 Fig. 3 Fig. 4 PMID:7777561

  3. DUF581 Is Plant Specific FCS-Like Zinc Finger Involved in Protein-Protein Interaction

    PubMed Central

    K, Muhammed Jamsheer; Laxmi, Ashverya

    2014-01-01

    Zinc fingers are a ubiquitous class of protein domain with considerable variation in structure and function. Zf-FCS is a highly diverged group of C2-C2 zinc finger which is present in animals, prokaryotes and viruses, but not in plants. In this study we identified that a plant specific domain of unknown function, DUF581 is a zf-FCS type zinc finger. Based on HMM-HMM comparison and signature motif similarity we named this domain as FCS-Like Zinc finger (FLZ) domain. A genome wide survey identified that FLZ domain containing genes are bryophytic in origin and this gene family is expanded in spermatophytes. Expression analysis of selected FLZ gene family members of A. thaliana identified an overlapping expression pattern suggesting a possible redundancy in their function. Unlike the zf-FCS domain, the FLZ domain found to be highly conserved in sequence and structure. Using a combination of bioinformatic and protein-protein interaction tools, we identified that FLZ domain is involved in protein-protein interaction. PMID:24901469

  4. Identification of Protein Interactions Involved in Cellular Signaling

    PubMed Central

    Westermarck, Jukka; Ivaska, Johanna; Corthals, Garry L.

    2013-01-01

    Protein-protein interactions drive biological processes. They are critical for all intra- and extracellular functions, and the technologies to analyze them are widely applied throughout the various fields of biological sciences. This study takes an in-depth view of some common principles of cellular regulation and provides a detailed account of approaches required to comprehensively map signaling protein-protein interactions in any particular cellular system or condition. We provide a critical review of the benefits and disadvantages of the yeast two-hybrid method and affinity purification coupled with mass spectrometric procedures for identification of signaling protein-protein interactions. In particular, we emphasize the quantitative and qualitative differences between tandem affinity and one-step purification (such as FLAG and Strep tag) methods. Although applicable to all types of interaction studies, a special section is devoted in this review to aspects that should be considered when attempting to identify signaling protein interactions that often are transient and weak by nature. Finally, we discuss shotgun and quantitative information that can be gleaned by MS-coupled methods for analysis of multiprotein complexes. PMID:23481661

  5. The VHL short variant involves in protein quality control.

    PubMed

    Liu, Yanbin; Yang, Haixia; Zuo, Feifei; Chen, Liang

    2016-09-01

    The von Hippel-Lindau (VHL) is the most important and frequently mutated gene in human clear cell renal cell carcinoma (ccRCC). In contrast to its long counterpart, the internal translational variant of VHL protein (VHLs) is evolutionarily conserved. Herein we present evidence that VHLs associates with ribosome complex via interaction with the large subunit 6 (RPL6). Manipulation of VHLs expression significantly alters protein synthesis, cell size and mitochondrial mass. VHLs deficiency leads to remarkable sensitivity to drug treatments eliciting nascent protein mis-folding and translational errors. The ubiquitination of nascent peptides are dramatically increased upon the ectopic over-expression of VHLs, which simultaneously co-localizes with proteasome and thus may facilitate the ubiquitin-proteasome mediated degradation. In summary, VHLs contributes to protein quality control in addition to its canonical function in maintaining homeostasis of hypoxia-induced factors alpha subunit (HIFα) in response to environmental oxygen supply. PMID:27196060

  6. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity

    PubMed Central

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; Camacho-Arroyo, Ignacio

    2015-01-01

    In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone. PMID:26635640

  7. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity.

    PubMed

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; Camacho-Arroyo, Ignacio

    2015-01-01

    In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone. PMID:26635640

  8. Differentiation of HL60 cells: involvement of protein phosphorylation

    SciTech Connect

    Spearman, T.N.; Fontana, J.A.; Butcher, F.R.; Durham, J.P.

    1986-05-01

    The addition of retinoic acid (RA) to the human promyelocytic leukemic cell line HL60 in culture results in the cessation of growth and the acquisition of a more mature phenotype. Previous work in these laboratories has demonstrated a concomitant increase in the activity of calcium-dependent, phospholipid-sensitive protein kinase (PK-C). HL60 cells were incubated with /sup 32/P-P/sub i/ in the absence and presence of RA, homogenized, and aliquots subjected to two-dimensional electrophoresis. A comparison of autoradiograms made from these gels revealed several phosphoproteins whose radiolabeling was affected by RA. The radiolabeling of one particular phosphoprotein (49kd, pI 4.8) was found to be increased prior to phenotypic evidence of differentiation. It was demonstrated via incubating HL60 cytosol with /sup 32/P -ATP and Ca/sup 2 +/ in the absence and presence of phosphatidylserine and resolving the labeled proteins as above that this protein is phosphorylated by PK-C. The labeling of this protein was also increased by RA in other leukemic cell lines which showed phenotypic evidence of differentiation while no effect was seen in HL60 sublines resistant to RA or in mature neutrophils (the end product of myeloid differentiation). These results suggest that this protein may be an important intermediate in myeloid differentiation.

  9. Protein Machineries Involved in the Attachment of Heme to Cytochrome c: Protein Structures and Molecular Mechanisms

    PubMed Central

    Travaglini-Allocatelli, Carlo

    2013-01-01

    Cytochromes c (Cyt c) are ubiquitous heme-containing proteins, mainly involved in electron transfer processes, whose structure and functions have been and still are intensely studied. Surprisingly, our understanding of the molecular mechanism whereby the heme group is covalently attached to the apoprotein (apoCyt) in the cell is still largely unknown. This posttranslational process, known as Cyt c biogenesis or Cyt c maturation, ensures the stereospecific formation of the thioether bonds between the heme vinyl groups and the cysteine thiols of the apoCyt heme binding motif. To accomplish this task, prokaryotic and eukaryotic cells have evolved distinctive protein machineries composed of different proteins. In this review, the structural and functional properties of the main maturation apparatuses found in gram-negative and gram-positive bacteria and in the mitochondria of eukaryotic cells will be presented, dissecting the Cyt c maturation process into three functional steps: (i) heme translocation and delivery, (ii) apoCyt thioreductive pathway, and (iii) apoCyt chaperoning and heme ligation. Moreover, current hypotheses and open questions about the molecular mechanisms of each of the three steps will be discussed, with special attention to System I, the maturation apparatus found in gram-negative bacteria. PMID:24455431

  10. Interactions of Dnd proteins involved in bacterial DNA phosphorothioate modification

    PubMed Central

    Xiong, Wei; Zhao, Gong; Yu, Hao; He, Xinyi

    2015-01-01

    DNA phosphorothioation (PT) is the first discovered physiological DNA backbone modification, in which a non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur atom in Rp (rectus for plane) configuration. PT modification is governed by a highly conserved gene cluster dndA/iscS-dndBCDE that is widespread across bacterial and archaeal species. However, little is known about how these proteins coordinately react with each other to perform oxygen–sulfur swap. We here demonstrated that IscS, DndC, DndD and DndE form a protein complex of which the molecular ratio for four proteins in the complex is approximate 1:1:1:1. DndB here displayed little or weak affinity to the complex and the constructs harboring dndACDE can confer the host in vivo PT modification. Using co-purification and pull down strategy, we demonstrated that the four proteins assemble into a pipeline in collinear to its gene organization, namely, IscS binding to DndC, DndC binding to DndD, and DndD binding to DndE. Moreover, weak interactions between DndE and IscS, DndE and DndC were also identified. PMID:26539172

  11. The Involvement of Transport Proteins in Transcriptional and Metabolic Regulation

    PubMed Central

    Västermark, Åke; Saier, Milton H.

    2014-01-01

    Transport proteins have sometimes gained secondary regulatory functions that influence gene expression and metabolism. These functions allow communication with the external world via mechanistically distinctive signal transduction pathways. In this brief review we focus on three transport systems in Escherichia coli that control and coordinate carbon, exogenous hexose-phosphate and phosphorous metabolism. The transport proteins that play central roles in these processes are (1) the phosphoenolpyruvate (PEP)-dependent phosphotransferase system, PTS, (2) the glucose-6-phosphate receptor, UhpC, and (3) the phosphate-specific transporter, PstSABC, respectively. While the PTS participates in multiple complex regulatory processes, three of which are discussed here, UhpC and the Pst transporters exemplify differing strategies. PMID:24513656

  12. Acanthamoeba castellanii: proteins involved in actin dynamics, glycolysis, and proteolysis are regulated during encystation.

    PubMed

    Bouyer, Sabrina; Rodier, Marie-Hélène; Guillot, Alain; Héchard, Yann

    2009-09-01

    Acanthamoeba castellanii is a pathogenic free-living amoeba. Cyst forms are particularly important in their pathogenicity, as they are more resistant to treatments and might protect pathogenic intracellular bacteria. However, encystation is poorly understood at the molecular level and global changes at the protein level have not been completely described. In this study, we performed two-dimensional gel electrophoresis to compare protein expression in trophozoite and cyst forms. Four proteins, specifically expressed in trophozoites, and four proteins, specifically expressed in cysts, were identified. Two proteins, enolase and fructose bisphosphate aldolase, are involved in the glycolytic pathway. Three proteins are likely actin-binding proteins, which is consistent with the dramatic morphological modifications of the cells during encystation. One protein belongs to the serine protease family and has been already linked to encystation in A. castellanii. In conclusion, this study found that the proteins whose expression was modified during encystation were likely involved in actin dynamics, glycolysis, and proteolysis. PMID:19523468

  13. P-proteins in Arabidopsis are heteromeric structures involved in rapid sieve tube sealing

    PubMed Central

    Jekat, Stephan B.; Ernst, Antonia M.; von Bohl, Andreas; Zielonka, Sascia; Twyman, Richard M.; Noll, Gundula A.; Prüfer, Dirk

    2013-01-01

    Structural phloem proteins (P-proteins) are characteristic components of the sieve elements in all dicotyledonous and many monocotyledonous angiosperms. Tobacco P-proteins were recently confirmed to be encoded by the widespread sieve element occlusion (SEO) gene family, and tobacco SEO proteins were shown to be directly involved in sieve tube sealing thus preventing the loss of photosynthate. Analysis of the two Arabidopsis SEO proteins (AtSEOa and AtSEOb) indicated that the corresponding P-protein subunits do not act in a redundant manner. However, there are still pending questions regarding the interaction properties and specific functions of AtSEOa and AtSEOb as well as the general function of structural P-proteins in Arabidopsis. In this study, we characterized the Arabidopsis P-proteins in more detail. We used in planta bimolecular fluorescence complementation assays to confirm the predicted heteromeric interactions between AtSEOa and AtSEOb. Arabidopsis mutants depleted for one or both AtSEO proteins lacked the typical P-protein structures normally found in sieve elements, underlining the identity of AtSEO proteins as P-proteins and furthermore providing the means to determine the role of Arabidopsis P-proteins in sieve tube sealing. We therefore developed an assay based on phloem exudation. Mutants with reduced AtSEO expression levels lost twice as much photosynthate following injury as comparable wild-type plants, confirming that Arabidopsis P-proteins are indeed involved in sieve tube sealing. PMID:23840197

  14. Involvement of protein kinase C activation in L-leucine-induced stimulation of protein synthesis in l6 myotubes.

    PubMed

    Yagasaki, Kazumi; Morisaki, Naoko; Kitahara, Yoshiro; Miura, Atsuhito; Funabiki, Ryuhei

    2003-11-01

    Effects of leucine and related compounds on protein synthesis were studied in L6 myotubes. The incorporation of [(3)H]tyrosine into cellular protein was measured as an index of protein synthesis. In leucine-depleted L6 myotubes, leucine and its keto acid, alpha-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipases A(2) and C, canceled stimulatory actions of L-leucine and KIC on protein synthesis. Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of proteinkinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. L-Leucine caused a rapid activation of protein kinase C in both cytosol and membrane fractions of the cells. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in L6 myotubes through activation of phospholipase C and protein kinase C. PMID:19003213

  15. Identification of the major lipoproteins in crayfish hemolymph as proteins involved in immune recognition and clotting.

    PubMed

    Hall, M; van Heusden, M C; Söderhäll, K

    1995-11-22

    Lipid-containing hemolymph proteins from males of the crayfish Pacifastacus leniusculus were isolated by density gradient ultracentrifugation. Two major lipoproteins, one high density lipoprotein (HDL) and one very high density lipoprotein (VHDL), were characterized. The HDL and the VHDL were found to be identical to two proteins previously studied for their roles in immune recognition and hemolymph clotting, namely the beta-1,3-glucan binding protein and the clotting protein. These results imply that crayfish lipoproteins have dual functions, and that they are involved in immunity, hemolymph clotting, and lipid transport in these animals. Also, the oxygen-transporting protein hemocyanin was found to have a small lipid content. PMID:7488215

  16. Dissection of the bifunctional ARGRII protein involved in the regulation of arginine anabolic and catabolic pathways.

    PubMed Central

    Qui, H F; Dubois, E; Messenguy, F

    1991-01-01

    ARGRII is a regulatory protein which regulates the arginine anabolic and catabolic pathways in combination with ARGRI and ARGRIII. We have investigated, by deletion analysis and fusion to LexA protein, the different domains of ARGRII protein. In contrast to other yeast regulatory proteins, 92% of ARGRII is necessary for its anabolic repression function and 80% is necessary for its catabolic activator function. We can define three domains in this protein: a putative DNA-binding domain containing a zinc finger motif, a region more involved in the repression activity located around the RNase-like sequence, and a large activation domain. Images PMID:2005903

  17. Involvement of Iron-Containing Proteins in Genome Integrity in Arabidopsis Thaliana

    PubMed Central

    Zhang, Caiguo

    2015-01-01

    The Arabidopsis genome encodes numerous iron-containing proteins such as iron-sulfur (Fe-S) cluster proteins and hemoproteins. These proteins generally utilize iron as a cofactor, and they perform critical roles in photosynthesis, genome stability, electron transfer, and oxidation-reduction reactions. Plants have evolved sophisticated mechanisms to maintain iron homeostasis for the assembly of functional iron-containing proteins, thereby ensuring genome stability, cell development, and plant growth. Over the past few years, our understanding of iron-containing proteins and their functions involved in genome stability has expanded enormously. In this review, I provide the current perspectives on iron homeostasis in Arabidopsis, followed by a summary of iron-containing protein functions involved in genome stability maintenance and a discussion of their possible molecular mechanisms. PMID:27330736

  18. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis.

    PubMed

    Tadini, Luca; Pesaresi, Paolo; Kleine, Tatjana; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Pribil, Mathias; Rothbart, Maxi; Hedtke, Boris; Grimm, Bernhard; Leister, Dario

    2016-03-01

    Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes. PMID:26823545

  19. The crystal structure of the thiocyanate-forming protein from Thlaspi arvense, a kelch protein involved in glucosinolate breakdown.

    PubMed

    Gumz, Frauke; Krausze, Joern; Eisenschmidt, Daniela; Backenköhler, Anita; Barleben, Leif; Brandt, Wolfgang; Wittstock, Ute

    2015-09-01

    Kelch repeat-containing proteins are involved in diverse cellular processes, but only a small subset of plant kelch proteins has been functionally characterized. Thiocyanate-forming protein (TFP) from field-penny cress, Thlaspi arvense (Brassicaceae), is a representative of specifier proteins, a group of kelch proteins involved in plant specialized metabolism. As components of the glucosinolate-myrosinase system of the Brassicaceae, specifier proteins determine the profile of bioactive products formed when plant tissue is disrupted and glucosinolates are hydrolyzed by myrosinases. Here, we describe the crystal structure of TaTFP at a resolution of 1.4 Å. TaTFP crystallized as homodimer. Each monomer forms a six-blade β-propeller with a wide "top" and a narrower "bottom" opening with distinct strand-connecting loops protruding far beyond the lower propeller surface. Molecular modeling and mutational analysis identified residues for glucosinolate aglucone and Fe(2+) cofactor binding within these loops. As the first experimentally determined structure of a plant kelch protein, the crystal structure of TaTFP not only enables more detailed mechanistic studies on glucosinolate breakdown product formation, but also provides a new basis for research on the diverse roles and mechanisms of other kelch proteins in plants. PMID:26260516

  20. Neuron Membrane Trafficking and Protein Kinases Involved in Autism and ADHD

    PubMed Central

    Kitagishi, Yasuko; Minami, Akari; Nakanishi, Atsuko; Ogura, Yasunori; Matsuda, Satoru

    2015-01-01

    A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention. PMID:25647412

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

    PubMed

    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

  2. Protein-Protein and Peptide-Protein Interactions of NudE-Like 1 (Ndel1): A Protein Involved in Schizophrenia.

    PubMed

    Hayashi, M A F; Felicori, L F; Fresqui, M A C; Yonamine, C M

    2015-01-01

    Schizophrenia (SCZ) is a devastating chronic mental disease determined by genetic and environmental factors, which susceptibility may involve an impaired neural migration during the neurodevelopmental process. Several candidate risk genes potentially associated with SCZ were related to the formation of protein complexes that ultimately mediate alterations in the neuroplasticity. The most studied SCZ risk gene is the Disrupted-in-Schizophrenia 1 (DISC1) gene, which functions seem to depend on the binding with cytoskeleton proteins, as the Nuclear-distribution gene E homolog like-1 (Ndel1) protein among others. Interestingly, Ndel1 is the only binding partner of DISC1 proteins with oligopeptidase activity, besides playing roles in multiple processes, including cytoskeletal organization, cell signaling, neuron migration, and neurite outgrowth. It is still not clear if the protein-protein interaction between Ndel1 and DISC1 is enough to explain all cellular functions attributed to these proteins, but there are several lines of evidence suggesting the importance of the catalytic activity of Ndel1 for the neurite outgrowth and neuron migration during embryogenesis. Recent works of the group have demonstrated the modulation of Ndel1 activity by DISC1, which is hypothetically impaired in SCZ patients. In fact, more recently, we also showed a lower Ndel1 activity in the plasma of SCZ patients compared to control health subjects, but the physiopathological significance of this feature is still unknown. Here we discuss Ndel1 ligands involved in protein-protein complex formations related to neurodevelopmental diseases, as (1) lissencephaly or Miller-Dieker Syndrome (MDS), which is characterized by the typical craniofacial features and abnormal smooth cerebral surface, and as (2) SCZ, since they both seem to be determined by defects in neuronal migration. Although impaired lissencephaly protein Lis1 complex formation with Ndel1 is the leading cause of lissencephaly, this

  3. Protein kinase C is involved in the regulation of several calreticulin posttranslational modifications.

    PubMed

    Cristina Castañeda-Patlán, M; Razo-Paredes, Roberto; Carrisoza-Gaytán, Rolando; González-Mariscal, Lorenza; Robles-Flores, Martha

    2010-01-01

    Calreticulin (CRT) is a highly versatile lectin-like chaperone that affects many cellular functions both inside and outside the endoplasmic reticulum lumen. We previously reported that calreticulin interacts with several protein kinase C isozymes both in vitro and in vivo. The aim of this study was to elucidate the molecular determinants involved in the association between these proteins and the biochemical significance of their interaction. Using full-length or CRT-domain constructs expressed as GST-fusion proteins, we found that protein kinase C binds to the CRT N domain in overlay and pull-down assays. Phosphorylation experiments showed that only this CRT domain is phosphorylated by the kinase. Lectin blot analysis demonstrated that CRT is modified by N-glycosylation, but this modification did not affect its interaction with protein kinase C. We also demonstrated that although both domains of protein kinase C theta can bind to CRT, it is the catalytic one that binds with higher affinity to CRT. Immunofluorescence studies showed that CRT and PKC co-localize mainly at the ER (estimated in 35%). Activation of protein kinase C induced caused transient changes in CRT localization, and unexpectedly, also induced changes in posttranslational modifications found in the protein: CRT N-glycosylation is abolished, whereas tyrosine phosphorylation and O-linked beta-N-acetylglucosamine modification are increased. Together, these findings suggest that protein kinase C is involved in the regulation of CRT function. PMID:19800981

  4. Cloning of two sea urchin DNA-binding proteins involved in mitochondrial DNA replication and transcription.

    PubMed

    Loguercio Polosa, Paola; Megli, Fiammetta; Di Ponzio, Barbara; Gadaleta, Maria Nicola; Cantatore, Palmiro; Roberti, Marina

    2002-03-01

    The cloning of the cDNA for two mitochondrial proteins involved in sea urchin mtDNA replication and transcription is reported here. The cDNA for the mitochondrial D-loop binding protein (mtDBP) from the sea urchin Strongylocentrotus purpuratus has been cloned by a polymerase chain reaction-based approach. The protein displays a very high similarity with the Paracentrotus lividus homologue as it contains also the two leucine zipper-like domains which are thought to be involved in intramolecular interactions needed to expose the two DNA binding domains in the correct position for contacting DNA. The cDNA for the mitochondrial single-stranded DNA-binding protein (mtSSB) from P. lividus has been also cloned by a similar approach. The precursor protein is 146 amino acids long with a presequence of 16 residues. The deduced amino acid sequence shows the highest homology with the Xenopus laevis protein and the lowest with the Drosophila mtSSB. The computer modeling of the tertiary structure of P. lividus mtSSB shows a structure very similar to that experimentally determined for human mtSSB, with the conservation of the main residues involved in protein tetramerization and in DNA binding. PMID:11943466

  5. Multiple Protein Interactions Involving Proposed Extracellular Loop Domains of the Tight Junction Protein Occludin

    PubMed Central

    Nusrat, Asma; Brown, G. Thomas; Tom, Jeffrey; Drake, Alex; Bui, Tam T.T.; Quan, Cliff; Mrsny, Randall J.

    2005-01-01

    Occludin is a tetraspan integral membrane protein in epithelial and endothelial tight junction (TJ) structures that is projected to have two extracellular loops. We have used peptides emulating central regions of human occludin's first and second loops, termed O-A:101–121 and O-B:210–228, respectively, to examine potential molecular interactions between these two regions of occludin and other TJ proteins. A superficial biophysical assessment of A:101–121 and O-B:210–228 showed them to have dissimilar solution conformation characteristics. Although O-A:101–121 failed to strongly interact with protein components of the human epithelial intestinal cell line T84, O-B:210–228 selectively associated with occludin, claudin-one and the junctional adhesion molecule (JAM)-A. Further, the presence of O-B:210–228, but not O-A:101–121, impeded the recovery of functional TJ structures. A scrambled peptide sequences of O-B:210–228 failed to influence TJ assembly. These studies demonstrate distinct properties for these two extracellular segments of the occludin protein and provide an improved understanding of how specific domains of occludin may interact with proteins present at TJ structures. PMID:15659655

  6. Proteomic Analysis of Differentially Expressed Proteins Involved in Peel Senescence in Harvested Mandarin Fruit.

    PubMed

    Li, Taotao; Zhang, Jingying; Zhu, Hong; Qu, Hongxia; You, Shulin; Duan, Xuewu; Jiang, Yueming

    2016-01-01

    Mandarin (Citrus reticulata), a non-climacteric fruit, is an economically important fruit worldwide. The mechanism underlying senescence of non-climacteric fruit is poorly understood. In this study, a gel-based proteomic study followed by LC-ESI-MS/MS analysis was carried out to investigate the proteomic changes involved in peel senescence in harvested mandarin "Shatangju" fruit stored for 18 days. Over the course of the storage period, the fruit gradually senesced, accompanied by a decreased respiration rate and increased chlorophyll degradation and disruption of membrane integrity. Sixty-three proteins spots that showed significant differences in abundance were identified. The up-regulated proteins were mainly associated with cell wall degradation, lipid degradation, protein degradation, senescence-related transcription factors, and transcription-related proteins. In contrast, most proteins associated with ATP synthesis and scavenging of reactive oxygen species were significantly down-regulated during peel senescence. Three thioredoxin proteins and three Ca(2+) signaling-related proteins were significantly up-regulated during peel senescence. It is suggested that mandarin peel senescence is associated with energy supply efficiency, decreased antioxidant capability, and increased protein and lipid degradation. In addition, activation of Ca(2+) signaling and transcription factors might be involved in cell wall degradation and primary or secondary metabolism. PMID:27303420

  7. Proteomic Analysis of Differentially Expressed Proteins Involved in Peel Senescence in Harvested Mandarin Fruit

    PubMed Central

    Li, Taotao; Zhang, Jingying; Zhu, Hong; Qu, Hongxia; You, Shulin; Duan, Xuewu; Jiang, Yueming

    2016-01-01

    Mandarin (Citrus reticulata), a non-climacteric fruit, is an economically important fruit worldwide. The mechanism underlying senescence of non-climacteric fruit is poorly understood. In this study, a gel-based proteomic study followed by LC-ESI-MS/MS analysis was carried out to investigate the proteomic changes involved in peel senescence in harvested mandarin “Shatangju” fruit stored for 18 days. Over the course of the storage period, the fruit gradually senesced, accompanied by a decreased respiration rate and increased chlorophyll degradation and disruption of membrane integrity. Sixty-three proteins spots that showed significant differences in abundance were identified. The up-regulated proteins were mainly associated with cell wall degradation, lipid degradation, protein degradation, senescence-related transcription factors, and transcription-related proteins. In contrast, most proteins associated with ATP synthesis and scavenging of reactive oxygen species were significantly down-regulated during peel senescence. Three thioredoxin proteins and three Ca2+ signaling-related proteins were significantly up-regulated during peel senescence. It is suggested that mandarin peel senescence is associated with energy supply efficiency, decreased antioxidant capability, and increased protein and lipid degradation. In addition, activation of Ca2+ signaling and transcription factors might be involved in cell wall degradation and primary or secondary metabolism. PMID:27303420

  8. Mapping of the Regions Involved in Homotypic Interactions of Tula Hantavirus N Protein

    PubMed Central

    Kaukinen, Pasi; Vaheri, Antti; Plyusnin, Alexander

    2003-01-01

    Hantavirus nucleocapsid (N) protein has been suggested to form homodimers and homotrimers that are further integrated into the nucleocapsid filaments around the viral RNA. Here we report detailed mapping of the regions involved in the homotypic N protein interactions in Tula hantavirus (TULV). Peptide scan screening was used to define the interaction regions, and the mammalian two-hybrid assay was used for the functional analysis of N protein mutants. To study linear regions responsible for N protein interaction(s), we used peptide scanning in which N peptides synthesized on membranes recognize recombinant TULV N protein. The data showed that the N protein bound to membrane-bound peptides comprising amino acids 13 to 30 and 41 to 57 in the N-terminal part and 340 to 379, 391 to 407, and 410 to 419 in the C-terminal part of the molecule. Further mapping of the interaction regions by alanine scanning indicated the importance of basic amino acids along the N protein and especially asparagine-394, histidine-395, and phenyalanine-396 in forming the binding interface. Analysis of truncated mutants in the mammalian two-hybrid assay showed that N-terminal amino acids 1 to 43 are involved in and C-terminal amino acids 393 to 398 (VNHFHL) are absolutely crucial for the homotypic interactions. Furthermore, our data suggested a tail-to-tail and head-to-head binding scheme for the N proteins. PMID:14512541

  9. Elucidating Protein Involvement in the Stabilization of the Biogenic Silver Nanoparticles.

    PubMed

    Ballottin, Daniela; Fulaz, Stephanie; Souza, Michele L; Corio, Paola; Rodrigues, Alexandre G; Souza, Ana O; Gaspari, Priscyla M; Gomes, Alexandre F; Gozzo, Fábio; Tasic, Ljubica

    2016-12-01

    Silver nanoparticles (AgNPs) have been broadly used as antibacterial and antiviral agents. Further, interests for green AgNP synthesis have increased in recent years and several results for AgNP biological synthesis have been reported using bacteria, fungi and plant extracts. The understanding of the role and nature of fungal proteins, their interaction with AgNPs and the subsequent stabilization of nanosilver is yet to be deeply investigated. Therefore, in an attempt to better understand biogenic AgNP stabilization with the extracellular fungal proteins and to describe these supramolecular interactions between proteins and silver nanoparticles, AgNPs, produced extracellularly by Aspergillus tubingensis-isolated as an endophytic fungus from Rizophora mangle-were characterized in order to study their physical characteristics, identify the involved proteins, and shed light into the interactions among protein-NPs by several techniques. AgNPs of around 35 nm in diameter as measured by TEM and a positive zeta potential of +8.48 mV were obtained. These AgNPs exhibited a surface plasmon resonance (SPR) band at 440 nm, indicating the nanoparticles formation, and another band at 280 nm, attributed to the electronic excitations in tryptophan, tyrosine, and/or phenylalanine residues in fungal proteins. Fungal proteins were covalently bounded to the AgNPs, mainly through S-Ag bonds due to cysteine residues (HS-) and with few N-Ag bonds from H2N- groups, as verified by Raman spectroscopy. Observed supramolecular interactions also occur by electrostatic and other protein-protein interactions. Furthermore, proteins that remain free on AgNP surface may perform hydrogen bonds with other proteins or water increasing thus the capping layer around the AgNPs and consequently expanding the hydrodynamic diameter of the particles (~264 nm, measured by DLS). FTIR results enabled us to state that proteins adsorbed to the AgNPs did not suffer relevant secondary structure alteration upon

  10. Elucidating Protein Involvement in the Stabilization of the Biogenic Silver Nanoparticles

    NASA Astrophysics Data System (ADS)

    Ballottin, Daniela; Fulaz, Stephanie; Souza, Michele L.; Corio, Paola; Rodrigues, Alexandre G.; Souza, Ana O.; Gaspari, Priscyla M.; Gomes, Alexandre F.; Gozzo, Fábio; Tasic, Ljubica

    2016-06-01

    Silver nanoparticles (AgNPs) have been broadly used as antibacterial and antiviral agents. Further, interests for green AgNP synthesis have increased in recent years and several results for AgNP biological synthesis have been reported using bacteria, fungi and plant extracts. The understanding of the role and nature of fungal proteins, their interaction with AgNPs and the subsequent stabilization of nanosilver is yet to be deeply investigated. Therefore, in an attempt to better understand biogenic AgNP stabilization with the extracellular fungal proteins and to describe these supramolecular interactions between proteins and silver nanoparticles, AgNPs, produced extracellularly by Aspergillus tubingensis—isolated as an endophytic fungus from Rizophora mangle—were characterized in order to study their physical characteristics, identify the involved proteins, and shed light into the interactions among protein-NPs by several techniques. AgNPs of around 35 nm in diameter as measured by TEM and a positive zeta potential of +8.48 mV were obtained. These AgNPs exhibited a surface plasmon resonance (SPR) band at 440 nm, indicating the nanoparticles formation, and another band at 280 nm, attributed to the electronic excitations in tryptophan, tyrosine, and/or phenylalanine residues in fungal proteins. Fungal proteins were covalently bounded to the AgNPs, mainly through S-Ag bonds due to cysteine residues (HS-) and with few N-Ag bonds from H2N- groups, as verified by Raman spectroscopy. Observed supramolecular interactions also occur by electrostatic and other protein-protein interactions. Furthermore, proteins that remain free on AgNP surface may perform hydrogen bonds with other proteins or water increasing thus the capping layer around the AgNPs and consequently expanding the hydrodynamic diameter of the particles (~264 nm, measured by DLS). FTIR results enabled us to state that proteins adsorbed to the AgNPs did not suffer relevant secondary structure alteration upon

  11. A Protein Involved in the Assembly of an Extracellular Calcium Storage Matrix*

    PubMed Central

    Glazer, Lilah; Shechter, Assaf; Tom, Moshe; Yudkovski, Yana; Weil, Simy; Aflalo, Eliahu David; Pamuru, Ramachandra Reddy; Khalaila, Isam; Bentov, Shmuel; Berman, Amir; Sagi, Amir

    2010-01-01

    Gastroliths, the calcium storage organs of crustaceans, consist of chitin-protein-mineral complexes in which the mineral component is stabilized amorphous calcium carbonate. To date, only three proteins, GAP 65, gastrolith matrix protein (GAMP), and orchestin, have been identified in gastroliths. Here, we report a novel protein, GAP 10, isolated from the gastrolith of the crayfish Cherax quadricarinatus and specifically expressed in its gastrolith disc. The encoding gene was cloned by partial sequencing of the protein extracted from the gastrolith matrix. Based on an assembled microarray cDNA chip, GAP 10 transcripts were found to be highly (12-fold) up-regulated in premolt gastrolith disc and significantly down-regulated in the hypodermis at the same molt stage. The deduced protein sequence of GAP 10 lacks chitin-binding domains and does not show homology to known proteins in the GenBankTM data base. It does, however, have an amino acid composition that has similarity to proteins extracted from invertebrate and ascidian-calcified extracellular matrices. The GAP 10 sequence contains a predicted signal peptide and predicted phosphorylation sites. In addition, the protein is phosphorylated and exhibits calcium-binding ability. Repeated daily injections of GAP 10 double strand RNA to premolt C. quadricarinatus resulted in a prolonged premolt stage and in the development of gastroliths with irregularly rough surfaces. These findings suggest that GAP 10 may be involved in the assembly of the gastrolith chitin-protein-mineral complex, particularly in the deposition of amorphous calcium carbonate. PMID:20150428

  12. A protein involved in the assembly of an extracellular calcium storage matrix.

    PubMed

    Glazer, Lilah; Shechter, Assaf; Tom, Moshe; Yudkovski, Yana; Weil, Simy; Aflalo, Eliahu David; Pamuru, Ramachandra Reddy; Khalaila, Isam; Bentov, Shmuel; Berman, Amir; Sagi, Amir

    2010-04-23

    Gastroliths, the calcium storage organs of crustaceans, consist of chitin-protein-mineral complexes in which the mineral component is stabilized amorphous calcium carbonate. To date, only three proteins, GAP 65, gastrolith matrix protein (GAMP), and orchestin, have been identified in gastroliths. Here, we report a novel protein, GAP 10, isolated from the gastrolith of the crayfish Cherax quadricarinatus and specifically expressed in its gastrolith disc. The encoding gene was cloned by partial sequencing of the protein extracted from the gastrolith matrix. Based on an assembled microarray cDNA chip, GAP 10 transcripts were found to be highly (12-fold) up-regulated in premolt gastrolith disc and significantly down-regulated in the hypodermis at the same molt stage. The deduced protein sequence of GAP 10 lacks chitin-binding domains and does not show homology to known proteins in the GenBank data base. It does, however, have an amino acid composition that has similarity to proteins extracted from invertebrate and ascidian-calcified extracellular matrices. The GAP 10 sequence contains a predicted signal peptide and predicted phosphorylation sites. In addition, the protein is phosphorylated and exhibits calcium-binding ability. Repeated daily injections of GAP 10 double strand RNA to premolt C. quadricarinatus resulted in a prolonged premolt stage and in the development of gastroliths with irregularly rough surfaces. These findings suggest that GAP 10 may be involved in the assembly of the gastrolith chitin-protein-mineral complex, particularly in the deposition of amorphous calcium carbonate. PMID:20150428

  13. Proteome analysis reveals protein candidates involved in early stages of brain regeneration of teleost fish.

    PubMed

    Ilieş, I; Zupanc, M M; Zupanc, G K H

    2012-09-01

    Exploration of the molecular dynamics underlying regeneration in the central nervous system of regeneration-competent organisms has received little attention thus far. By combining a cerebellar lesion paradigm with differential proteome analysis at a post-lesion survival time of 30 min, we screened for protein candidates involved in the early stages of regeneration in the cerebellum of such an organism, the teleost fish Apteronotus leptorhynchus. Out of 769 protein spots, the intensity of 26 spots was significantly increased by a factor of at least 1.5 in the lesioned hemisphere, relative to the intact hemisphere. The intensity of 9 protein spots was significantly reduced by a factor of at least 1.5. The proteins associated with 15 of the spots were identified by peptide mass fingerprinting and/or tandem mass spectrometry, resulting in the identification of a total of 11 proteins. Proteins whose abundance was significantly increased include: erythrocyte membrane protein 4.1N, fibrinogen gamma polypeptide, fructose-biphosphate aldolase C, alpha-internexin neuronal intermediate filament protein, major histocompatibility complex class I heavy chain, 26S proteasome non-ATPase regulatory subunit 8, tubulin alpha-1C chain, and ubiquitin-specific protease 5. Proteins with significantly decreased levels of abundance include: brain glycogen phosphorylase, neuron-specific calcium-binding protein hippocalcin, and spectrin alpha 2. We hypothesize that these proteins are involved in energy metabolism, blood clotting, electron transfer in oxidative reactions, cytoskeleton degradation, apoptotic cell death, synaptic plasticity, axonal regeneration, and promotion of mitotic activity. PMID:22659563

  14. [Protein quality control and psychiatric disorder--involvement of sigma-1 receptor].

    PubMed

    Kudo, Takashi

    2014-01-01

    The protein quality control mechanism in the endoplasmic reticulum is referred to as the unfolded protein response (UPR), and its failure may be involved in the onset of some psychiatric disorders. We showed that induction of the sigma-1 receptor plays a role in the UPR, and suggested the possibility that this mechanism is impaired in disorders such as schizophrenia. We also demonstrated that fluvoxamine induces expression of the sigma-1 receptor. Therefore, it has the potential to be developed as a drug which exerts an anti-ER-stress effect, i. e., protein quality control effect. PMID:25672212

  15. Vesicular trafficking in characean green algae and the possible involvement of a VAMP72-family protein.

    PubMed

    Hoepflinger, Marion C; Hametner, Christina; Ueda, Takashi; Foissner, Ilse

    2014-01-01

    The RAB5 GTPase ARA6 of Arabidopsis thaliana is known to be involved in endosomal trafficking by targeting vesicles to the plasma membrane. During this process AtARA6 is working in close relationship with the SNARE protein VAMP727 (vesicle associated membrane protein 727). Recently, ARA6 of the characean green algae Chara australis (CaARA6) was shown to have properties similar to AtARA6, pointing to similar trafficking pathways. In order to gain further insight into the vesicle trafficking machinery of Characeae, C. australis was analyzed for homologous proteins of the VAMP72-family. A CaVAMP72 protein was detected and classified by protein sequence alignment and phylogenetic analyses. PMID:25764429

  16. Vesicular trafficking in characean green algae and the possible involvement of a VAMP72-family protein.

    PubMed

    Hoepflinger, Marion; Hametner, Christina; Ueda, Takashi; Foissner, Ilse

    2014-01-01

    The RAB5 GTPase ARA6 (AtARA6) of Arabidopsis thaliana is known to be involved in endosomal trafficking by targeting vesicles to the plasma membrane. During this process AtARA6 is working in close relationship with the SNARE protein VAMP727 (vesicle associated membrane protein 727). Recently, ARA6 of the characean green algae Chara australis (CaARA6) was shown to have properties similar to AtARA6, pointing to similar trafficking pathways. In order to gain further insight into the vesicle trafficking machinery of characeae, C. australis was analyzed for homologous proteins of the VAMP72-family. A CaVAMP72 protein was detected and classified by protein sequence alignment and phylogenetic analyses. PMID:24614164

  17. Phylogenomic analysis of the Chlamydomonas genome unmasks proteins potentially involved in photosynthetic function and regulation

    PubMed Central

    Karpowicz, Steven J.; Heinnickel, Mark; Dewez, David; Hamel, Blaise; Dent, Rachel; Niyogi, Krishna K.; Johnson, Xenie; Alric, Jean; Wollman, Francis-André; Li, Huiying; Merchant, Sabeeha S.

    2010-01-01

    Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus. PMID:20490922

  18. Vesicular trafficking in characean green algae and the possible involvement of a VAMP72-family protein

    PubMed Central

    Hoepflinger, Marion C; Hametner, Christina; Ueda, Takashi; Foissner, Ilse

    2014-01-01

    The RAB5 GTPase ARA6 of Arabidopsis thaliana is known to be involved in endosomal trafficking by targeting vesicles to the plasma membrane. During this process AtARA6 is working in close relationship with the SNARE protein VAMP727 (vesicle associated membrane protein 727). Recently, ARA6 of the characean green algae Chara australis (CaARA6) was shown to have properties similar to AtARA6, pointing to similar trafficking pathways. In order to gain further insight into the vesicle trafficking machinery of Characeae, C. australis was analyzed for homologous proteins of the VAMP72-family. A CaVAMP72 protein was detected and classified by protein sequence alignment and phylogenetic analyses. PMID:24614164

  19. RNA-binding proteins involved in post-transcriptional regulation in bacteria

    PubMed Central

    Van Assche, Elke; Van Puyvelde, Sandra; Vanderleyden, Jos; Steenackers, Hans P.

    2015-01-01

    Post-transcriptional regulation is a very important mechanism to control gene expression in changing environments. In the past decade, a lot of interest has been directed toward the role of small RNAs (sRNAs) in bacterial post-transcriptional regulation. However, sRNAs are not the only molecules controlling gene expression at this level, RNA-binding proteins (RBPs) play an important role as well. CsrA and Hfq are the two best studied bacterial proteins of this type, but recently, additional proteins involved in post-transcriptional control have been identified. This review focuses on the general working mechanisms of post-transcriptionally active RBPs, which include (i) adaptation of the susceptibility of mRNAs and sRNAs to RNases, (ii) modulating the accessibility of the ribosome binding site of mRNAs, (iii) recruiting and assisting in the interaction of mRNAs with other molecules and (iv) regulating transcription terminator/antiterminator formation, and gives an overview of both the well-studied and the newly identified proteins that are involved in post-transcriptional regulatory processes. Additionally, the post-transcriptional mechanisms by which the expression or the activity of these proteins is regulated, are described. For many of the newly identified proteins, however, mechanistic questions remain. Most likely, more post-transcriptionally active proteins will be identified in the future. PMID:25784899

  20. Olive seed protein bodies store degrading enzymes involved in mobilization of oil bodies

    PubMed Central

    Rodríguez-García, María Isabel

    2014-01-01

    The major seed storage reserves in oilseeds are accumulated in protein bodies and oil bodies, and serve as an energy, carbon, and nitrogen source during germination. Here, the spatio-temporal relationships between protein bodies and several key enzymes (phospholipase A, lipase, and lipoxygenase) involved in storage lipid mobilization in cotyledon cells was analysed during in vitro seed germination. Enzyme activities were assayed in-gel and their cellular localization were determined using microscopy techniques. At seed maturity, phospholipase A and triacylglycerol lipase activities were found exclusively in protein bodies. However, after seed imbibition, these activities were shifted to the cytoplasm and the surface of the oil bodies. The activity of neutral lipases was detected by using α-naphthyl palmitate and it was associated mainly with protein bodies during the whole course of germination. This pattern of distribution was highly similar to the localization of neutral lipids, which progressively appeared in protein bodies. Lipoxygenase activity was found in both the protein bodies and on the surface of the oil bodies during the initial phase of seed germination. The association of lipoxygenase with oil bodies was temporally correlated with the appearance of phospholipase A and lipase activities on the surface of oil bodies. It is concluded that protein bodies not only serve as simple storage structures, but are also dynamic and multifunctional organelles directly involved in storage lipid mobilization during olive seed germination. PMID:24170742

  1. The TSG101 protein binds to connexins and is involved in connexin degradation

    SciTech Connect

    Auth, Tanja Schlueter, Sharazad; Urschel, Stephanie; Kussmann, Petra; Sonntag, Stephan; Hoeher, Thorsten; Kreuzberg, Maria M.; Dobrowolski, Radoslaw; Willecke, Klaus

    2009-04-01

    Gap junctions mediate electrical and metabolic communication between cells in almost all tissues and are proposed to play important roles in cellular growth control, differentiation and embryonic development. Gap junctional communication and channel assembly were suggested to be regulated by interaction of connexins with different proteins including kinases and phosphatases. Here, we identified the tumor susceptibility gene 101 (TSG101) protein to bind to the carboxyterminal tail of connexin45 in a yeast two-hybrid protein interaction screen. Glutathione S-transferase pull down experiments and immunoprecipitation revealed that not only connexin45 but also connexin30.2, -36, and -43 carboxyterminal regions were associated with TSG101 protein in pull down analyses and that connexin31, -43 and -45 co-precipitate with endogenous TSG101 protein in lysates from HM1 embryonic stem cells. TSG101 has been shown to be involved in cell cycle control, transcriptional regulation and turnover of endocytosed proteins. Thus, we decided to study the functional role of this interaction. SiRNA mediated knock down of TSG101 in HM1 embryonic stem cells led to increased levels of connexin43 and -45, prolonged half life of these connexins and increased transfer of microinjected Lucifer yellow. Our results suggest that TSG101 is involved in the degradation of connexins via interaction with connexin proteins.

  2. HOPS: a novel cAMP-dependent shuttling protein involved in protein synthesis regulation.

    PubMed

    Della Fazia, Maria Agnese; Castelli, Marilena; Bartoli, Daniela; Pieroni, Stefania; Pettirossi, Valentina; Piobbico, Danilo; Viola-Magni, Mariapia; Servillo, Giuseppe

    2005-07-15

    The liver has the ability to autonomously regulate growth and mass. Following partial hepatectomy, hormones, growth factors, cytokines and their coupled signal transduction pathways have been implicated in hepatocyte proliferation. To understand the mechanisms responsible for the proliferative response, we studied liver regeneration by characterization of novel genes that are activated in residual hepatocytes. A regenerating liver cDNA library screening was performed with cDNA-subtracted probes derived from regenerating and normal liver. Here, we describe the biology of Hops (for hepatocyte odd protein shuttling). HOPS is a novel shuttling protein that contains an ubiquitin-like domain, a putative NES and a proline-rich region. HOPS is rapidly exported from the nucleus and is overexpressed during liver regeneration. Evidence shows that cAMP governs HOPS export in hepatocytes of normal and regenerating liver and is mediated via CRM-1. We demonstrate that HOPS binds to elongation factor eEF-1A and interferes in protein synthesis. HOPS overexpression in H-35-hepatoma and 3T3-NIH cells strongly reduces proliferation. PMID:16014383

  3. The promoter of filamentation (POF1) protein from Saccharomyces cerevisiae is an ATPase involved in the protein quality control process

    PubMed Central

    2011-01-01

    Background The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene. Results Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo. Conclusions Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation. PMID:22204397

  4. Vitamin D receptor regulates intestinal proteins involved in cell proliferation, migration and stress response

    PubMed Central

    2014-01-01

    Background Genome-wide association studies found low plasma levels of 25-hydroxyvitamin D and vitamin D receptor (VDR) polymorphisms associated with a higher prevalence of pathological changes in the intestine such as chronic inflammatory bowel diseases. Methods In this study, a proteomic approach was applied to understand the overall physiological importance of vitamin D in the small intestine, beyond its function in calcium and phosphate absorption. Results In total, 569 protein spots could be detected by two-dimensional-difference in-gel electrophoresis (2D-DIGE), and 82 proteins were considered as differentially regulated in the intestinal mucosa of VDR-deficient mice compared to that of wildtype (WT) mice. Fourteen clearly detectable proteins were identified by MS/MS and further analyzed by western blot and/or real-time RT-PCR. The differentially expressed proteins are functionally involved in cell proliferation, cell adhesion and cell migration, stress response and lipid transport. Mice lacking VDR revealed higher levels of intestinal proteins associated with proliferation and migration such as the 37/67 kDa laminin receptor, collagen type VI (alpha 1 chain), keratin-19, tropomyosin-3, adseverin and higher levels of proteins involved in protein trafficking and stress response than WT mice. In contrast, proteins that are involved in transport of bile and fatty acids were down-regulated in small intestine of mice lacking VDR compared to WT mice. However, plasma and liver concentrations of cholesterol and triglycerides were not different between the two groups of mice. Conclusion Collectively, these data imply VDR as an important factor for controlling cell proliferation, migration and stress response in the small intestine. PMID:24641763

  5. Spermidine-Induced Improvement of Reconsolidation of Memory Involves Calcium-Dependent Protein Kinase in Rats

    ERIC Educational Resources Information Center

    Girardi, Bruna Amanda; Ribeiro, Daniela Aymone; Signor, Cristiane; Muller, Michele; Gais, Mayara Ana; Mello, Carlos Fernando; Rubin, Maribel Antonello

    2016-01-01

    In this study, we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by the intrahippocampal administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4-mA footshock as an unconditioned stimulus.…

  6. Expression of proteins involved in host plant defense against greenbug infestation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The greenbug, Schizaphis graminum (Rondani), has been recognized as a major pest of small grains, including sorghum and wheat. To understand the molecular mechanisms involved in host plant defense against greenbug aphids, a proteomic analysis of greenbug-induced proteins in the seedlings of sorghum...

  7. Pdsg1 and Pdsg2, novel proteins involved in developmental genome remodelling in Paramecium.

    PubMed

    Arambasic, Miroslav; Sandoval, Pamela Y; Hoehener, Cristina; Singh, Aditi; Swart, Estienne C; Nowacki, Mariusz

    2014-01-01

    The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization. PMID:25397898

  8. Protein-protein interactions involving voltage-gated sodium channels: Post-translational regulation, intracellular trafficking and functional expression.

    PubMed

    Shao, Dongmin; Okuse, Kenji; Djamgoz, Mustafa B A

    2009-07-01

    Voltage-gated sodium channels (VGSCs), classically known to play a central role in excitability and signalling in nerves and muscles, have also been found to be expressed in a range of 'non-excitable' cells, including lymphocytes, fibroblasts and endothelia. VGSC abnormalities are associated with various diseases including epilepsy, long-QT syndrome 3, Brugada syndrome, sudden infant death syndrome and, more recently, various human cancers. Given their pivotal role in a wide range of physiological and pathophysiological processes, regulation of functional VGSC expression has been the subject of intense study. An emerging theme is post-translational regulation and macro-molecular complexing by protein-protein interactions and intracellular trafficking, leading to changes in functional VGSC expression in plasma membrane. This partially involves endoplasmic reticulum associated degradation and ubiquitin-proteasome system. Several proteins have been shown to associate with VGSCs. Here, we review the interactions involving VGSCs and the following proteins: p11, ankyrin, syntrophin, beta-subunit of VGSC, papin, ERM and Nedd4 proteins. Protein kinases A and C, as well as Ca(2+)-calmodulin dependent kinase II that have also been shown to regulate intracellular trafficking of VGSCs by changing the balance of externalization vs. internalization, and an effort is made to separate these effects from the short-term phosphorylation of mature proteins in plasma membrane. Two further modulatory mechanisms are reciprocal interactions with the cytoskeleton and, late-stage, activity-dependent regulation. Thus, the review gives an updated account of the range of post-translational molecular mechanisms regulating functional VGSC expression. However, many details of VGSC subtype-specific regulation and pathophysiological aspects remain unknown and these are highlighted throughout for completeness. PMID:19401147

  9. Hermes RNA-binding protein targets RNAs-encoding proteins involved in meiotic maturation, early cleavage, and germline development.

    PubMed

    Song, Hye-Won; Cauffman, Karen; Chan, Agnes P; Zhou, Yi; King, Mary Lou; Etkin, Laurence D; Kloc, Malgorzata

    2007-07-01

    The early development of metazoans is mainly regulated by differential translation and localization of maternal mRNAs in the embryo. In general, these processes are orchestrated by RNA-binding proteins interacting with specific sequence motifs in the 3'-untranslated region (UTR) of their target RNAs. Hermes is an RNA-binding protein, which contains a single RNA recognition motif (RRM) and is found in various vertebrate species from fish to human. In Xenopus laevis, Hermes mRNA and protein are localized in the vegetal region of oocytes. A subpopulation of Hermes protein is concentrated in a specific structure in the vegetal cortex, called the germ plasm (believed to contain determinants of the germ cell fate) where Hermes protein co-localizes with Xcat2 and RINGO/Spy mRNAs. The level of total Hermes protein decreases during maturation. The precocious depletion of Hermes protein by injection of Hermes antisense morpholino oligonucleotide (HE-MO) accelerates the process of maturation and results in cleavage defects in vegetal blastomeres of the embryo. It is known that several maternal mRNAs including RINGO/Spy and Mos are regulated at the translational level during meiotic maturation and early cleavage in Xenopus. The ectopic expression of RINGO/Spy or Mos causes resumption of meiotic maturation and cleavage arrests, which resemble the loss of Hermes phenotypes. We found that the injection of HE-MO enhances the acceleration of maturation caused by the injection of RINGO/Spy mRNA, and that Hermes protein is present as mRNP complex containing RINGO/Spy, Mos, and Xcat2 mRNAs in vivo. We propose that as an RNA-binding protein, Hermes may be involved in maturation, cleavage events at the vegetal pole and germ cell development by negatively regulating the expression of RINGO/Spy, Mos, and Xcat2 mRNAs. PMID:17309605

  10. Hsp90 is involved in the regulation of cytosolic precursor protein abundance in tomato.

    PubMed

    Tillmann, Bodo; Röth, Sascha; Bublak, Daniela; Sommer, Manuel; Stelzer, Ernst H K; Scharf, Klaus-Dieter; Schleiff, Enrico

    2015-02-01

    Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the families of heat stress proteins 70 (Hsp70) and 90 (Hsp90) assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins with respect to the cytosolic chaperone-dependent regulation. Some preproteins such as pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins such as pSSU is more strongly dependent on Hsp70. The E3 ligase, C-terminus of Hsp70-interacting protein (Chip), appears to have a more general role in the control of cytosolic protein abundance. We discuss why the different reaction modes are comparable with the cytosolic unfolded protein response. PMID:25619681

  11. Towards identifying Brassica proteins involved in mediating resistance to Leptosphaeria maculans: a proteomics-based approach.

    PubMed

    Sharma, Nidhi; Hotte, Naomi; Rahman, Muhammad H; Mohammadi, Mohsen; Deyholos, Michael K; Kav, Nat N V

    2008-09-01

    To better understand the pathogen-stress response of Brassica species against the ubiquitous hemi-biotroph fungus Leptosphaeria maculans, we conducted a comparative proteomic analysis between blackleg-susceptible Brassica napus and blackleg-resistant Brassica carinata following pathogen inoculation. We examined temporal changes (6, 12, 24, 48 and 72 h) in protein profiles of both species subjected to pathogen-challenge using two-dimensional gel electrophoresis. A total of 64 proteins were found to be significantly affected by the pathogen in the two species, out of which 51 protein spots were identified using tandem mass spectrometry. The proteins identified included antioxidant enzymes, photosynthetic and metabolic enzymes, and those involved in protein processing and signaling. Specifically, we observed that in the tolerant B. carinata, enzymes involved in the detoxification of free radicals increased in response to the pathogen whereas no such increase was observed in the susceptible B. napus. The expression of genes encoding four selected proteins was validated using quantitative real-time PCR and an additional one by Western blotting. Our findings are discussed with respect to tolerance or susceptibility of these species to the pathogen. PMID:18668695

  12. Identification and Characterization of Proteins Involved in Rice Urea and Arginine Catabolism1[W

    PubMed Central

    Cao, Feng-Qiu; Werner, Andrea K.; Dahncke, Kathleen; Romeis, Tina; Liu, Lai-Hua; Witte, Claus-Peter

    2010-01-01

    Rice (Oryza sativa) production relies strongly on nitrogen (N) fertilization with urea, but the proteins involved in rice urea metabolism have not yet been characterized. Coding sequences for rice arginase, urease, and the urease accessory proteins D (UreD), F (UreF), and G (UreG) involved in urease activation were identified and cloned. The functionality of urease and the urease accessory proteins was demonstrated by complementing corresponding Arabidopsis (Arabidopsis thaliana) mutants and by multiple transient coexpression of the rice proteins in Nicotiana benthamiana. Secondary structure models of rice (plant) UreD and UreF proteins revealed a possible functional conservation to bacterial orthologs, especially for UreF. Using amino-terminally StrepII-tagged urease accessory proteins, an interaction between rice UreD and urease could be shown. Prokaryotic and eukaryotic urease activation complexes seem conserved despite limited protein sequence conservation for UreF and UreD. In plant metabolism, urea is generated by the arginase reaction. Rice arginase was transiently expressed as a carboxyl-terminally StrepII-tagged fusion protein in N. benthamiana, purified, and biochemically characterized (Km = 67 mm, kcat = 490 s−1). The activity depended on the presence of manganese (Kd = 1.3 μm). In physiological experiments, urease and arginase activities were not influenced by the external N source, but sole urea nutrition imbalanced the plant amino acid profile, leading to the accumulation of asparagine and glutamine in the roots. Our data indicate that reduced plant performance with urea as N source is not a direct result of insufficient urea metabolism but may in part be caused by an imbalance of N distribution. PMID:20631318

  13. Gene expression profiling to identify eggshell proteins involved in physical defense of the chicken egg

    PubMed Central

    2010-01-01

    Background As uricoletic animals, chickens produce cleidoic eggs, which are self-contained bacteria-resistant biological packages for extra-uterine development of the chick embryo. The eggshell constitutes a natural physical barrier against bacterial penetration if it forms correctly and remains intact. The eggshell's remarkable mechanical properties are due to interactions among mineral components and the organic matrix proteins. The purpose of our study was to identify novel eggshell proteins by examining the transcriptome of the uterus during calcification of the eggshell. An extensive bioinformatic analysis on genes over-expressed in the uterus allowed us to identify novel eggshell proteins that contribute to the egg's natural defenses. Results Our 14 K Del-Mar Chicken Integrated Systems microarray was used for transcriptional profiling in the hen's uterus during eggshell deposition. A total of 605 transcripts were over-expressed in the uterus compared with the magnum or white isthmus across a wide range of abundance (1.1- to 79.4-fold difference). The 605 highly-expressed uterine transcripts correspond to 469 unique genes, which encode 437 different proteins. Gene Ontology (GO) analysis was used for interpretation of protein function. The most over-represented GO terms are related to genes encoding ion transport proteins, which provide eggshell mineral precursors. Signal peptide sequence was found for 54 putative proteins secreted by the uterus during eggshell formation. Many functional proteins are involved in calcium binding or biomineralization--prerequisites for interacting with the mineral phase during eggshell fabrication. While another large group of proteins could be involved in proper folding of the eggshell matrix. Many secreted uterine proteins possess antibacterial properties, which would protect the egg against microbial invasion. A final group includes proteases and protease inhibitors that regulate protein activity in the acellular uterine fluid

  14. Evidence against the involvement of ionically bound cell wall proteins in pea epicotyl growth

    NASA Technical Reports Server (NTRS)

    Melan, M. A.; Cosgrove, D. J.

    1988-01-01

    Ionically bound cell wall proteins were extracted from 7 day old etiolated pea (Pisum sativum L. cv Alaska) epicotyls with 3 molar LiCl. Polyclonal antiserum was raised in rabbits against the cell wall proteins. Growth assays showed that treatment of growing region segments (5-7 millimeters) of peas with either dialyzed serum, serum globulin fraction, affinity purified immunoglobulin, or papain-cleaved antibody fragments had no effect on growth. Immunofluorescence microscopy confirmed antibody binding to cell walls and penetration of the antibodies into the tissues. Western blot analysis, immunoassay results, and affinity chromatography utilizing Sepharose-bound antibodies confirmed recognition of the protein preparation by the antibodies. Experiments employing in vitro extension as a screening measure indicated no effect upon extension by antibodies, by 50 millimolar LiCl perfusion of the apoplast or by 3 molar LiCl extraction. Addition of cell wall protein to protease pretreated segments did not restore extension nor did addition of cell wall protein to untreated segments increase extension. It is concluded that, although evidence suggests that protein is responsible for the process of extension, the class(es) of proteins which are extracted from pea cell walls with 3 molar LiCl are probably not involved in this process.

  15. Hsp90 is involved in the regulation of cytosolic precursor protein abundance in tomato.

    PubMed

    Tillmann, Bodo; Röth, Sascha; Bublak, Daniela; Sommer, Manuel; Stelzer, Ernst H K; Scharf, Klaus-Dieter; Schleiff, Enrico

    2014-10-20

    Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the heat stress protein 70 and 90 (Hsp70 or Hsp90) families assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins in respect to the cytosolic chaperone dependent regulation. Some preproteins like pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins like pSSU is more strongly dependent on Hsp70. The E3 ligase Chip appears to have a more general role in the control of cytosolic protein abundance. We discuss why the different reaction modes are comparable to the cytosolic unfolded protein response. PMID:25336566

  16. Identification of host proteins involved in rickettsial invasion of tick cells.

    PubMed

    Petchampai, Natthida; Sunyakumthorn, Piyanate; Banajee, Kaikhushroo H; Verhoeve, Victoria I; Kearney, Michael T; Macaluso, Kevin R

    2015-03-01

    Tick-borne spotted fever group (SFG) Rickettsia species are obligate intracellular bacteria capable of infecting both vertebrate and invertebrate host cells, an essential process for subsequent bacterial survival in distinct hosts. The host cell signaling molecules involved in the uptake of Rickettsia into mammalian and Drosophila cells have been identified; however, invasion into tick cells is understudied. Considering the movement of SFG Rickettsia between vertebrate and invertebrate hosts, the hypothesis is that conserved mechanisms are utilized for host cell invasion. The current study employed biochemical inhibition assays to determine the tick proteins involved in Rickettsia montanensis infection of tick-derived cells from a natural host, Dermacentor variabilis. The results revealed several tick proteins important for rickettsial invasion, including actin filaments, actin-related protein 2/3 complex, phosphatidylinositol-3'-kinase, protein tyrosine kinases (PTKs), Src family PTK, focal adhesion kinase, Rho GTPase Rac1, and neural Wiskott-Aldrich syndrome protein. Delineating the molecular mechanisms of rickettsial infection is critical to a thorough understanding of rickettsial transmission in tick populations and the ecology of tick-borne rickettsial diseases. PMID:25547795

  17. The Stimulatory Gαs Protein Is Involved in Olfactory Signal Transduction in Drosophila

    PubMed Central

    Deng, Ying; Zhang, Weiyi; Farhat, Katja; Oberland, Sonja; Gisselmann, Günter; Neuhaus, Eva M.

    2011-01-01

    Seven-transmembrane receptors typically mediate olfactory signal transduction by coupling to G-proteins. Although insect odorant receptors have seven transmembrane domains like G-protein coupled receptors, they have an inverted membrane topology, constituting a key difference between the olfactory systems of insects and other animals. While heteromeric insect ORs form ligand-activated non-selective cation channels in recombinant expression systems, the evidence for an involvement of cyclic nucleotides and G-proteins in odor reception is inconsistent. We addressed this question in vivo by analyzing the role of G-proteins in olfactory signaling using electrophysiological recordings. We found that Gαs plays a crucial role for odorant induced signal transduction in OR83b expressing olfactory sensory neurons, but not in neurons expressing CO2 responsive proteins GR21a/GR63a. Moreover, signaling of Drosophila ORs involved Gαs also in a heterologous expression system. In agreement with these observations was the finding that elevated levels of cAMP result in increased firing rates, demonstrating the existence of a cAMP dependent excitatory signaling pathway in the sensory neurons. Together, we provide evidence that Gαs plays a role in the OR mediated signaling cascade in Drosophila. PMID:21490930

  18. Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein.

    PubMed Central

    Wahlberg, J M; Bron, R; Wilschut, J; Garoff, H

    1992-01-01

    Infection of cells with enveloped viruses is accomplished through membrane fusion. The binding and fusion processes are mediated by the spike proteins in the envelope of the virus particle and usually involve a series of conformational changes in these proteins. We have studied the low-pH-mediated fusion process of the alphavirus Semliki Forest virus (SFV). The spike protein of SFV is composed of three copies of the protein heterodimer E2E1. This structure is resistant to solubilization in mild detergents such as Nonidet P-40 (NP40). We have recently shown that the spike structure is reorganized during virus entry into acidic endosomes (J. M. Wahlberg and H. Garoff, J. Cell Biol. 116:339-348, 1992). The original NP40-resistant heterodimer is dissociated, and the E1 subunits form new NP40-resistant protein oligomers. Here, we show that the new oligomer is represented by an E1 trimer. From studies that use an in vitro assay for fusion of SFV with liposomes, we show that the E1 trimer is efficiently expressed during virus-mediated membrane fusion. Time course studies show that both E1 trimer formation and fusion are fast processes, occurring in seconds. It was also possible to inhibit virus binding and fusion with a monoclonal antibody directed toward the trimeric E1. These results give support for a model in which the E1 trimeric structure is involved in the SFV-mediated fusion reaction. Images PMID:1433520

  19. Identification of Host Proteins Involved in Rickettsial Invasion of Tick Cells

    PubMed Central

    Sunyakumthorn, Piyanate; Banajee, Kaikhushroo H.; Verhoeve, Victoria I.; Kearney, Michael T.; Macaluso, Kevin R.

    2014-01-01

    Tick-borne spotted fever group (SFG) Rickettsia species are obligate intracellular bacteria capable of infecting both vertebrate and invertebrate host cells, an essential process for subsequent bacterial survival in distinct hosts. The host cell signaling molecules involved in the uptake of Rickettsia into mammalian and Drosophila cells have been identified; however, invasion into tick cells is understudied. Considering the movement of SFG Rickettsia between vertebrate and invertebrate hosts, the hypothesis is that conserved mechanisms are utilized for host cell invasion. The current study employed biochemical inhibition assays to determine the tick proteins involved in Rickettsia montanensis infection of tick-derived cells from a natural host, Dermacentor variabilis. The results revealed several tick proteins important for rickettsial invasion, including actin filaments, actin-related protein 2/3 complex, phosphatidylinositol-3′-kinase, protein tyrosine kinases (PTKs), Src family PTK, focal adhesion kinase, Rho GTPase Rac1, and neural Wiskott-Aldrich syndrome protein. Delineating the molecular mechanisms of rickettsial infection is critical to a thorough understanding of rickettsial transmission in tick populations and the ecology of tick-borne rickettsial diseases. PMID:25547795

  20. SEORious business: structural proteins in sieve tubes and their involvement in sieve element occlusion.

    PubMed

    Knoblauch, Michael; Froelich, Daniel R; Pickard, William F; Peters, Winfried S

    2014-04-01

    The phloem provides a network of sieve tubes for long-distance translocation of photosynthates. For over a century, structural proteins in sieve tubes have presented a conundrum since they presumably increase the hydraulic resistance of the tubes while no potential function other than sieve tube or wound sealing in the case of injury has been suggested. Here we summarize and critically evaluate current speculations regarding the roles of these proteins. Our understanding suffers from the suggestive power of images; what looks like a sieve tube plug on micrographs may not actually impede translocation very much. Recent reports of an involvement of SEOR (sieve element occlusion-related) proteins, a class of P-proteins, in the sealing of injured sieve tubes are inconclusive; various lines of evidence suggest that, in neither intact nor injured plants, are SEORs determinative of translocation stoppage. Similarly, the popular notion that P-proteins serve in the defence against phloem sap-feeding insects is unsupported by empirical facts; it is conceivable that in functional sieve tubes, aphids actually could benefit from inducing a plug. The idea that rising cytosolic Ca(2+) generally triggers sieve tube blockage by P-proteins appears widely accepted, despite lacking experimental support. Even in forisomes, P-protein assemblages restricted to one single plant family and the only Ca(2+)-responsive P-proteins known, the available evidence does not unequivocally suggest that plug formation is the cause rather than a consequence of translocation stoppage. We conclude that the physiological roles of structural P-proteins remain elusive, and that in vivo studies of their dynamics in continuous sieve tube networks combined with flow velocity measurements will be required to (hopefully) resolve this scientific roadblock. PMID:24591057

  1. A Bacillus thuringiensis S-Layer Protein Involved in Toxicity against Epilachna varivestis (Coleoptera: Coccinellidae)

    PubMed Central

    Peña, Guadalupe; Miranda-Rios, Juan; de la Riva, Gustavo; Pardo-López, Liliana; Soberón, Mario; Bravo, Alejandra

    2006-01-01

    The use of Bacillus thuringiensis as a biopesticide is a viable alternative for insect control since the insecticidal Cry proteins produced by these bacteria are highly specific; harmless to humans, vertebrates, and plants; and completely biodegradable. In addition to Cry proteins, B. thuringiensis produces a number of extracellular compounds, including S-layer proteins (SLP), that contribute to virulence. The S layer is an ordered structure representing a proteinaceous paracrystalline array which completely covers the surfaces of many pathogenic bacteria. In this work, we report the identification of an S-layer protein by the screening of B. thuringiensis strains for activity against the coleopteran pest Epilachna varivestis (Mexican bean beetle; Coleoptera: Coccinellidae). We screened two B. thuringiensis strain collections containing unidentified Cry proteins and also strains isolated from dead insects. Some of the B. thuringiensis strains assayed against E. varivestis showed moderate toxicity. However, a B. thuringiensis strain (GP1) that was isolated from a dead insect showed a remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against E. varivestis but not against the lepidopteran Manduca sexta or Spodoptera frugiperda or against the dipteran Aedes aegypti. The gene encoding this protein was cloned and sequenced. It corresponded to an S-layer protein highly similar to previously described SLP in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). The phylogenetic relationships among SLP from different bacteria showed that these proteins from Bacillus cereus, Bacillus sphaericus, B. anthracis, B. licheniformis, and B. thuringiensis are arranged in the same main group, suggesting similar origins. This is the first report that demonstrates that an S-layer protein is directly involved in toxicity to a coleopteran pest. PMID:16391064

  2. The RND protein is involved in the vulnibactin export system in Vibrio vulnificus M2799.

    PubMed

    Kawano, Hiroaki; Miyamoto, Katsushiro; Yasunobe, Megumi; Murata, Masahiro; Myojin, Tomoka; Tsuchiya, Takahiro; Tanabe, Tomotaka; Funahashi, Tatsuya; Sato, Takaji; Azuma, Takashi; Mino, Yoshiki; Tsujibo, Hiroshi

    2014-10-01

    Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium exports vulnibactin for iron acquisition from the environment. The mechanisms of vulnibactin biosynthesis and ferric-vulnibactin uptake systems have recently been reported, while the vulnibactin export system has not been reported. Mutant growth under low-iron concentration conditions and a bioassay of the culture supernatant indicate that the VV1_0612 protein plays a crucial role in the vulnibactin secretion as a component of the resistance-nodulation-division (RND)-type efflux system in V. vulnificus M2799. To identify which RND protein(s) together with VV1_0612 TolC constituted the RND efflux system for vulnibactin secretion, deletion mutants of 11 RND protein-encoding genes were constructed. The growth inhibition of a multiple mutant (Δ11) of the RND protein-encoding genes was observed 6 h after the beginning of the culture. Furthermore, ΔVV1_1681 exhibited a growth curve that was similar to that of Δ11, while the multiple mutant except ΔVV1_1681 showed the same growth as the wild-type strain. These results indicate that the VV1_1681 protein is involved in the vulnibactin export system of V. vulnificus M2799. This is the first genetic evidence that vulnibactin is secreted through the RND-type efflux systems in V. vulnificus. PMID:25205089

  3. Fission yeast pkl1 is a kinesin-related protein involved in mitotic spindle function.

    PubMed Central

    Pidoux, A L; LeDizet, M; Cande, W Z

    1996-01-01

    We have used anti-peptide antibodies raised against highly conserved regions of the kinesin motor domain to identify kinesin-related proteins in the fission yeast Schizosaccharomyces pombe. Here we report the identification of a new kinesin-related protein, which we have named pkl1. Sequence homology and domain organization place pkl1 in the Kar3/ncd subfamily of kinesin-related proteins. Bacterially expressed pkl1 fusion proteins display microtubule-stimulated ATPase activity, nucleotide-sensitive binding, and bundling of microtubules. Immunofluorescence studies with affinity-purified antibodies indicate that the pkl1 protein localizes to the nucleus and the mitotic spindle. Pkl1 null mutants are viable but have increased sensitivity to microtubule-disrupting drugs. Disruption of pkl1+ suppresses mutations in another kinesin-related protein, cut7, which is known to act in the spindle. Overexpression of pkl1 to very high levels causes a similar phenotype to that seen in cut7 mutants: V-shaped and star-shaped microtubule structures are observed, which we interpret to be spindles with unseparated spindle poles. These observations suggest that pkl1 and cut7 provide opposing forces in the spindle. We propose that pkl1 functions as a microtubule-dependent motor that is involved in microtubule organization in the mitotic spindle. Images PMID:8898367

  4. Involvement of Fis protein in replication of the Escherichia coli chromosome.

    PubMed Central

    Filutowicz, M; Ross, W; Wild, J; Gourse, R L

    1992-01-01

    We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo. At high temperatures, fis null mutants form filamentous cells, show aberrant nucleoid segregation, and are unable to form single colonies. DNA synthesis is inhibited in these fis mutant strains following upshift to 44 degrees C. The pattern of DNA synthesis inhibition upon temperature upshift and the requirement for RNA synthesis, but not protein synthesis, for resumed DNA synthesis upon downshift to 32 degrees C indicate that synthesis is affected in the initiation phase. fis mutations act synergistically with gyrB alleles known to affect initiation. oriC-dependent plasmids are poorly established and maintained in fis mutant strains. Finally, purified Fis protein interacts in vitro with sites in oriC. These interactions could be involved in mediating the effect of Fis on DNA synthesis in vivo. Images PMID:1309527

  5. Characterization of berberine transport into Coptis japonica cells and the involvement of ABC protein.

    PubMed

    Sakai, Kyoko; Shitan, Nobukazu; Sato, Fumihiko; Ueda, Kazumitsu; Yazaki, Kazufumi

    2002-09-01

    Cultured Coptis japonica cells are able to take up berberine, a benzylisoquinoline alkaloid, from the medium and transport it exclusively into the vacuoles. Uptake activity depends on the growth phase of the cultured cells whereas the culture medium had no effect on uptake. Treatment with several inhibitors suggested that berberine uptake depended on the ATP level. Some inhibitors of P-glycoprotein, an ABC transporter involved in multiple drug resistance in cancer cells, strongly inhibited berberine uptake, whereas a specific inhibitor for glutathione biosynthesis and vacuolar ATPase, bafilomycin A1, had little effect. Vanadate-induced ATP trap experiments to detect ABC proteins expressed in C. japonica cells showed that three membrane proteins of between 120 and 150 kDa were photolabelled with 8-azido-[alpha-32P] ATP. Two revealed the same photoaffinity-labelling pattern as P-glycoprotein, and the interaction of these proteins with berberine was also demonstrated. These results suggest that ABC proteins of the MDR-type are involved in the uptake of berberine from the medium. PMID:12177126

  6. Involvement of Bcl-2-associated athanogene (BAG)-family proteins in the neuroprotection by rasagiline

    PubMed Central

    Guo, Ji-Feng; He, Shuang; Kang, Ji-Feng; Xu, Qian; Hu, Ya-Cen; Zhang, Hai-Nan; Wang, Chun-Yu; Yan, Xin-Xiang; Tang, Bei-Sha

    2015-01-01

    Rasagiline, a novel monoamine oxidase (MAO)-B inhibitor, has a mild to moderate effect in relieving Parkinson’s disease (PD) symptoms as well as unique neuroprotective effects. Previous studies demonstrated rasagiline protect neurons by regulating Bcl-2 family proteins. Our study aimed to study whether Bcl-2-associated athanogene (BAG)-family proteins, which were reported closely associated with neurodegenerative disease, were involved in the neuroprotective effect of rasagiline. We found that after the administration of 1-methy1-4-phenvl-1,2,3,6-tetrahvdropvridine (MPTP), BAG2 and BAG5 proteins were up-regulated in the substantia nigra dopaminergic neurons of PD mouse model. A further increase of BAG2 and BAG5 was detected after intragastric administration of rasagiline to post-MPTP lesioned mice. Thus, the current study proved the association of BAG family proteins with PD, and suggested the involvement and a positive role of BAG2, BAG5 in the neuroprotection of rasagiline. These preliminary results implicate a novel pathway for further study on neuroprotection of rasagiline. PMID:26770414

  7. Myomegalin is a novel A-kinase anchoring protein involved in the phosphorylation of cardiac myosin binding protein C

    PubMed Central

    2011-01-01

    Background Cardiac contractility is regulated by dynamic phosphorylation of sarcomeric proteins by kinases such as cAMP-activated protein kinase A (PKA). Efficient phosphorylation requires that PKA be anchored close to its targets by A-kinase anchoring proteins (AKAPs). Cardiac Myosin Binding Protein-C (cMyBPC) and cardiac troponin I (cTNI) are hypertrophic cardiomyopathy (HCM)-causing sarcomeric proteins which regulate contractility in response to PKA phosphorylation. Results During a yeast 2-hybrid (Y2H) library screen using a trisphosphorylation mimic of the C1-C2 region of cMyBPC, we identified isoform 4 of myomegalin (MMGL) as an interactor of this N-terminal cMyBPC region. As MMGL has previously been shown to interact with phosphodiesterase 4D, we speculated that it may be a PKA-anchoring protein (AKAP). To investigate this possibility, we assessed the ability of MMGL isoform 4 to interact with PKA regulatory subunits R1A and R2A using Y2H-based direct protein-protein interaction assays. Additionally, to further elucidate the function of MMGL, we used it as bait to screen a cardiac cDNA library. Other PKA targets, viz. CARP, COMMD4, ENO1, ENO3 and cTNI were identified as putative interactors, with cTNI being the most frequent interactor. We further assessed and confirmed these interactions by fluorescent 3D-co-localization in differentiated H9C2 cells as well as by in vivo co-immunoprecipitation. We also showed that quantitatively more interaction occurs between MMGL and cTNI under β-adrenergic stress. Moreover, siRNA-mediated knockdown of MMGL leads to reduction of cMyBPC levels under conditions of adrenergic stress, indicating that MMGL-assisted phosphorylation is requisite for protection of cMyBPC against proteolytic cleavage. Conclusions This study ascribes a novel function to MMGL isoform 4: it meets all criteria for classification as an AKAP, and we show that is involved in the phosphorylation of cMyBPC as well as cTNI, hence MMGL is an important

  8. Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking ▿

    PubMed Central

    Takeuchi, Hiroshi; Takeuchi, Takako; Gao, Jing; Cantley, Lewis C.; Hirata, Masato

    2010-01-01

    The phox homology (PX) domain is a phosphoinositide-binding module that typically binds phosphatidylinositol 3-phosphate. Out of 47 mammalian proteins containing PX domains, more than 30 are denoted sorting nexins and several of these have been implicated in internalization of cell surface proteins to the endosome, where phosphatidylinositol-3-phosphate is concentrated. Here we investigated a multimodular protein termed PXK, composed of a PX domain, a protein kinase-like domain, and a WASP homology 2 domain. We show that the PX domain of PXK localizes this protein to the endosomal membrane via binding to phosphatidylinositol 3-phosphate. PXK expression in COS7 cells accelerated the ligand-induced internalization and degradation of epidermal growth factor receptors by a mechanism requiring phosphatidylinositol 3-phosphate binding but not involving the WASP homology 2 domain. Conversely, depletion of PXK using RNA interference decreased the rate of epidermal growth factor receptor internalization and degradation. Ubiquitination of epidermal growth factor receptor by the ligand stimulation was enhanced in PXK-expressing cells. These results indicate that PXK plays a critical role in epidermal growth factor receptor trafficking through modulating ligand-induced ubiquitination of the receptor. PMID:20086096

  9. A histidine protein kinase is involved in polar organelle development in Caulobacter crescentus.

    PubMed Central

    Wang, S P; Sharma, P L; Schoenlein, P V; Ely, B

    1993-01-01

    Mutations having pleiotropic effects on polar organelle development (pod) in Caulobacter crescentus have been identified and shown to occur in at least 13 genes scattered throughout the genome. Mutations at each locus affect a unique combination of polar traits, suggesting that complex interactions occur among these genes. The DNA sequence of one of these genes, pleC, indicates that it is homologous to members of the family of histidine protein kinase genes. Membes of this family include the senor components of the bacterial two-component regulatory systems. Furthermore, in vitro experiments demonstrated that the PleC protein was capable of autophosphorylation. These results suggest that the PleC protein (and perhaps the proteins encoded by the other pod genes as well) regulates the expression of genes involved in polar organelle development through the phosphorylation of key regulatory proteins. The use of a phosphorelay system cued to internal changes in the cell would provide a mechanism for coordinating major changes in gene expression with the completion of specific cell cycle events. Images PMID:8421698

  10. Microtubule-severing proteins are involved in flagellar length control and mitosis in Trypanosomatids.

    PubMed

    Casanova, Magali; Crobu, Lucien; Blaineau, Christine; Bourgeois, Nathalie; Bastien, Patrick; Pagès, Michel

    2009-03-01

    Microtubules are key players in the biology of Trypanosomatid parasites, not only as classical components of the mitotic spindle, microtubule-organizing centres and flagellum but also as the essential constituent of the cytoskeleton. Their length dynamics are regulated by, among others, microtubule-severing proteins. Four and six genes encoding microtubule-severing proteins can be found bioinformatically in the Leishmania major and Trypanosoma brucei genome respectively. We investigated all these proteins in these organisms, which include the katanin, katanin-like, spastin and fidgetin, and looked at their subcellular localization as well as their putative function by examining 'loss-of-function' phenotypes. The katanin-like KAT60b was found implicated in flagellar length reduction, but not in its size increase, while the katanin p80 subunit appeared clearly involved in cytokinesis. Fidgetin and spastin homologues were both localized in the nucleus: the first as a discrete and variable number of dots during most of the cell cycle, redistributing to the spindle and midbody during mitosis; the second concentrated as < or = 5 perinucleolar punctuations, similar to the electron-dense plaques identified in T. brucei, which were assimilated to kinetochores. This first study of microtubule-severing proteins in 'divergent' eukaryotes gives further insight into the multiple functions of these proteins identified in the hitherto studied models. PMID:19183280

  11. Identification of an Atypical Membrane Protein Involved in the Formation of Protein Disulfide Bonds in Oxygenic Photosynthetic Organisms*S⃞

    PubMed Central

    Singh, Abhay K.; Bhattacharyya-Pakrasi, Maitrayee; Pakrasi, Himadri B.

    2008-01-01

    The evolution of oxygenic photosynthesis in cyanobacteria nearly three billion years ago provided abundant reducing power and facilitated the elaboration of numerous oxygen-dependent reactions in our biosphere. Cyanobacteria contain an internal thylakoid membrane system, the site of photosynthesis, and a typical Gram-negative envelope membrane system. Like other organisms, the extracytoplasmic space in cyanobacteria houses numerous cysteine-containing proteins. However, the existence of a biochemical system for disulfide bond formation in cyanobacteria remains to be determined. Extracytoplasmic disulfide bond formation in non-photosynthetic organisms is catalyzed by coordinated interaction between two proteins, a disulfide carrier and a disulfide generator. Here we describe a novel gene, SyndsbAB, required for disulfide bond formation in the extracytoplasmic space of cyanobacteria. The SynDsbAB orthologs are present in most cyanobacteria and chloroplasts of higher plants with fully sequenced genomes. The SynDsbAB protein contains two distinct catalytic domains that display significant similarity to proteins involved in disulfide bond formation in Escherichia coli and eukaryotes. Importantly, SyndsbAB complements E. coli strains defective in disulfide bond formation. In addition, the activity of E. coli alkaline phosphatase localized to the periplasm of Synechocystis 6803 is dependent on the function of SynDsbAB. Deletion of SyndsbAB in Synechocystis 6803 causes significant growth impairment under photoautotrophic conditions and results in hyper-sensitivity to dithiothreitol, a reductant, whereas diamide, an oxidant had no effect on the growth of the mutant strains. We conclude that SynDsbAB is a critical protein for disulfide bond formation in oxygenic photosynthetic organisms and required for their optimal photoautotrophic growth. PMID:18413314

  12. Interferon-inducible GTPase: a novel viral response protein involved in rabies virus infection.

    PubMed

    Li, Ling; Wang, Hualei; Jin, Hongli; Cao, Zengguo; Feng, Na; Zhao, Yongkun; Zheng, Xuexing; Wang, Jianzhong; Li, Qian; Zhao, Guoxing; Yan, Feihu; Wang, Lina; Wang, Tiecheng; Gao, Yuwei; Tu, Changchun; Yang, Songtao; Xia, Xianzhu

    2016-05-01

    Rabies virus infection is a major public health concern because of its wide host-interference spectrum and nearly 100 % lethality. However, the interactions between host and virus remain unclear. To decipher the authentic response in the central nervous system after rabies virus infection, a dynamic analysis of brain proteome alteration was performed. In this study, 104 significantly differentially expressed proteins were identified, and intermediate filament, interferon-inducible GTPases, and leucine-rich repeat-containing protein 16C were the three outstanding groups among these proteins. Interferon-inducible GTPases were prominent because of their strong upregulation. Moreover, quantitative real-time PCR showed distinct upregulation of interferon-inducible GTPases at the level of transcription. Several studies have shown that interferon-inducible GTPases are involved in many biological processes, such as viral infection, endoplasmic reticulum stress response, and autophagy. These findings indicate that interferon-inducible GTPases are likely to be a potential target involved in rabies pathogenesis or the antiviral process. PMID:26906695

  13. Homeodomain Protein Scr Regulates the Transcription of Genes Involved in Juvenile Hormone Biosynthesis in the Silkworm

    PubMed Central

    Meng, Meng; Liu, Chun; Peng, Jian; Qian, Wenliang; Qian, Heying; Tian, Ling; Li, Jiarui; Dai, Dandan; Xu, Anying; Li, Sheng; Xia, Qingyou; Cheng, Daojun

    2015-01-01

    The silkworm Dominant trimolting (Moltinism, M3) mutant undergoes three larval molts and exhibits precocious metamorphosis. In this study, we found that compared with the wild-type (WT) that undergoes four larval molts, both the juvenile hormone (JH) concentration and the expression of the JH-responsive gene Krüppel homolog 1 (Kr-h1) began to be greater in the second instar of the M3 mutant. A positional cloning analysis revealed that only the homeodomain transcription factor gene Sex combs reduced (Scr) is located in the genomic region that is tightly linked to the M3 locus. The expression level of the Scr gene in the brain-corpora cardiaca-corpora allata (Br-CC-CA) complex, which controls the synthesis of JH, was very low in the final larval instar of both the M3 and WT larvae, and exhibited a positive correlation with JH titer changes. Importantly, luciferase reporter analysis and electrophoretic mobility shift assay (EMSA) demonstrated that the Scr protein could promote the transcription of genes involved in JH biosynthesis by directly binding to the cis-regulatory elements (CREs) of homeodomain protein on their promoters. These results conclude that the homeodomain protein Scr is transcriptionally involved in the regulation of JH biosynthesis in the silkworm. PMID:26540044

  14. Assessment of cholesteryl ester transfer protein inhibitors for interaction with proteins involved in the immune response to infection.

    PubMed

    Clark, Ronald W; Cunningham, David; Cong, Yang; Subashi, Timothy A; Tkalcevic, George T; Lloyd, David B; Boyd, James G; Chrunyk, Boris A; Karam, George A; Qiu, Xiayang; Wang, Ing-Kae; Francone, Omar L

    2010-05-01

    The CETP inhibitor, torcetrapib, was prematurely terminated from phase 3 clinical trials due to an increase in cardiovascular and noncardiovascular mortality. Because nearly half of the latter deaths involved patients with infection, we have tested torcetrapib and other CETPIs to see if they interfere with lipopolysaccharide binding protein (LBP) or bactericidal/permeability increasing protein (BPI). No effect of these potent CETPIs on LPS binding to either protein was detected. Purified CETP itself bound weakly to LPS with a Kd >or= 25 microM compared with 0.8 and 0.5 nM for LBP and BPI, respectively, and this binding was not blocked by torcetrapib. In whole blood, LPS induced tumor necrosis factor-alpha normally in the presence of torcetrapib. Furthermore, LPS had no effect on CETP activity. We conclude that the sepsis-related mortality of the ILLUMINATE trial was unlikely due to a direct effect of torcetrapib on LBP or BPI function, nor to inhibition of an interaction of CETP with LPS. Instead, we speculate that the negative outcome seen for patients with infections might be related to the changes in plasma lipoprotein composition and metabolism, or alternatively to the known off-target effects of torcetrapib, such as aldosterone elevation, which may have aggravated the effects of sepsis. PMID:19965592

  15. Assessment of cholesteryl ester transfer protein inhibitors for interaction with proteins involved in the immune response to infection[S

    PubMed Central

    Clark, Ronald W.; Cunningham, David; Cong, Yang; Subashi, Timothy A.; Tkalcevic, George T.; Lloyd, David B.; Boyd, James G.; Chrunyk, Boris A.; Karam, George A.; Qiu, Xiayang; Wang, Ing-Kae; Francone, Omar L.

    2010-01-01

    The CETP inhibitor, torcetrapib, was prematurely terminated from phase 3 clinical trials due to an increase in cardiovascular and noncardiovascular mortality. Because nearly half of the latter deaths involved patients with infection, we have tested torcetrapib and other CETPIs to see if they interfere with lipopolysaccharide binding protein (LBP) or bactericidal/permeability increasing protein (BPI). No effect of these potent CETPIs on LPS binding to either protein was detected. Purified CETP itself bound weakly to LPS with a Kd ≥ 25 uM compared with 0.8 and 0.5 nM for LBP and BPI, respectively, and this binding was not blocked by torcetrapib. In whole blood, LPS induced tumor necrosis factor-α normally in the presence of torcetrapib. Furthermore, LPS had no effect on CETP activity. We conclude that the sepsis-related mortality of the ILLUMINATE trial was unlikely due to a direct effect of torcetrapib on LBP or BPI function, nor to inhibition of an interaction of CETP with LPS. Instead, we speculate that the negative outcome seen for patients with infections might be related to the changes in plasma lipoprotein composition and metabolism, or alternatively to the known off-target effects of torcetrapib, such as aldosterone elevation, which may have aggravated the effects of sepsis. PMID:19965592

  16. The Prediction of Key Cytoskeleton Components Involved in Glomerular Diseases Based on a Protein-Protein Interaction Network

    PubMed Central

    Ju, Wenjun; Li, Xuejuan; Li, Shao; Ding, Jie

    2016-01-01

    Maintenance of the physiological morphologies of different types of cells and tissues is essential for the normal functioning of each system in the human body. Dynamic variations in cell and tissue morphologies depend on accurate adjustments of the cytoskeletal system. The cytoskeletal system in the glomerulus plays a key role in the normal process of kidney filtration. To enhance the understanding of the possible roles of the cytoskeleton in glomerular diseases, we constructed the Glomerular Cytoskeleton Network (GCNet), which shows the protein-protein interaction network in the glomerulus, and identified several possible key cytoskeletal components involved in glomerular diseases. In this study, genes/proteins annotated to the cytoskeleton were detected by Gene Ontology analysis, and glomerulus-enriched genes were selected from nine available glomerular expression datasets. Then, the GCNet was generated by combining these two sets of information. To predict the possible key cytoskeleton components in glomerular diseases, we then examined the common regulation of the genes in GCNet in the context of five glomerular diseases based on their transcriptomic data. As a result, twenty-one cytoskeleton components as potential candidate were highlighted for consistently down- or up-regulating in all five glomerular diseases. And then, these candidates were examined in relation to existing known glomerular diseases and genes to determine their possible functions and interactions. In addition, the mRNA levels of these candidates were also validated in a puromycin aminonucleoside(PAN) induced rat nephropathy model and were also matched with existing Diabetic Nephropathy (DN) transcriptomic data. As a result, there are 15 of 21 candidates in PAN induced nephropathy model were consistent with our predication and also 12 of 21 candidates were matched with differentially expressed genes in the DN transcriptomic data. By providing a novel interaction network and prediction, GCNet

  17. GidA is an FAD-binding protein involved in development of Myxococcus xanthus.

    PubMed

    White, D J; Merod, R; Thomasson, B; Hartzell, P L

    2001-10-01

    A gene encoding a homologue of the Escherichia coli GidA protein (glucose-inhibited division protein A) lies immediately upstream of aglU, a gene encoding a WD-repeat protein required for motility and development in Myxococcus xanthus. The GidA protein of M. xanthus shares about 48% identity overall with the small (approximately equal to 450 amino acid) form of GidA from eubacteria and about 24% identity overall with the large (approximately equal to 620 amino acid) form of GidA from eubacteria and eukaryotes. Each of these proteins has a conserved dinucleotide-binding motif at the N-terminus. To determine if GidA binds dinucleotide, the M. xanthus gene was expressed with a His6 tag in E. coli cells. Purified rGidA is a yellow protein that absorbs maximally at 374 and 450 nm, consistent with FAD or FMN. Thin-layer chromatography (TLC) showed that rGidA contains an FAD cofactor. Fractionation and immunocytochemical localization show that full length GidA protein is present in the cytoplasm and transported to the periplasm of vegetative-grown M. xanthus cells. In cells that have been starved for nutrients, GidA is found in the cytoplasm. Although GidA lacks an obvious signal sequence, it contains a twin arginine transport (Tat) motif, which is conserved among proteins that bind cofactors in the cytoplasm and are transported to the periplasm as folded proteins. To determine if GidA, like AglU, is involved in motility and development, the gidA gene was disrupted. The gidA- mutant has wild-type gliding motility and initially is able to form fruiting bodies like the wild type when starved for nutrients. However, after several generations, a stable derivative arises, gidA*, which is indistinguishable from the gidA- parent on vegetative medium, but is no longer able to form fruiting bodies. The gidA* mutant releases a heat-stable, protease-resistant, small molecular weight molecule that acts in trans to inhibit aggregation and gene expression of wild-type cells during

  18. Water-soluble chlorophyll protein is involved in herbivore resistance activation during greening of Arabidopsis thaliana

    PubMed Central

    Boex-Fontvieille, Edouard; Rustgi, Sachin; von Wettstein, Diter; Reinbothe, Steffen; Reinbothe, Christiane

    2015-01-01

    Water-soluble chlorophyll proteins (WSCPs) constitute a small family of unusual chlorophyll (Chl)-binding proteins that possess a Kunitz-type protease inhibitor domain. In Arabidopsis thaliana, a WSCP has been identified, named AtWSCP, that forms complexes with Chl and the Chl precursor chlorophyllide (Chlide) in vitro. AtWSCP exhibits a quite unexpected expression pattern for a Chl binding protein and accumulated to high levels in the apical hook of etiolated plants. AtWSCP expression was negatively light-regulated. Transgenic expression of AtWSCP fused to green fluorescent protein (GFP) revealed that AtWSCP is localized to cell walls/apoplastic spaces. Biochemical assays identified AtWSCP as interacting with RD21 (RESPONSIVE TO DESICCATION 21), a granulin domain-containing cysteine protease implicated in stress responses and defense. Reconstitution experiments showed tight interactions between RD21 and WSCP that were relieved upon Chlide binding. Laboratory feeding experiments with two herbivorous isopod crustaceans, Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug), identified the apical hook as Achilles’ heel of etiolated plants and that this was protected by RD21 during greening. Because Chlide is formed in the apical hook during seedling emergence from the soil, our data suggest an unprecedented mechanism of herbivore resistance activation that is triggered by light and involves AtWSCP. PMID:26016527

  19. Water-soluble chlorophyll protein is involved in herbivore resistance activation during greening of Arabidopsis thaliana.

    PubMed

    Boex-Fontvieille, Edouard; Rustgi, Sachin; von Wettstein, Diter; Reinbothe, Steffen; Reinbothe, Christiane

    2015-06-01

    Water-soluble chlorophyll proteins (WSCPs) constitute a small family of unusual chlorophyll (Chl)-binding proteins that possess a Kunitz-type protease inhibitor domain. In Arabidopsis thaliana, a WSCP has been identified, named AtWSCP, that forms complexes with Chl and the Chl precursor chlorophyllide (Chlide) in vitro. AtWSCP exhibits a quite unexpected expression pattern for a Chl binding protein and accumulated to high levels in the apical hook of etiolated plants. AtWSCP expression was negatively light-regulated. Transgenic expression of AtWSCP fused to green fluorescent protein (GFP) revealed that AtWSCP is localized to cell walls/apoplastic spaces. Biochemical assays identified AtWSCP as interacting with RD21 (responsive to desiccation 21), a granulin domain-containing cysteine protease implicated in stress responses and defense. Reconstitution experiments showed tight interactions between RD21 and WSCP that were relieved upon Chlide binding. Laboratory feeding experiments with two herbivorous isopod crustaceans, Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug), identified the apical hook as Achilles' heel of etiolated plants and that this was protected by RD21 during greening. Because Chlide is formed in the apical hook during seedling emergence from the soil, our data suggest an unprecedented mechanism of herbivore resistance activation that is triggered by light and involves AtWSCP. PMID:26016527

  20. Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in benzylisoquinoline alkaloid biosynthesis

    PubMed Central

    Yamada, Yasuyuki; Sato, Fumihiko

    2016-01-01

    Benzylisoquinoline alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKYY115E phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs. PMID:27552928

  1. Possible involvement of lipoic acid in binding protein-dependent transport systems in Escherichia coli.

    PubMed

    Richarme, G

    1985-04-01

    We describe the properties of the binding protein dependent-transport of ribose, galactose, and maltose and of the lactose permease, and the phosphoenolpyruvate-glucose phosphotransferase transport systems in a strain of Escherichia coli which is deficient in the synthesis of lipoic acid, a cofactor involved in alpha-keto acid dehydrogenation. Such a strain can grow in the absence of lipoic acid in minimal medium supplemented with acetate and succinate. Although the lactose permease and the phosphoenolypyruvate-glucose phosphotransferase are not affected by lipoic acid deprivation, the binding protein-dependent transports are reduced by 70% in conditions of lipoic acid deprivation when compared with their activity in conditions of lipoic acid supply. The remaining transport is not affected by arsenate but is inhibited by the uncoupler carbonylcyanide-m-chlorophenylhydrazone; however the lipoic acid-dependent transport is completely inhibited by arsenate and only weakly inhibited by carbonylcyanide-m-chlorophenylhydrazone. The known inhibitor of alpha-keto acid dehydrogenases, 5-methoxyindole-2-carboxylic acid, completely inhibits all binding protein-dependent transports whether in conditions of lipoic supply or deprivation; the results suggest a possible relation between binding protein-dependent transport and alpha-keto acid dehydrogenases and shed light on the inhibition of these transports by arsenicals and uncouplers. PMID:3920206

  2. Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in benzylisoquinoline alkaloid biosynthesis.

    PubMed

    Yamada, Yasuyuki; Sato, Fumihiko

    2016-01-01

    Benzylisoquinoline alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKY(Y115E) phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs. PMID:27552928

  3. Intrahippocampal infusion of spermidine improves memory persistence: Involvement of protein kinase A.

    PubMed

    Signor, Cristiane; Temp, Fernanda R; Mello, Carlos F; Oliveira, Mauro S; Girardi, Bruna A; Gais, Mayara A; Funck, Vinicius R; Rubin, Maribel A

    2016-05-01

    Spermidine (SPD) is an endogenous aliphatic amine that modulates GluN2B-containing NMDA receptors and improves memory. Recent evidence suggests that systemic SPD improves the persistence of the long term memory of fear. However, the role of hippocampal polyamines and its binding sites in the persistence of fear memory is to be determined, as well as its putative underlying mechanisms. This study investigated whether the intrahippocampal (i.h.) infusion of spermidine or arcaine, modulators of polyamine binding site at GluN2B-containing NMDA receptors, alters the persistence of the memory of contextual fear conditioning task in rats. We also investigated whether protein synthesis and cAMP dependent protein kinase (PKA) play a role in SPD-induced improvement of the fear memory persistence. While 12h post-training infusion of spermidine facilitated, arcaine and the inhibitor of protein synthesis (anisomycin) impaired the memory of fear assessed 7days after training. The infusion of arcaine, anisomycin or a selective PKA inhibitor (H-89), at doses that have no effect on memory per se, prevented the SPD-induced improvement of memory persistence. H-89 prevented the stimulatory effect of SPD on phospho-PKA/total-PKA ratio. These results suggests that the improvement of fear memory persistence induced by spermidine involves GluN2B-containing NMDA receptors, PKA pathway and protein synthesis in rats. PMID:26968655

  4. Identification of proteins involved in desiccation tolerance in the red seaweed Pyropia orbicularis (Rhodophyta, Bangiales).

    PubMed

    López-Cristoffanini, Camilo; Zapata, Javier; Gaillard, Fanny; Potin, Philippe; Correa, Juan A; Contreras-Porcia, Loretto

    2015-12-01

    Extreme reduction in cellular water content leads to desiccation, which, if persistent, affects the physiology of organisms, mainly through oxidative stress. Some organisms are highly tolerant to desiccation, including resurrection plants and certain intertidal seaweeds. One such species is Pyropia orbicularis, a rhodophycean that colonizes upper intertidal zones along the Chilean coast. Despite long, daily periods of air exposure due to tides, this alga is highly tolerant to desiccation. The present study examined the proteome of P. orbicularis by 2DE and LC-MS/MS analyses to determine the proteins associated with desiccation tolerance (DT). The results showed that, under natural conditions, there were significant changes in the protein profile during low tide as compared to naturally hydrated plants at high tide. These changes were mainly in newly appeared proteins spots such as chaperones, monodehydroascorbate reductase, and manganese superoxide dismutase, among others. Previously undescribed proteins under desiccation conditions included phycobiliproteins, glyoxalase I, and phosphomannomutase. These changes evidenced that several physiological responses involved in DT are activated during low tide, including decreased photosynthetic activity, increased antioxidant capacity, and the preservation of cell physiology by regulating water content, cell wall structure, and cell volume. Similar responses have been observed in resurrection plants and bryophytes exposed to desiccation. Therefore, the coordinated activation of different desiccation tolerance pathways in P. orbicularis could explain the successful biological performance of this seaweed in the upper intertidal rocky zones. PMID:26154304

  5. Centlein, a novel microtubule-associated protein stabilizing microtubules and involved in neurite formation.

    PubMed

    Jing, Zhenli; Yin, Huilong; Wang, Pan; Gao, Juntao; Yuan, Li

    2016-04-01

    We have previously reported that the centriolar protein centlein functions as a molecular link between C-Nap1 and Cep68 to maintain centrosome cohesion [1]. In this study, we identified centlein as a novel microtubule-associated protein (MAP), directly binding to purified microtubules (MTs) via its longest coiled-coil domain. Overexpression of centlein caused profound nocodazole- and cold-resistant MT bundles, which also relied on its MT-binding domain. siRNA-mediated centlein depletion resulted in a significant reduction in tubulin acetylation level and overall fluorescence intensity of cytoplasmic MT acetylation. Centlein was further characterized in neurons. We found that centlein overexpression inhibited neurite formation in retinoic acid (RA)-induced SH-SY5Y and N2a cells. Taken together, we propose that centlein is involved in MT stability and neuritogenesis in vivo. PMID:26915804

  6. Yeast Irc22 Is a Novel Dsk2-Interacting Protein that Is Involved in Salt Tolerance

    PubMed Central

    Ishii, Takashi; Funakoshi, Minoru; Kobayashi, Hideki; Sekiguchi, Takeshi

    2014-01-01

    The yeast ubiquitin-like and ubiquitin-associated protein Dsk2 is one of the ubiquitin receptors that function in the ubiquitin-proteasome pathway. We screened the Dsk2-interacting proteins in Saccharomyces cerevisiae by a two-hybrid assay and identified a novel Dsk2-interacting protein, Irc22, the gene locus of which has previously been described as YEL001C, but the function of which is unknown. IRC22/YEL001C encodes 225 amino acid residues with a calculated molecular weight of 25 kDa. The Irc22 protein was detected in yeast cells. IRC22 was a nonessential gene for yeast growth, and its homologs were found among ascomycetous yeasts. Irc22 interacted with Dsk2 in yeast cells, but not with Rad23 and Ddi1. Ubiquitin-dependent degradation was impaired mildly by over-expression or disruption of IRC22. Compared with the wild-type strain, dsk2Δ exhibited salt sensitivity while irc22Δ exhibited salt tolerance at high temperatures. The salt-tolerant phenotype that was observed in irc22Δ disappeared in the dsk2Δirc22Δ double disruptant, indicating that DSK2 is positively and IRC22 is negatively involved in salt stress tolerance. IRC22 disruption did not affect any responses to DNA damage and oxidative stress when comparing the irc22Δ and wild-type strains. Collectively, these results suggest that Dsk2 and Irc22 are involved in salt stress tolerance in yeast. PMID:24709957

  7. Development of neurodevelopmental disorders: a regulatory mechanism involving bromodomain-containing proteins.

    PubMed

    Li, Junlin; Zhao, Guifang; Gao, Xiaocai

    2013-01-01

    Neurodevelopmental disorders are classified as diseases that cause abnormal functions of the brain or central nervous system. Children with neurodevelopmental disorders show impaired language and speech abilities, learning and memory damage, and poor motor skills. However, we still know very little about the molecular etiology of these disorders. Recent evidence implicates the bromodomain-containing proteins (BCPs) in the initiation and development of neurodevelopmental disorders. BCPs have a particular domain, the bromodomain (Brd), which was originally identified as specifically binding acetyl-lysine residues at the N-terminus of histone proteins in vitro and in vivo. Other domains of BCPs are responsible for binding partner proteins to form regulatory complexes. Once these complexes are assembled, BCPs alter chromosomal states and regulate gene expression. Some BCP complexes bind nucleosomes, are involved in basal transcription regulation, and influence the transcription of many genes. However, most BCPs are involved in targeting. For example, some BCPs function as a recruitment platform or scaffold through their Brds-binding targeting sites. Others are recruited to form a complex to bind the targeting sites of their partners. The regulation mediated by these proteins is especially critical during normal and abnormal development. Mutant BCPs or dysfunctional BCP-containing complexes are implicated in the initiation and development of neurodevelopmental disorders. However, the pathogenic molecular mechanisms are not fully understood. In this review, we focus on the roles of regulatory BCPs associated with neurodevelopmental disorders, including mental retardation, Fragile X syndrome (FRX), Williams syndrome (WS), Rett syndrome and Rubinstein-Taybi syndrome (RTS). A better understanding of the molecular pathogenesis, based upon the roles of BCPs, will lead to screening of targets for the treatment of neurodevelopmental disorders. PMID:23425632

  8. Development of neurodevelopmental disorders: a regulatory mechanism involving bromodomain-containing proteins

    PubMed Central

    2013-01-01

    Neurodevelopmental disorders are classified as diseases that cause abnormal functions of the brain or central nervous system. Children with neurodevelopmental disorders show impaired language and speech abilities, learning and memory damage, and poor motor skills. However, we still know very little about the molecular etiology of these disorders. Recent evidence implicates the bromodomain-containing proteins (BCPs) in the initiation and development of neurodevelopmental disorders. BCPs have a particular domain, the bromodomain (Brd), which was originally identified as specifically binding acetyl-lysine residues at the N-terminus of histone proteins in vitro and in vivo. Other domains of BCPs are responsible for binding partner proteins to form regulatory complexes. Once these complexes are assembled, BCPs alter chromosomal states and regulate gene expression. Some BCP complexes bind nucleosomes, are involved in basal transcription regulation, and influence the transcription of many genes. However, most BCPs are involved in targeting. For example, some BCPs function as a recruitment platform or scaffold through their Brds-binding targeting sites. Others are recruited to form a complex to bind the targeting sites of their partners. The regulation mediated by these proteins is especially critical during normal and abnormal development. Mutant BCPs or dysfunctional BCP-containing complexes are implicated in the initiation and development of neurodevelopmental disorders. However, the pathogenic molecular mechanisms are not fully understood. In this review, we focus on the roles of regulatory BCPs associated with neurodevelopmental disorders, including mental retardation, Fragile X syndrome (FRX), Williams syndrome (WS), Rett syndrome and Rubinstein-Taybi syndrome (RTS). A better understanding of the molecular pathogenesis, based upon the roles of BCPs, will lead to screening of targets for the treatment of neurodevelopmental disorders. PMID:23425632

  9. Mouse neuron navigator 1, a novel microtubule-associated protein involved in neuronal migration.

    PubMed

    Martínez-López, María José; Alcántara, Soledad; Mascaró, Cristina; Pérez-Brangulí, Francesc; Ruiz-Lozano, Pilar; Maes, Tamara; Soriano, Eduardo; Buesa, Carlos

    2005-04-01

    The development of the nervous system (NS) requires the coordinated migration of multiple waves of neurons and subsequent processes of neurite maturation, both involving selective guidance mechanisms. In Caenorhabditis elegans, unc-53 codes for a new multidomain protein involved in the directional migration of a subset of cells. We describe here the first functional characterization of the mouse homologue, mouse Neuron navigator 1 (mNAV1), whose expression is largely restricted to the NS during development. EGFP-mNAV1 associates with microtubules (MTs) plus ends present in the growth cone through a new microtubule-binding (MTB) domain. Moreover, its overexpression in transfected cells leads to MT bundling. The abolition of mNAV1 causes loss of directionality in the leading processes of pontine-migrating cells, providing evidence for a role of mNAV1 in mediating Netrin-1-induced directional migration. PMID:15797708

  10. STAT5 proteins are involved in down-regulation of iron regulatory protein 1 gene expression by nitric oxide.

    PubMed

    Starzynski, Rafal Radoslaw; Gonçalves, Ana Sofia; Muzeau, Françoise; Tyrolczyk, Zofia; Smuda, Ewa; Drapier, Jean-Claude; Beaumont, Carole; Lipinski, Pawel

    2006-12-01

    RNA-binding activity of IRP1 (iron regulatory protein 1) is regulated by the insertion/extrusion of a [4Fe-4S] cluster into/from the IRP1 molecule. NO (nitic oxide), whose ability to activate IRP1 by removing its [4Fe-4S] cluster is well known, has also been shown to down-regulate expression of the IRP1 gene. In the present study, we examine whether this regulation occurs at the transcriptional level. Analysis of the mouse IRP1 promoter sequence revealed two conserved putative binding sites for transcription factor(s) regulated by NO and/or changes in intracellular iron level: Sp1 (promoter-selective transcription factor 1) and MTF1 (metal transcription factor 1), plus GAS (interferon-gamma-activated sequence), a binding site for STAT (signal transducer and activator of transcription) proteins. In order to define the functional activity of these sequences, reporter constructs were generated through the insertion of overlapping fragments of the mouse IRP1 promoter upstream of the luciferase gene. Transient expression assays following transfection of HuH7 cells with these plasmids revealed that while both the Sp1 and GAS sequences are involved in basal transcriptional activity of the IRP1 promoter, the role of the latter is predominant. Analysis of protein binding to these sequences in EMSAs (electrophoretic mobility-shift assays) using nuclear extracts from mouse RAW 264.7 macrophages stimulated to synthesize NO showed a significant decrease in the formation of Sp1-DNA and STAT-DNA complexes, compared with controls. We have also demonstrated that the GAS sequence is involved in NO-dependent down-regulation of IRP1 transcription. Further analysis revealed that levels of STAT5a and STAT5b in the nucleus and cytosol of NO-producing macrophages are substantially lower than in control cells. These findings provide evidence that STAT5 proteins play a role in NO-mediated down-regulation of IRP1 gene expression. PMID:16886906

  11. STAT5 proteins are involved in down-regulation of iron regulatory protein 1 gene expression by nitric oxide

    PubMed Central

    Starzynski, Rafal Radoslaw; Gonçalves, Ana Sofia; Muzeau, Françoise; Tyrolczyk, Zofia; Smuda, Ewa; Drapier, Jean-Claude; Beaumont, Carole; Lipinski, Pawel

    2006-01-01

    RNA-binding activity of IRP1 (iron regulatory protein 1) is regulated by the insertion/extrusion of a [4Fe-4S] cluster into/from the IRP1 molecule. NO (nitic oxide), whose ability to activate IRP1 by removing its [4Fe-4S] cluster is well known, has also been shown to down-regulate expression of the IRP1 gene. In the present study, we examine whether this regulation occurs at the transcriptional level. Analysis of the mouse IRP1 promoter sequence revealed two conserved putative binding sites for transcription factor(s) regulated by NO and/or changes in intracellular iron level: Sp1 (promoter-selective transcription factor 1) and MTF1 (metal transcription factor 1), plus GAS (interferon-γ-activated sequence), a binding site for STAT (signal transducer and activator of transcription) proteins. In order to define the functional activity of these sequences, reporter constructs were generated through the insertion of overlapping fragments of the mouse IRP1 promoter upstream of the luciferase gene. Transient expression assays following transfection of HuH7 cells with these plasmids revealed that while both the Sp1 and GAS sequences are involved in basal transcriptional activity of the IRP1 promoter, the role of the latter is predominant. Analysis of protein binding to these sequences in EMSAs (electrophoretic mobility-shift assays) using nuclear extracts from mouse RAW 264.7 macrophages stimulated to synthesize NO showed a significant decrease in the formation of Sp1–DNA and STAT–DNA complexes, compared with controls. We have also demonstrated that the GAS sequence is involved in NO-dependent down-regulation of IRP1 transcription. Further analysis revealed that levels of STAT5a and STAT5b in the nucleus and cytosol of NO-producing macrophages are substantially lower than in control cells. These findings provide evidence that STAT5 proteins play a role in NO-mediated down-regulation of IRP1 gene expression. PMID:16886906

  12. Involvement of calmodulin and calmodulin-like proteins in plant responses to abiotic stresses

    PubMed Central

    Zeng, Houqing; Xu, Luqin; Singh, Amarjeet; Wang, Huizhong; Du, Liqun; Poovaiah, B. W.

    2015-01-01

    Transient changes in intracellular Ca2+ concentration have been well recognized to act as cell signals coupling various environmental stimuli to appropriate physiological responses with accuracy and specificity in plants. Calmodulin (CaM) and calmodulin-like proteins (CMLs) are major Ca2+ sensors, playing critical roles in interpreting encrypted Ca2+ signals. Ca2+-loaded CaM/CMLs interact and regulate a broad spectrum of target proteins such as channels/pumps/antiporters for various ions, transcription factors, protein kinases, protein phosphatases, metabolic enzymes, and proteins with unknown biochemical functions. Many of the target proteins of CaM/CMLs directly or indirectly regulate plant responses to environmental stresses. Basic information about stimulus-induced Ca2+ signal and overview of Ca2+ signal perception and transduction are briefly discussed in the beginning of this review. How CaM/CMLs are involved in regulating plant responses to abiotic stresses are emphasized in this review. Exciting progress has been made in the past several years, such as the elucidation of Ca2+/CaM-mediated regulation of AtSR1/CAMTA3 and plant responses to chilling and freezing stresses, Ca2+/CaM-mediated regulation of CAT3, MAPK8 and MKP1 in homeostasis control of reactive oxygen species signals, discovery of CaM7 as a DNA-binding transcription factor regulating plant response to light signals. However, many key questions in Ca2+/CaM-mediated signaling warrant further investigation. Ca2+/CaM-mediated regulation of most of the known target proteins is presumed based on their interaction. The downstream targets of CMLs are mostly unknown, and how specificity of Ca2+ signaling could be realized through the actions of CaM/CMLs and their target proteins is largely unknown. Future breakthroughs in Ca2+/CaM-mediated signaling will not only improve our understanding of how plants respond to environmental stresses, but also provide the knowledge base to improve stress-tolerance of

  13. Presynaptic kainate receptor facilitation of glutamate release involves protein kinase A in the rat hippocampus

    PubMed Central

    Rodríguez-Moreno, Antonio; Sihra, Talvinder S

    2004-01-01

    We have explored the mechanisms involved in the facilitation of glutamate release mediated by the activation of kainate receptors in the rat hippocampus using isolated nerve terminal (synaptosome) and slice preparations. In hippocampal nerve terminals, kainate (KA) produced an increase of glutamate release at concentrations of agonist ranging from 10 to 1000 μm. In hippocampal slices, KA at low nanomolar concentrations (20–50 nm) also produced an increase of evoked excitatory postsynaptic currents (eEPSCs) at mossy fibre–CA3 synapses. In both, synaptosomes and slices, the effect of KA was antagonized by CNQX, and persisted after pretreatment with a cocktail of antagonists for other receptors whose activation could potentially have produced facilitation of release. These data indicate that the facilitation of glutamate release observed is mediated by the activation of presynaptic glutamate receptors of the kainate type. Mechanistically, the observed effects of KA appear to be the same in synaptosomal and slice preparations. Thus, the effect of KA on glutamate release and mossy fibre–CA3 synaptic transmission was occluded by the stimulation of adenylyl cyclase by forskolin and suppressed by the inhibition of protein kinase A by H-89 or Rp-Br-cAMP. We conclude that kainate receptors present at presynaptic terminals in the rat hippocampus mediate the facilitation of glutamate release through a mechanism involving the activation of an adenylyl cyclase–second messenger cAMP–protein kinase A signalling cascade. PMID:15107475

  14. Identification and Characterization of Anaplasma phagocytophilum Proteins Involved in Infection of the Tick Vector, Ixodes scapularis

    PubMed Central

    Kocan, Katherine M.; Bonzón-Kulichenko, Elena; Alberdi, Pilar; Blouin, Edmour F.; Weisheit, Sabine; Mateos-Hernández, Lourdes; Cabezas-Cruz, Alejandro; Bell-Sakyi, Lesley; Vancová, Marie; Bílý, Tomáš; Meyer, Damien F.; Sterba, Jan; Contreras, Marinela; Rudenko, Nataliia; Grubhoffer, Libor; Vázquez, Jesús; de la Fuente, José

    2015-01-01

    Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10–15% and 65–71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface

  15. A VAMP-associated protein, PVA31 is involved in leaf senescence in Arabidopsis

    PubMed Central

    Ichikawa, Mie; Nakai, Yusuke; Arima, Keita; Nishiyama, Sayo; Hirano, Tomoko; Sato, Masa H

    2015-01-01

    VAMP-associated proteins (VAPs) are highly conserved among eukaryotes. Here, we report a functional analysis of one of the VAPs, PVA31, and demonstrate its novel function on leaf senescence in Arabidopsis. The expression of PVA31 is highly induced in senescence leaves, and localizes to the plasma membrane as well as the ARA7-positive endosomes. Yeast two-hybrid analysis demonstrates that PVA31 is interacted with the plasma membrane localized-VAMP proteins, VAMP721/722/724 but not with the endosome-localized VAMPs, VAMP711 and VAMP727, indicating that PVA31 is associated with VAMP721/722/724 on the plasma membrane. Strong constitutive expression of PVA31 under the control of the Cauliflower mosaic virus 35S promoter induces the typical symptom of leaf senescence earlier than WT in normal growth and an artificially induced senescence conditions. In addition, the marker genes for the SA-mediated signaling pathways, PR-1, is promptly expressed with elicitor application. These data indicate that PVA31-overexpressing plants exhibit the early senescence phenotype in their leaves, and suggest that PVA31 is involved in the SA-mediated programmed cell death process during leaf senescence and PR-protein secretion during pathogen infection in Arabidopsis. PMID:25897470

  16. Bioinformatic analysis of functional proteins involved in obesity associated with diabetes.

    PubMed

    Rao, Allam Appa; Tayaru, N Manga; Thota, Hanuman; Changalasetty, Suresh Babu; Thota, Lalitha Saroja; Gedela, Srinubabu

    2008-03-01

    The twin epidemic of diabetes and obesity pose daunting challenges worldwide. The dramatic rise in obesity-associated diabetes resulted in an alarming increase in the incidence and prevalence of obesity an important complication of diabetes. Differences among individuals in their susceptibility to both these conditions probably reflect their genetic constitutions. The dramatic improvements in genomic and bioinformatic resources are accelerating the pace of gene discovery. It is tempting to speculate the key susceptible genes/proteins that bridges diabetes mellitus and obesity. In this regard, we evaluated the role of several genes/proteins that are believed to be involved in the evolution of obesity associated diabetes by employing multiple sequence alignment using ClustalW tool and constructed a phylogram tree using functional protein sequences extracted from NCBI. Phylogram was constructed using Neighbor-Joining Algorithm a bioinformatic tool. Our bioinformatic analysis reports resistin gene as ominous link with obesity associated diabetes. This bioinformatic study will be useful for future studies towards therapeutic inventions of obesity associated type 2 diabetes. PMID:23675069

  17. Xanthorrhizol induced DNA fragmentation in HepG2 cells involving Bcl-2 family proteins

    SciTech Connect

    Tee, Thiam-Tsui; Cheah, Yew-Hoong; Meenakshii, Nallappan; Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer We isolated xanthorrhizol, a sesquiterpenoid compound from Curcuma xanthorrhiza. Black-Right-Pointing-Pointer Xanthorrhizol induced apoptosis in HepG2 cells as observed using SEM. Black-Right-Pointing-Pointer Apoptosis in xanthorrhizol-treated HepG2 cells involved Bcl-2 family proteins. Black-Right-Pointing-Pointer DNA fragmentation was observed in xanthorrhizol-treated HepG2 cells. Black-Right-Pointing-Pointer DNA fragmentation maybe due to cleavage of PARP and DFF45/ICAD proteins. -- Abstract: Xanthorrhizol is a plant-derived pharmacologically active sesquiterpenoid compound isolated from Curcuma xanthorrhiza. Previously, we have reported that xanthorrhizol inhibited the proliferation of HepG2 human hepatoma cells by inducing apoptotic cell death via caspase activation. Here, we attempt to further elucidate the mode of action of xanthorrhizol. Apoptosis in xanthorrhizol-treated HepG2 cells as observed by scanning electron microscopy was accompanied by truncation of BID; reduction of both anti-apoptotic Bcl-2 and Bcl-X{sub L} expression; cleavage of PARP and DFF45/ICAD proteins and DNA fragmentation. Taken together, these results suggest xanthorrhizol as a potent antiproliferative agent on HepG2 cells by inducing apoptosis via Bcl-2 family members. Hence we proposed that xanthorrhizol could be used as an anti-liver cancer drug for future studies.

  18. Chemical genetic screen for AMPKα2 substrates uncovers a network of proteins involved in mitosis

    PubMed Central

    Banko, Max R.; Allen, Jasmina J.; Schaffer, Bethany E.; Wilker, Erik W.; Tsou, Peiling; White, Jamie L.; Villén, Judit; Wang, Beatrice; Kim, Sara R.; Sakamoto, Kei; Gygi, Steven P.; Cantley, Lewis C.; Yaffe, Michael B.; Shokat, Kevan M.; Brunet, Anne

    2011-01-01

    SUMMARY The energy-sensing AMP-activated protein kinase (AMPK) is activated by low nutrient levels. Functions of AMPK, other than its role in cellular metabolism, are just beginning to emerge. Here we use a chemical genetics screen to identify direct substrates of AMPK in human cells. We find that AMPK phosphorylates 28 previously unidentified substrates, several of which are involved in mitosis and cytokinesis. We identify the residues phosphorylated by AMPK in vivo in several substrates, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21 -activated protein kinase (PAK2). AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain. Both AMPK activity and PPP1R12C phosphorylation are increased in mitotic cells and are important for mitosis completion. These findings suggest that AMPK coordinates nutrient status with mitosis completion, which may be critical for the organism’s response to low nutrients during development, or in adult stem and cancer cells. PMID:22137581

  19. Interatomic Coulombic Decay Effects in Theoretical DNA Recombination Systems Involving Protein Interaction Sites

    NASA Astrophysics Data System (ADS)

    Vargas, E. L.; Rivas, D. A.; Duot, A. C.; Hovey, R. T.; Andrianarijaona, V. M.

    2015-03-01

    DNA replication is the basis for all biological reproduction. A strand of DNA will ``unzip'' and bind with a complimentary strand, creating two identical strands. In this study, we are considering how this process is affected by Interatomic Coulombic Decay (ICD), specifically how ICD affects the individual coding proteins' ability to hold together. ICD mainly deals with how the electron returns to its original state after excitation and how this affects its immediate atomic environment, sometimes affecting the connectivity between interaction sites on proteins involved in the DNA coding process. Biological heredity is fundamentally controlled by DNA and its replication therefore it affects every living thing. The small nature of the proteins (within the range of nanometers) makes it a good candidate for research of this scale. Understanding how ICD affects DNA molecules can give us invaluable insight into the human genetic code and the processes behind cell mutations that can lead to cancer. Authors wish to give special thanks to Pacific Union College Student Senate in Angwin, California, for their financial support.

  20. Noc2, a putative zinc finger protein involved in exocytosis in endocrine cells.

    PubMed

    Kotake, K; Ozaki, N; Mizuta, M; Sekiya, S; Inagaki, N; Seino, S

    1997-11-21

    We have cloned a cDNA encoding a novel protein of 302 amino acids (designated Noc2, no C2 domain) that has 40.7% amino acid identity with and 77.9% similarity to the N-terminal region of rabphilin-3A, a target molecule of Rab3A. However, unlike rabphilin-3A, Noc2 lacks two C2 domains that are thought to interact with Ca2+ and phospholipids. Noc2 is expressed predominantly in endocrine tissues and hormone-secreting cell lines and at very low levels in brain. Immunoblot analysis of subcellular fractions of the insulin-secreting cell line MIN6 and immunocytochemistry reveal that Noc2 is a 38-kDa protein present in the cytoplasm. Overexpression of Noc2 in PC12 cells cotransfected with growth hormone enhances high K+-induced growth hormone secretion. Screening a mouse embryonic cDNA library with the yeast two-hybrid system shows that Noc2 interacts with the LIM domain-containing protein zyxin, a component of the cytoskeleton, and this interaction is further confirmed by the coimmunoprecipitation experiment. Accordingly, Noc2 is probably involved in regulated exocytosis in endocrine cells by interacting with the cytoskeleton. PMID:9367993

  1. Five RecA-like proteins of Schizosaccharomyces pombe are involved in meiotic recombination.

    PubMed Central

    Grishchuk, A L; Kohli, J

    2003-01-01

    The genome of Schizosaccharomyces pombe contains five genes that code for proteins with sequence similarity to the Escherichia coli recombination protein RecA: rad51+, rhp55+, rhp57+, rlp1+, and dmc1+. We analyzed the effect of deletion of each of these genes on meiotic recombination and viability of spores. Meiotic recombination levels were different from wild type in all recA-related mutants in several genetic intervals, suggesting that all five RecA homologs of S. pombe are required for normal levels of meiotic recombination. Spore viability was reduced in rad51, rhp55, and rhp57 mutants, but not in rlp1 and dmc1. It is argued that reduction of crossover is not the only cause for the observed reduction of spore viability. Analysis of double and triple mutants revealed that Rad51 and Dmc1 play major and partially overlapping roles in meiotic recombination, while Rhp55, Rhp57, and Rlp1 play accessory roles. Remarkably, deletion of Rlp1 decreases the frequency of intergenic recombination (crossovers), but increases intragenic recombination (gene conversion). On the basis of our results, we present a model for the involvement of five RecA-like proteins of S. pombe in meiotic recombination and discuss their respective roles. PMID:14668362

  2. Abrogation of TNF-mediated cytotoxicity by space flight involves protein kinase C

    NASA Technical Reports Server (NTRS)

    Woods, K. M.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Experiments conducted on STS-50 indicated that space flight significantly inhibited tumor necrosis factor (TNF)-mediated killing of LM929 cells compared to ground controls. In ground-based studies, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) also inhibited TNF-mediated killing of LM929 cells. Therefore, we used PKC inhibitors to determine if the inhibitory effects of spaceflight on TNF-mediated cytotoxicity involved the activation of PKC. In experiments conducted onboard space shuttle mission STS-54, we saw that in the presence of the protein kinase C inhibitors H7 and H8, TNF-mediated cytotoxicity was restored to levels of those observed in the ground controls. Subsequent experiments done during the STS-57 mission tested the dose response of two protein kinase inhibitors, H7 and HA1004. We again saw that killing was restored in a dose-dependent manner, with inhibitor concentrations known to inhibit PKC being most effective. These data suggest that space flight ameliorates the action of TNF by affecting PKC in target cells.

  3. Involvement of the nuclear cap-binding protein complex in alternative splicing in Arabidopsis thaliana

    PubMed Central

    Raczynska, Katarzyna Dorota; Simpson, Craig G.; Ciesiolka, Adam; Szewc, Lukasz; Lewandowska, Dominika; McNicol, Jim; Szweykowska-Kulinska, Zofia; Brown, John W. S.; Jarmolowski, Artur

    2010-01-01

    The nuclear cap-binding protein complex (CBC) participates in 5′ splice site selection of introns that are proximal to the mRNA cap. However, it is not known whether CBC has a role in alternative splicing. Using an RT–PCR alternative splicing panel, we analysed 435 alternative splicing events in Arabidopsis thaliana genes, encoding mainly transcription factors, splicing factors and stress-related proteins. Splicing profiles were determined in wild type plants, the cbp20 and cbp80(abh1) single mutants and the cbp20/80 double mutant. The alternative splicing events included alternative 5′ and 3′ splice site selection, exon skipping and intron retention. Significant changes in the ratios of alternative splicing isoforms were found in 101 genes. Of these, 41% were common to all three CBC mutants and 15% were observed only in the double mutant. The cbp80(abh1) and cbp20/80 mutants had many more changes in alternative splicing in common than did cbp20 and cbp20/80 suggesting that CBP80 plays a more significant role in alternative splicing than CBP20, probably being a platform for interactions with other splicing factors. Cap-binding proteins and the CBC are therefore directly involved in alternative splicing of some Arabidopsis genes and in most cases influenced alternative splicing of the first intron, particularly at the 5′ splice site. PMID:19864257

  4. Ras family proteins: new players involved in the diplotene arrest of Xenopus oocytes.

    PubMed

    Jessus, C; Rime, H; Ozon, R

    1998-11-01

    Oogonia undergo numerous mitotic cell cycles before completing the last DNA replication and entering the meiotic prophase I. After chromosome pairing and chromatid exchanges between paired chromosomes, the oocyte I remains arrested at the diplotene stage of the first meiotic prophase. Oocyte growth then occurs independently of cell division; indeed, during this growth period, oocytes (4n DNA) are prevented from completing the meiotic divisions. How is the prophase arrest regulated? One of the players of the prophase block is the high level of intracellular cAMP, maintained by an active adenylate cyclase. By using lethal toxin from Clostridium sordellii (LT), a glucosyltransferase that glucosylates and inactivates small G proteins of the Ras subfamily, we have shown that inhibition of either Ras or Rap or both proteins is sufficient to release the prophase block of Xenopus oocytes in a cAMP-dependent manner. The implications of Ras family proteins as new players involved in the prophase arrest of Xenopus oocytes will be discussed here. PMID:10069002

  5. Unconventional N-H…N Hydrogen Bonds Involving Proline Backbone Nitrogen in Protein Structures.

    PubMed

    Deepak, R N V Krishna; Sankararamakrishnan, Ramasubbu

    2016-05-10

    Contrary to DNA double-helical structures, hydrogen bonds (H-bonds) involving nitrogen as the acceptor are not common in protein structures. We systematically searched N-H…N H-bonds in two different sets of protein structures. Data set I consists of neutron diffraction and ultrahigh-resolution x-ray structures (0.9 Å resolution or better) and the hydrogen atom positions in these structures were determined experimentally. Data set II contains structures determined using x-ray diffraction (resolution ≤ 1.8 Å) and the positions of hydrogen atoms were generated using a computational method. We identified 114 and 14,347 potential N-H…N H-bonds from these two data sets, respectively, and 56-66% of these were of the Ni+1-Hi+1…Ni type, with Ni being the proline backbone nitrogen. To further understand the nature of such unusual contacts, we performed quantum chemical calculations on the model compound N-acetyl-L-proline-N-methylamide (Ace-Pro-NMe) with coordinates taken from the experimentally determined structures. A potential energy profile generated by varying the ψ dihedral angle in Ace-Pro-NMe indicates that the conformation with the N-H…N H-bond is the most stable. An analysis of H-bond-forming proline residues reveals that more than 30% of the proline carbonyl groups are also involved in n → π(∗) interactions with the carbonyl carbon of the preceding residue. Natural bond orbital analyses demonstrate that the strength of N-H…N H-bonds is less than half of that observed for a conventional H-bond. This study clearly establishes the H-bonding capability of proline nitrogen and its prevalence in protein structures. We found many proteins with multiple instances of H-bond-forming prolines. With more than 15% of all proline residues participating in N-H…N H-bonds, we suggest a new, to our knowledge, structural role for proline in providing stability to loops and capping regions of secondary structures in proteins. PMID:27166805

  6. Macrophage Replication Screen Identifies a Novel Francisella Hydroperoxide Resistance Protein Involved in Virulence

    PubMed Central

    Llewellyn, Anna C.; Bina, James E.; Weiss, David S.

    2011-01-01

    Francisella tularensis is a Gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI), validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is

  7. Involvement of breast cancer resistance protein (ABCG2) in the biliary excretion mechanism of fluoroquinolones.

    PubMed

    Ando, Tomohiro; Kusuhara, Hiroyuki; Merino, Gracia; Alvarez, Ana I; Schinkel, Alfred H; Sugiyama, Yuichi

    2007-10-01

    Fluoroquinolones are effective antibiotics for the treatment of bile duct infections. It has been shown that the biliary excretion of grepafloxacin is partly accounted for by multidrug resistance-associated protein 2 (MRP2/ABCC2), whereas neither MRP2 nor P-glycoprotein is involved in the biliary excretion of ulifloxacin. In the present study, we examined the involvement of breast cancer resistance protein (BCRP/ABCG2) in the biliary excretion of fluoroquinolones (grepafloxacin, ulifloxacin, ciprofloxacin, and ofloxacin). In Madin-Darby canine kidney II cells expressing human BCRP or mouse Bcrp, the basal-to-apical transport of grepafloxacin and ulifloxacin was greater than that of the mock control, which was inhibited by a BCRP inhibitor, 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143). Plasma and bile concentrations of fluoroquinolones were determined in wild-type and Bcrp(-/-) mice after i.v. bolus injection. The cumulative biliary excretion of fluoroquinolones was significantly reduced in Bcrp(-/-) mice, resulting in a reduction of the biliary excretion clearances to 86, 50, 40, and 16 of the control values, for ciprofloxacin, grepafloxacin, ofloxacin, and ulifloxacin, respectively. Preinfusion of sulfobromophthalein significantly inhibited the biliary excretion of grepafloxacin in Bcrp(-/-) mice. There was no change in the tissue/plasma concentration ratios of fluoroquinolones in the liver or brain, whereas those in the kidney were increased 3.6- and 1.5-fold for ciprofloxacin and grepafloxacin, respectively, in Bcrp(-/-) mice but were unchanged for ofloxacin and ulifloxacin. The present study shows that BCRP mediates the biliary excretion of fluoroquinolones and suggests that it is also involved in the tubular secretion of ciprofloxacin and grepafloxacin. PMID:17639028

  8. Tau pathology involves protein phosphatase 2A in parkinsonism-dementia of Guam.

    PubMed

    Arif, Mohammad; Kazim, Syed Faraz; Grundke-Iqbal, Inge; Garruto, Ralph M; Iqbal, Khalid

    2014-01-21

    Parkinsonism-dementia (PD) of Guam is a neurodegenerative disease with parkinsonism and early-onset Alzheimer-like dementia associated with neurofibrillary tangles composed of hyperphosphorylated microtubule-associated protein, tau. β-N-methylamino-l-alanine (BMAA) has been suspected of being involved in the etiology of PD, but the mechanism by which BMAA leads to tau hyperphosphorylation is not known. We found a decrease in protein phosphatase 2A (PP2A) activity associated with an increase in inhibitory phosphorylation of its catalytic subunit PP2Ac at Tyr(307) and abnormal hyperphosphorylation of tau in brains of patients who had Guam PD. To test the possible involvement of BMAA in the etiopathogenesis of PD, we studied the effect of this environmental neurotoxin on PP2A activity and tau hyperphosphorylation in mouse primary neuronal cultures and metabolically active rat brain slices. BMAA treatment significantly decreased PP2A activity, with a concomitant increase in tau kinase activity resulting in elevated tau hyperphosphorylation at PP2A favorable sites. Moreover, we found an increase in the phosphorylation of PP2Ac at Tyr(307) in BMAA-treated rat brains. Pretreatment with metabotropic glutamate receptor 5 (mGluR5) and Src antagonists blocked the BMAA-induced inhibition of PP2A and the abnormal hyperphosphorylation of tau, indicating the involvement of an Src-dependent PP2A pathway. Coimmunoprecipitation experiments showed that BMAA treatment dissociated PP2Ac from mGluR5, making it available for phosphorylation at Tyr(307). These findings suggest a scenario in which BMAA can lead to tau pathology by inhibiting PP2A through the activation of mGluR5, the consequent release of PP2Ac from the mGluR5-PP2A complex, and its phosphorylation at Tyr(307) by Src. PMID:24395787

  9. Tau pathology involves protein phosphatase 2A in Parkinsonism-dementia of Guam

    PubMed Central

    Arif, Mohammad; Kazim, Syed Faraz; Grundke-Iqbal, Inge; Garruto, Ralph M.; Iqbal, Khalid

    2014-01-01

    Parkinsonism-dementia (PD) of Guam is a neurodegenerative disease with parkinsonism and early-onset Alzheimer-like dementia associated with neurofibrillary tangles composed of hyperphosphorylated microtubule-associated protein, tau. β-N-methylamino-l-alanine (BMAA) has been suspected of being involved in the etiology of PD, but the mechanism by which BMAA leads to tau hyperphosphorylation is not known. We found a decrease in protein phosphatase 2A (PP2A) activity associated with an increase in inhibitory phosphorylation of its catalytic subunit PP2Ac at Tyr307 and abnormal hyperphosphorylation of tau in brains of patients who had Guam PD. To test the possible involvement of BMAA in the etiopathogenesis of PD, we studied the effect of this environmental neurotoxin on PP2A activity and tau hyperphosphorylation in mouse primary neuronal cultures and metabolically active rat brain slices. BMAA treatment significantly decreased PP2A activity, with a concomitant increase in tau kinase activity resulting in elevated tau hyperphosphorylation at PP2A favorable sites. Moreover, we found an increase in the phosphorylation of PP2Ac at Tyr307 in BMAA-treated rat brains. Pretreatment with metabotropic glutamate receptor 5 (mGluR5) and Src antagonists blocked the BMAA-induced inhibition of PP2A and the abnormal hyperphosphorylation of tau, indicating the involvement of an Src-dependent PP2A pathway. Coimmunoprecipitation experiments showed that BMAA treatment dissociated PP2Ac from mGluR5, making it available for phosphorylation at Tyr307. These findings suggest a scenario in which BMAA can lead to tau pathology by inhibiting PP2A through the activation of mGluR5, the consequent release of PP2Ac from the mGluR5–PP2A complex, and its phosphorylation at Tyr307 by Src. PMID:24395787

  10. Vaccination with proteins involved in tick-pathogen interactions reduces vector infestations and pathogen infection.

    PubMed

    Merino, Octavio; Antunes, Sandra; Mosqueda, Juan; Moreno-Cid, Juan A; Pérez de la Lastra, José M; Rosario-Cruz, Rodrigo; Rodríguez, Sergio; Domingos, Ana; de la Fuente, José

    2013-12-01

    Tick-borne pathogens cause diseases that greatly impact animal health and production worldwide. The ultimate goal of tick vaccines is to protect against tick-borne diseases through the control of vector infestations and reducing pathogen infection and transmission. Tick genetic traits are involved in vector-pathogen interactions and some of these molecules such as Subolesin (SUB) have been shown to protect against vector infestations and pathogen infection. Based on these premises, herein we characterized the efficacy of cattle vaccination with tick proteins involved in vector-pathogen interactions, TROSPA, SILK, and Q38 for the control of cattle tick, Rhipicephalus (Boophilus) microplus infestations and infection with Anaplasma marginale and Babesia bigemina. SUB and adjuvant/saline placebo were used as positive and negative controls, respectively. The results showed that vaccination with Q38, SILK and SUB reduced tick infestations and oviposition with vaccine efficacies of 75% (Q38), 62% (SILK) and 60% (SUB) with respect to ticks fed on placebo control cattle. Vaccination with TROSPA did not have a significant effect on any of the tick parameters analyzed. The results also showed that vaccination with Q38, TROSPA and SUB reduced B. bigemina DNA levels in ticks while vaccination with SILK and SUB resulted in lower A. marginale DNA levels when compared to ticks fed on placebo control cattle. The positive correlation between antigen-specific antibody titers and reduction of tick infestations and pathogen infection strongly suggested that the effect of the vaccine was the result of the antibody response in vaccinated cattle. Vaccination and co-infection with A. marginale and B. bigemina also affected the expression of genes encoding for vaccine antigens in ticks fed on cattle. These results showed that vaccines using tick proteins involved in vector-pathogen interactions could be used for the dual control of tick infestations and pathogen infection. PMID:24084474

  11. Homologies between the Salmonella typhimurium CheY protein and proteins involved in the regulation of chemotaxis, membrane protein synthesis, and sporulation.

    PubMed Central

    Stock, A; Koshland, D E; Stock, J

    1985-01-01

    Chemotactic receptors at the bacterial cell surface communicate with flagellar basal structures to elicit appropriate motor behavior in response to extracellular stimuli. Genetic and physiological studies indicate that the product of the cheY gene interacts directly with components of the flagellar motor to control swimming behavior. We have purified and characterized the Salmonella typhimurium CheY protein and have determined the nucleotide sequence of the cheY gene. Amino acid sequence comparisons showed CheY to be homologous over its entire length (129 residues) to the N-terminal regulatory domain of another protein involved in chemotaxis, the CheB methyl esterase. The entire CheY protein and the regulatory domain of CheB also homologous to the N-terminal portions of the Escherichia coli OmpR and Dye proteins and the Bacillus subtilis Spo0A protein. These homologies suggest an evolutionary and functional relationship between the chemotaxis system and systems that are thought to regulate gene expression in response to changing environmental conditions. Images PMID:2999789

  12. Effects of Radiation and Dietary Iron on Expression of Genes and Proteins Involved in Drug Metabolism

    NASA Technical Reports Server (NTRS)

    Faust, K. M.; Wotring, V. E.

    2014-01-01

    Liver function, especially the rate of metabolic enzyme activities, determines the concentration of circulating drugs and the duration of their efficacy. Most pharmaceuticals are metabolized by the liver, and clinically-used medication doses are given with normal liver function in mind. A drug overdose can result in the case of a liver that is damaged and removing pharmaceuticals from the circulation at a rate slower than normal. Alternatively, if liver function is elevated and removing drugs from the system more quickly than usual, it would be as if too little drug had been given for effective treatment. Because of the importance of the liver in drug metabolism, we want to understand any effects of spaceflight on the enzymes of the liver. Dietary factors and exposure to radiation are aspects of spaceflight that are potential oxidative stressors and both can be modeled in ground experiments. In this experiment, we examined the effects of high dietary iron and low dose gamma radiation (individually and combined) on the gene expression of enzymes involved in drug metabolism, redox homeostasis, and DNA repair. METHODS All procedures were approved by the JSC Animal Care and Use Committee. Male Sprague-Dawley rats were divided into 4 groups (n=8); control, high Fe diet (650 mg iron/kg), radiation (fractionated 3 Gy exposure from a Cs- 137 source) and combined high Fe diet + radiation exposure. Animals were euthanized 24h after the last treatment of radiation; livers were removed immediately and flash -frozen in liquid nitrogen. Expression of genes thought to be involved in redox homeostasis, drug metabolism and DNA damage repair was measured by RT-qPCR. Where possible, protein expression of the same genes was measured by western blotting. All data are expressed as % change in expression normalized to reference gene expression; comparisons were then made of each treatment group to the sham exposed/ normal diet control group. Data was considered significant at p< 0

  13. NMR studies of conformational states of proteins involved in biosynthesis of iron-sulfur clusters

    NASA Astrophysics Data System (ADS)

    Dai, Ziqi

    Iron-sulfur (Fe-S) clusters are the most ancient and ubiquitous cofactors that exist throughout evolution. The most important biosynthetic system of the cluster in both prokaryotes and eukaryotes is the ISC system. Defects in this system can be lethal and have been associated with a number of human diseases. Previous works show that a number of proteins are involved in the [Fe-S] biosynthetic processes and the structural flexibility may play an important role. For example, it was shown that apo-IscU, the scaffold protein, from Escherichia coli populates two functionally important conformational states, one dynamically disordered (D-state) and the other more structured (S-state) (Kim et al., 2009; Kim et al., 2012c). To further investigate the characteristics and transition of the conformational states of proteins involved in this system, I performed extensive NMR studies. Here, I present the findings based on my studies of two important players of the ISC system, IscU and HscB. In this research, I find that a peptidyl-prolyl cis/trans isomerization might account for the slow step in the S-D interconversion of IscU. More specifically, P14 and P101 are trans in the S-state, but become cis in the D-state. In addition, I discover that IscU is very responsive to pH changes, and I postulate that this response is correlated to conserved histidine residues, H10 and H105. Moreover, my thermodynamic analyses reveal that the S-D equilibrium of IscU is also very sensitive to change in temperature, pressure, and amino acid sequence compared to other proteins. In the study, I also discovered a novel state of IscU, the unfolded U-state. I suspect that this state may serve as an intermediate of interconversion between IscU S-/D-states. Finally, I extended the effort to HscB, and find that it may possess more conformational flexibility than expected earlier. I postulate that this flexibility may be the cause of the line-broadening observed during interaction of HscB with Isc

  14. TEC protein tyrosine kinase is involved in the Erk signaling pathway induced by HGF

    SciTech Connect

    Li, Feifei; Jiang, Yinan; Zheng, Qiping; Yang, Xiaoming; Wang, Siying

    2011-01-07

    Research highlights: {yields} TEC is rapidly tyrosine-phosphorylated and activated by HGF-stimulation in vivo or after partial hepatectomy in mice. {yields} TEC enhances the activity of Elk and serum response element (SRE) in HGF signaling pathway in hepatocyte. {yields} TEC promotes hepatocyte proliferation through the Erk-MAPK pathway. -- Abstract: Background/aims: TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involved in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation. Methods: We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC{sup KM}) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by {sup 3}H-TdR incorporation. Results: TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk. Conclusions: TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.

  15. AN ODORANT-BINDING PROTEIN INVOLVED IN PERCEPTION OF HOST PLANT ODORANTS IN LOCUST Locusta migratoria.

    PubMed

    Li, Jia; Zhang, Long; Wang, Xiaoqi

    2016-04-01

    Locusts, Locusta migratoria (Orthoptera: Acrididae), are extremely destructive agricultural pests, but very little is known of their molecular aspects of perception to host plant odorants including related odorant-binding proteins (OBPs), though several OBPs have been identified in locust. To elucidate the function of LmigOBP1, the first OBP identified from locust, RNA interference was employed in this study to silence LmigOBP1, which was achieved by injection of dsRNA targeting LmigOBP1 into the hemolymph of male nymphs. Compared with LmigOBP1 normal nymphs, LmigOBP1 knockdown nymphs significantly decreased food (maize leaf, Zea mays) consumption and electro-antennography responses to five maize leaf volatiles, ((Z)-3-hexenol, linalool, nonanal, decanal, and (Z)-3-hexenyl acetate). These suggest that LmigOBP1 is involved in perception of host plant odorants. PMID:26864243

  16. The collagen-binding protein of Streptococcus mutans is involved in haemorrhagic stroke

    PubMed Central

    Nakano, Kazuhiko; Hokamura, Kazuya; Taniguchi, Naho; Wada, Koichiro; Kudo, Chiho; Nomura, Ryota; Kojima, Ayuchi; Naka, Shuhei; Muranaka, Yoshinori; Thura, Min; Nakajima, Atsushi; Masuda, Katsuhiko; Nakagawa, Ichiro; Speziale, Pietro; Shimada, Nobumitsu; Amano, Atsuo; Kamisaki, Yoshinori; Tanaka, Tokutaro; Umemura, Kazuo; Ooshima, Takashi

    2011-01-01

    Although several risk factors for stroke have been identified, one-third remain unexplained. Here we show that infection with Streptococcus mutans expressing collagen-binding protein (CBP) is a potential risk factor for haemorrhagic stroke. Infection with serotype k S. mutans, but not a standard strain, aggravates cerebral haemorrhage in mice. Serotype k S. mutans accumulates in the damaged, but not the contralateral hemisphere, indicating an interaction of bacteria with injured blood vessels. The most important factor for high-virulence is expression of CBP, which is a common property of most serotype k strains. The detection frequency of CBP-expressing S. mutans in haemorrhagic stroke patients is significantly higher than in control subjects. Strains isolated from haemorrhagic stroke patients aggravate haemorrhage in a mouse model, indicating that they are haemorrhagic stroke-associated. Administration of recombinant CBP causes aggravation of haemorrhage. Our data suggest that CBP of S. mutans is directly involved in haemorrhagic stroke. PMID:21952219

  17. Cellular COPII Proteins Are Involved in Production of the Vesicles That Form the Poliovirus Replication Complex

    PubMed Central

    Rust, René C.; Landmann, Lukas; Gosert, Rainer; Tang, Bor Luen; Hong, Wanjin; Hauri, Hans-Peter; Egger, Denise; Bienz, Kurt

    2001-01-01

    Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway. PMID:11559814

  18. Charged MVB protein 5 is involved in T-cell receptor signaling.

    PubMed

    Wi, Sae Mi; Min, Yoon; Lee, Ki-Young

    2016-01-01

    Charged multivesicular body protein 5 (CHMP5) has a key role in multivesicular body biogenesis and a critical role in the downregulation of signaling pathways through receptor degradation. However, the role of CHMP5 in T-cell receptor (TCR)-mediated signaling has not been previously investigated. In this study, we utilized a short hairpin RNA-based RNA interference approach to investigate the functional role of CHMP5. Upon TCR stimulation, CHMP5-knockdown (CHMP5(KD)) Jurkat T cells exhibited activation of TCR downstream signaling molecules, such as PKCθ and IKKαβ, and resulted in the activation of nuclear factor-κB and the marked upregulation of TCR-induced gene expression. Moreover, we found that activator protein-1 and nuclear factor of activated T-cells transcriptional factors were markedly activated in CHMP5(KD) Jurkat cells in response to TCR stimulation, which led to a significant increase in interleukin-2 secretion. Biochemical studies revealed that CHMP5 endogenously forms high-molecular-weight complexes, including TCR molecules, and specifically interacts with TCRβ. Interestingly, flow cytometry analysis also revealed that CHMP5(KD) Jurkat T cells exhibit upregulation of TCR expression on the cell surface compared with control Jurkat T cells. Taken together, these findings demonstrated that CHMP5 might be involved in the homeostatic regulation of TCR on the cell surface, presumably through TCR recycling or degradation. Thus CHMP5 is implicated in TCR-mediated signaling. PMID:26821576

  19. Charged MVB protein 5 is involved in T-cell receptor signaling

    PubMed Central

    Wi, Sae Mi; Min, Yoon; Lee, Ki-Young

    2016-01-01

    Charged multivesicular body protein 5 (CHMP5) has a key role in multivesicular body biogenesis and a critical role in the downregulation of signaling pathways through receptor degradation. However, the role of CHMP5 in T-cell receptor (TCR)–mediated signaling has not been previously investigated. In this study, we utilized a short hairpin RNA-based RNA interference approach to investigate the functional role of CHMP5. Upon TCR stimulation, CHMP5-knockdown (CHMP5KD) Jurkat T cells exhibited activation of TCR downstream signaling molecules, such as PKCθ and IKKαβ, and resulted in the activation of nuclear factor-κB and the marked upregulation of TCR-induced gene expression. Moreover, we found that activator protein-1 and nuclear factor of activated T-cells transcriptional factors were markedly activated in CHMP5KD Jurkat cells in response to TCR stimulation, which led to a significant increase in interleukin-2 secretion. Biochemical studies revealed that CHMP5 endogenously forms high-molecular-weight complexes, including TCR molecules, and specifically interacts with TCRβ. Interestingly, flow cytometry analysis also revealed that CHMP5KD Jurkat T cells exhibit upregulation of TCR expression on the cell surface compared with control Jurkat T cells. Taken together, these findings demonstrated that CHMP5 might be involved in the homeostatic regulation of TCR on the cell surface, presumably through TCR recycling or degradation. Thus CHMP5 is implicated in TCR-mediated signaling. PMID:26821576

  20. Hepatitis B virus-induced calreticulin protein is involved in IFN resistance.

    PubMed

    Yue, Xin; Wang, Hui; Zhao, Fanpeng; Liu, Shi; Wu, Jianguo; Ren, Wendan; Zhu, Ying

    2012-07-01

    IFN-α is a widely used treatment for hepatitis B virus (HBV) infection, and IFN resistance caused by viral and/or host factors is currently a challenging clinical problem. A better understanding of the molecular mechanisms underlying IFN immunotherapy in the treatment of viral infection would be very beneficial clinically and is of immense clinical importance. Calreticulin (CRT) is an endoplasmic reticulum luminal calcium-binding chaperone that is involved in the regulation of calcium homoeostasis, the folding of newly synthesized proteins, and many other cellular functions. However, little is known about the role of CRT in HBV infection. In this study, we observed high levels of CRT expression in the sera and PBMCs of patients with HBV relative to those of healthy individuals. HBV upregulated the expression of CRT at the transcriptional level. Further investigation showed that HBV-induced CRT enhanced HBV replication by antagonizing the IFN pathway. CRT suppressed the production of endogenous IFN-α by reducing the nuclear translocation of IFN regulatory factor-7 but not IFN regulatory factor-3. Furthermore, CRT also suppressed the antiviral activity of IFN-α by inhibiting the phosphorylation of STAT1 and decreasing the expression of two IFN-α downstream effectors, protein kinase R and 2',5'-oligoadenylate synthetase. Our results offer new insights into the pathogenesis of HBV infection and may provide potential targets for anti-HBV therapy. PMID:22661095

  1. Simiate is an Actin binding protein involved in filopodia dynamics and arborization of neurons

    PubMed Central

    Derlig, Kristin; Ehrhardt, Toni; Gießl, Andreas; Brandstätter, Johann H.; Enz, Ralf; Dahlhaus, Regina

    2014-01-01

    The Actin cytoskeleton constitutes the functional base for a multitude of cellular processes extending from motility and migration to cell mechanics and morphogenesis. The latter is particularly important to neuronal cells since the accurate functioning of the brain crucially depends on the correct arborization of neurons, a process that requires the formation of several dozens to hundreds of dendritic branches. Recently, a model was proposed where different transcription factors are detailed to distinct facets and phases of dendritogenesis and exert their function by acting on the Actin cytoskeleton, however, the proteins involved as well as the underlying molecular mechanisms are largely unknown. Here, we demonstrate that Simiate, a protein previously indicated to activate transcription, directly associates with both, G- and F-Actin and in doing so, affects Actin polymerization and Actin turnover in living cells. Imaging studies illustrate that Simiate particularly influences filopodia dynamics and specifically increases the branching of proximal, but not distal dendrites of developing neurons. The data suggests that Simiate functions as a direct molecular link between transcription regulation on one side, and dendritogenesis on the other, wherein Simiate serves to coordinate the development of proximal and distal dendrites by acting on the Actin cytoskeleton of filopodia and on transcription regulation, hence supporting the novel model. PMID:24782708

  2. A Wiskott-Aldrich syndrome protein is involved in endocytosis in Aspergillus nidulans.

    PubMed

    Hoshi, Hiro-Omi; Zheng, Lu; Ohta, Akinori; Horiuchi, Hiroyuki

    2016-09-01

    Endocytosis is vital for hyphal tip growth in filamentous fungi and is involved in the tip localization of various membrane proteins. To investigate the function of a Wiskott-Aldrich syndrome protein (WASP) in endocytosis of filamentous fungi, we identified a WASP ortholog-encoding gene, wspA, in Aspergillus nidulans and characterized it. The wspA product, WspA, localized to the tips of germ tubes during germination and actin rings in the subapical regions of mature hyphae. wspA is essential for the growth and functioned in the polarity establishment and maintenance during germination of conidia. We also investigated its function in endocytosis and revealed that endocytosis of SynA, a synaptobrevin ortholog that is known to be endocytosed at the subapical regions of hyphal tips in A. nidulans, did not occur when wspA expression was repressed. These results suggest that WspA plays roles in endocytosis at hyphal tips and polarity establishment during germination. PMID:26927610

  3. Increased protein nitration in mitochondrial diseases: evidence for vessel wall involvement.

    PubMed

    Vattemi, Gaetano; Mechref, Yehia; Marini, Matteo; Tonin, Paola; Minuz, Pietro; Grigoli, Laura; Guglielmi, Valeria; Klouckova, Iveta; Chiamulera, Cristiano; Meneguzzi, Alessandra; Di Chio, Marzia; Tedesco, Vincenzo; Lovato, Laura; Degan, Maurizio; Arcaro, Guido; Lechi, Alessandro; Novotny, Milos V; Tomelleri, Giuliano

    2011-04-01

    Mitochondrial diseases (MD) are heterogeneous disorders because of impairment of respiratory chain function leading to oxidative stress. We hypothesized that in MD the vascular endothelium may be affected by increased oxidative/nitrative stress causing a reduction of nitric oxide availability. We therefore, investigated the pathobiology of vasculature in MD patients by assaying the presence of 3-nitrotyrosine in muscle biopsies followed by the proteomic identification of proteins which undergo tyrosine nitration. We then measured the flow-mediated vasodilatation as a proof of altered nitric oxide generation/bioactivity. Here, we show that 3-nitrotyrosine staining is specifically located in the small vessels of muscle tissue and that the reaction is stronger and more evident in a significant percentage of vessels from MD patients as compared with controls. Eleven specific proteins which are nitrated under pathological conditions were identified; most of them are involved in energy metabolism and are located mainly in mitochondria. In MD patients the flow-mediated vasodilatation was reduced whereas baseline arterial diameters, blood flow velocity and endothelium-independent vasodilatation were similar to controls. The present results provide evidence that in MD the vessel wall is a target of increased oxidative/nitrative stress. PMID:21156839

  4. The Ku70 DNA-repair protein is involved in centromere function in a grasshopper species.

    PubMed

    Cabrero, Josefa; Bakkali, Mohammed; Navarro-Domínguez, Beatriz; Ruíz-Ruano, Francisco J; Martín-Blázquez, Rubén; López-León, María Dolores; Camacho, Juan Pedro M

    2013-06-25

    The Ku70 protein is involved in numerous cell functions, the nonhomologous end joining (NHEJ) DNA repair pathway being the best known. Here, we report a novel function for this protein in the grasshopper Eyprepocnemis plorans. We observed the presence of large Ku70 foci on the centromeres of meiotic and mitotic chromosomes during the cell cycle stages showing the highest centromeric activity (i.e., metaphase and anaphase). The fact that colchicine treatment prevented centromeric location of Ku70, suggests a microtubule-dependent centromeric function for Ku70. Likewise, the absence of Ku70 at metaphase-anaphase centromeres from three males whose Ku70 gene had been knocked down using interference RNA, and the dramatic increase in the frequency of polyploid spermatids observed in these males, suggest that the centromeric presence of Ku70 is required for normal cytokinesis in this species. The centromeric function of Ku70 was not observed in 14 other grasshopper and locust species, or in the mouse, thus suggesting that it is an autapomorphy in E. plorans. PMID:23797468

  5. Gene expression in primary cultured astrocytes affected by aluminum: alteration of chaperons involved in protein folding

    PubMed Central

    Aremu, David A.; Ezomo, Ojeiru F.

    2010-01-01

    Objectives Aluminum is notorious as a neurotoxic metal. The aim of our study was to determine whether endoplasmic reticulum (ER) stress is involved in aluminum-induced apoptosis in astrocytes. Methods Mitochondrial RNA (mRNA) was analyzed by reverse transcription (RT)-PCR following pulse exposure of aluminum glycinate to primary cultured astrocytes. Tunicamycin was used as a positive control. Results Gene expression analysis revealed that Ire1β was up-regulated in astrocytes exposed to aluminum while Ire1α was up-regulated by tunicamycin. Exposure to aluminum glycinate, in contrast to tunicamycin, seemed to down-regulate mRNA expression of many genes, including the ER resident molecular chaperone BiP/Grp78 and Ca2+-binding chaperones (calnexin and calreticulin), as well as stanniocalcin 2 and OASIS. The down-regulation or non-activation of the molecular chaperons, whose expressions are known to be protective by increasing protein folding, may spell doom for the adaptive response. Exposure to aluminum did not have any significant effects on the expression of Bax and Bcl2 in astrocytes. Conclusions The results of this study demonstrate that aluminum may induce apoptosis in astrocytes via ER stress by impairing the protein-folding machinery. PMID:21432213

  6. Identification of novel residues involved in nuclear localization of a baculovirus polyhedrin protein.

    PubMed

    Katsuma, S; Deng, D X; Zhou, C L; Iwanaga, M; Noguchi, Y; Kobayashi, M; Maeda, S

    2000-10-01

    A baculovirus polyhedrin protein has proven to possess a nuclear localization signal (NLS) sequence and a domain required for supramolecular assembly. Here we investigated five Bombyx mori nucleopolyhedrovirus (BmNPV) mutants that did not produce polyhedra. Two of five mutants were generated during routine baculoviral expression vector screening, and three were isolated by treatment with the mutagen 5-bromo-2'-deoxyuridine (BrdU). Marker rescue mapping and nucleotide sequence analysis showed that mutations in the polyhedrin gene caused the altered phenotype of these mutants. Biochemical fractionation indicated that cells infected with these mutants exhibited polyhedrin protein in both the nucleus and the cytoplasm. Electron microscopic observation revealed that polyhedrin produced by these mutants ocurred in both the nucleus and the cytoplasm, but did not form a crystalline lattice. Despite the incompleteness of polyhedrin nuclear localization, the NLSs of the five mutants were unchanged, although some of the mutations occurred within residues just outside of the domain reported to be required for polyhedron assembly (4). This result suggests that (a) the polyhedrin NLS directs polyhedrin to the nucleus, but the efficiency of this localization is regulated by regions other than the NLS (probably, polyhedrin conformation and its association with the nucleus are also involved), and (b) formation of a crystalline lattice may also be determined by several domains within polyhedrin. PMID:11129641

  7. Equatorial Segment Protein (ESP) Is a Human Alloantigen Involved in Sperm-Egg Binding and Fusion

    PubMed Central

    Wolkowicz, M. J.; Digilio, L.; Klotz, K.; Shetty, J.; Flickinger, C. J.; Herr, J. C.

    2010-01-01

    The equatorial segment of the sperm head is known to play a role in fertilization; however, the specific sperm molecules contributing to the integrity of the equatorial segment and in binding and fusion at the oolemma remain incomplete. Moreover, identification of molecular mediators of fertilization that are also immunogenic in humans is predicted to advance both the diagnosis and treatment of immune infertility. We previously reported the cloning of Equatorial Segment Protein (ESP), a protein localized to the equatorial segment of ejaculated human sperm. ESP is a biomarker for a subcompartment of the acrosomal matrix that can be traced through all stages of acrosome biogenesis (Wolkowicz et al, 2003). In the present study, ESP immunoreacted on Western blots with 4 (27%) of 15 antisperm antibody (ASA)–positive serum samples from infertile male patients and 2 (40%) of 5 ASA-positive female sera. Immunofluorescent studies revealed ESP in the equatorial segment of 89% of acrosome-reacted sperm. ESP persisted as a defined equatorial segment band on 100% of sperm tightly bound to the oolemma of hamster eggs. Antisera to recombinant human ESP inhibited both oolemmal binding and fusion of human sperm in the hamster egg penetration assay. The results indicate that ESP is a human alloantigen involved in sperm-egg binding and fusion. Defined recombinant sperm immunogens, such as ESP, may offer opportunities for differential diagnosis of immune infertility. PMID:17978344

  8. Increased Protein Nitration in Mitochondrial Diseases: Evidence for Vessel Wall Involvement

    PubMed Central

    Vattemi, Gaetano; Mechref, Yehia; Marini, Matteo; Tonin, Paola; Minuz, Pietro; Grigoli, Laura; Guglielmi, Valeria; Klouckova, Iveta; Chiamulera, Cristiano; Meneguzzi, Alessandra; Di Chio, Marzia; Tedesco, Vincenzo; Lovato, Laura; Degan, Maurizio; Arcaro, Guido; Lechi, Alessandro; Novotny, Milos V.; Tomelleri, Giuliano

    2011-01-01

    Mitochondrial diseases (MD) are heterogeneous disorders because of impairment of respiratory chain function leading to oxidative stress. We hypothesized that in MD the vascular endothelium may be affected by increased oxidative/nitrative stress causing a reduction of nitric oxide availability. We therefore, investigated the pathobiology of vasculature in MD patients by assaying the presence of 3-nitrotyrosine in muscle biopsies followed by the proteomic identification of proteins which undergo tyrosine nitration. We then measured the flow-mediated vasodilatation as a proof of altered nitric oxide generation/bioactivity. Here, we show that 3-nitrotyrosine staining is specifically located in the small vessels of muscle tissue and that the reaction is stronger and more evident in a significant percentage of vessels from MD patients as compared with controls. Eleven specific proteins which are nitrated under pathological conditions were identified; most of them are involved in energy metabolism and are located mainly in mitochondria. In MD patients the flow-mediated vasodilatation was reduced whereas baseline arterial diameters, blood flow velocity and endothelium-independent vasodilatation were similar to controls. The present results provide evidence that in MD the vessel wall is a target of increased oxidative/nitrative stress. PMID:21156839

  9. Small G proteins in peroxisome biogenesis: the potential involvement of ADP-ribosylation factor 6

    PubMed Central

    2009-01-01

    Background Peroxisomes execute diverse and vital functions in virtually every eukaryote. New peroxisomes form by budding from pre-existing organelles or de novo by vesiculation of the ER. It has been suggested that ADP-ribosylation factors and COPI coatomer complexes are involved in these processes. Results Here we show that all viable Saccharomyces cerevisiae strains deficient in one of the small GTPases which have an important role in the regulation of vesicular transport contain functional peroxisomes, and that the number of these organelles in oleate-grown cells is significantly upregulated in the arf1 and arf3 null strains compared to the wild-type strain. In addition, we provide evidence that a portion of endogenous Arf6, the mammalian orthologue of yeast Arf3, is associated with the cytoplasmic face of rat liver peroxisomes. Despite this, ablation of Arf6 did neither influence the regulation of peroxisome abundance nor affect the localization of peroxisomal proteins in cultured fetal hepatocytes. However, co-overexpression of wild-type, GTP hydrolysis-defective or (dominant-negative) GTP binding-defective forms of Arf1 and Arf6 caused mislocalization of newly-synthesized peroxisomal proteins and resulted in an alteration of peroxisome morphology. Conclusion These observations suggest that Arf6 is a key player in mammalian peroxisome biogenesis. In addition, they also lend strong support to and extend the concept that specific Arf isoform pairs may act in tandem to regulate exclusive trafficking pathways. PMID:19686593

  10. Involvement of Arabidopsis RACK1 in Protein Translation and Its Regulation by Abscisic Acid

    SciTech Connect

    Guo, Jianjun; Wang, Shucai; Valerius, Oliver; Hall, Hardy; Zeng, Qingning; Li, Jian-Feng; Weston, David; Ellis, Brian; Chen, Jay

    2011-01-01

    Earlier studies have shown that RACK1 functions as a negative regulator of ABA responses in Arabidopsis, but the molecular mechanism of the action of RACK1 in these processes remains elusive. Global gene expression profiling revealed that approximately 40% of the genes affected by ABA treatment were affected in a similar manner by the rack1 mutation, supporting the view that RACK1 is an important regulator of ABA responses. On the other hand, co-expression analysis revealed that >80% of the genes co-expressed with RACK1 encode ribosome proteins, implying a close relationship between RACK1 s function and the ribosome complex. These results implied that the regulatory role for RACK1 in ABA responses may be partially due to its putative function in protein translation, which is one of the major cellular processes that mammalian and yeast RACK1 is involved in. Consistently, all three Arabidopsis RACK1 homologous genes, namely RACK1A, RACK1B and RACK1C, complemented the growth defects of the S. cerevisiae cpc2/rack1 mutant. In addition, RACK1 physically interacts with Arabidopsis Eukaryotic Initiation Factor 6 (eIF6), whose mammalian homologue is a key regulator of 80S ribosome assembly. Moreover, rack1 mutants displayed hypersensitivity to anisomycin, an inhibitor of protein translation, and displayed characteristics of impaired 80S functional ribosome assembly and 60S ribosomal subunit biogenesis in a ribosome profiling assay. Gene expression analysis revealed that ABA inhibits the expression of both RACK1 and eIF6. Taken together, these results suggest that RACK1 may be required for normal production of 60S and 80S ribosomes and that its action in these processes may be regulated by ABA.

  11. Quantitative characterization of protein–protein complexes involved in base excision DNA repair

    PubMed Central

    Moor, Nina A.; Vasil'eva, Inna A.; Anarbaev, Rashid O.; Antson, Alfred A.; Lavrik, Olga I.

    2015-01-01

    Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein–protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process. PMID:26013813

  12. Involvement of calcitonin gene-related peptide and receptor component protein in experimental autoimmune encephalomyelitis

    PubMed Central

    Sardi, Claudia; Zambusi, Laura; Finardi, Annamaria; Ruffini, Francesca; Tolun, Adviye A.; Dickerson, Ian M.; Righi, Marco; Zacchetti, Daniele; Grohovaz, Fabio; Provini, Luciano; Furlan, Roberto; Morara, Stefano

    2015-01-01

    Calcitonin Gene-Related Peptide (CGRP) inhibits microglia inflammatory activation in vitro. We here analyzed the involvement of CGRP and Receptor Component Protein (RCP) in experimental autoimmune encephalomyelitis (EAE). Alpha-CGRP deficiency increased EAE scores which followed the scale alpha-CGRP null > heterozygote > wild type. In wild type mice, CGRP delivery into the cerebrospinal fluid (CSF) 1) reduced chronic EAE (C-EAE) signs, 2) inhibited microglia activation (revealed by quantitative shape analysis), and 3) did not alter GFAP expression, cell density, lymphocyte infiltration, and peripheral lymphocyte production of IFN-gamma, TNF-alpha, IL-17, IL-2, and IL-4. RCP (probe for receptor involvement) was expressed in white matter microglia, astrocytes, oligodendrocytes, and vascular-endothelial cells: in EAE, also in infiltrating lymphocytes. In relapsing–remitting EAE (R-EAE) RCP increased during relapse, without correlation with lymphocyte density. RCP nuclear localization (stimulated by CGRP in vitro) was I) increased in microglia and decreased in astrocytes (R-EAE), and II) increased in microglia by CGRP CSF delivery (C-EAE). Calcitonin like receptor was rarely localized in nuclei of control and relapse mice. CGRP increased in motoneurons. In conclusion, CGRP can inhibit microglia activation in vivo in EAE. CGRP and its receptor may represent novel protective factors in EAE, apparently acting through the differential cell-specific intracellular translocationof RCP. PMID:24746422

  13. Cotton photosynthesis-related PSAK1 protein is involved in plant response to aphid attack.

    PubMed

    Zhang, Jian-Min; Huang, Geng-Qing; Li, Yang; Zheng, Yong; Li, Xue-Bao

    2014-05-01

    It is believed that hundreds of genes, including photosynthesis-related genes, are typically involved in plant response to aphid feeding. Up to now, however, it is little known on the relationship between the photosynthesis-related genes and plant response to herbivores. In this study, we identified a cotton photosynthesis-related gene (GhPSAK1) which belongs to PSI-PSAK family and encodes a putative protein of 162 amino acids. RT-PCR analysis revealed that GhPSAK1 transcripts in leaves were increased at 12-24 h, but decreased at 48-72 h after cotton aphid attack or wounding induction. Choice assay and no-choice assay demonstrated that overexpression of GhPSAK1 in Arabidopsis improved plant tolerance to green peach aphids (Myzus persicae). The defense response genes related to salicylic acid signaling pathway were enhanced in the GhPSAK1 overexpressing transgenic plants. In addition, the callose amount in transgenic Arabidopsis leaves was more than that of wild type. Contents of the soluble sugars and total amino acids were also altered in leaves of transgenic Arabidopsis plants. Activities of superoxide dismutase and peroxidase in transgenic leaves were higher than those of wild type. These results suggested that GhPSAK1 may be involved in regulation of cotton response and tolerance to aphid attack. PMID:24469731

  14. Protein kinase C is involved in resistance to myocardial infarction induced by heat stress.

    PubMed

    Joyeux, M; Baxter, G F; Thomas, D L; Ribuot, C; Yellon, D M

    1997-12-01

    Heat stress (HS) is known to protect against mechanical dysfunction and myocardial necrosis in myocardial ischemia-reperfusion models both in vivo and in vitro. However, the mechanisms involved in this form of cardioprotection remain unclear. Protein kinase C (PKC) and tyrosine kinase activation have both been shown to be involved in the delayed phase of protection following ischemic preconditioning, a phenomenon which appears to be analogous to HS-induced protection. Therefore, we investigated the role of PKC and tyrosine kinase in HS-induced resistance to myocardial infarction, in the isolated rat heart. The selective inhibitors chelerythrine (Che) and genistein (Gen) were used to inhibit PKC and tyrosine kinase, respectively. Rats were treated with Che (5 mg/kg, i.p.) or Gen (5 mg/kg, i.p.) or vehicle before they were either heat stressed (42 degrees C for 15 min) or sham anesthetized. Twenty-four h later their hearts were isolated, retrogradely perfused, and subjected to 35-min occlusion of the left coronary artery followed by 120-min of reperfusion. Infarct-to-risk ratio was significantly reduced in HS (19.9+/-1.1%) compared to sham (43.1+/-1.1%) hearts. This reduction in infarct size was abolished in chelerythrine-treated groups (43.8+/-1.9% in HS+Che v 44.9+/-2.0% in sham+Che), but was conserved in genistein-treated groups (17.7+/-0.9% in HS+Gen v 36.4+/-2.8% in sham+Gen). In order to confirm that genistein at this dose was effectively inhibiting tyrosine kinase activity, we observed the ability of the agent to prevent the hypoglycemic responses to insulin in a separate group of anesthetised rats receiving an i.v. insulin infusion. Western blot analysis of the myocardial hsp72 showed a HS-induced increase of this protein, which was modified by neither the PKC inhibitor, chelerythrine, nor the tyrosine kinase inhibitor, genistein. We conclude that the activation of PKC, but not of tyrosine kinase, appears to play a role in the functional cardioprotection

  15. Inner Membrane Protein YhcB Interacts with RodZ Involved in Cell Shape Maintenance in Escherichia coli

    PubMed Central

    Li, Gaochi; Hamamoto, Kentaro; Kitakawa, Madoka

    2012-01-01

    Depletion of YhcB, an inner membrane protein of Escherichia coli, inhibited the growth of rodZ deletion mutant showing that the loss of both YhcB and RodZ is synthetically lethal. Furthermore, YhcB was demonstrated to interact with RodZ as well as several other proteins involved in cell shape maintenance and an inner membrane protein YciS of unknown function, using bacterial two-hybrid system. These observations seem to indicate that YhcB is involved in the biogenesis of cell envelope and the maintenance of cell shape together with RodZ.

  16. cDNA Library Screening Identifies Protein Interactors Potentially Involved in Non-Telomeric Roles of Arabidopsis Telomerase

    PubMed Central

    Dokládal, Ladislav; Honys, David; Rana, Rajiv; Lee, Lan-Ying; Gelvin, Stanton B.; Sýkorová, Eva

    2015-01-01

    Telomerase-reverse transcriptase (TERT) plays an essential catalytic role in maintaining telomeres. However, in animal systems telomerase plays additional non-telomeric functional roles. We previously screened an Arabidopsis cDNA library for proteins that interact with the C-terminal extension (CTE) TERT domain and identified a nuclear-localized protein that contains an RNA recognition motif (RRM). This RRM-protein forms homodimers in both plants and yeast. Mutation of the gene encoding the RRM-protein had no detectable effect on plant growth and development, nor did it affect telomerase activity or telomere length in vivo, suggesting a non-telomeric role for TERT/RRM-protein complexes. The gene encoding the RRM-protein is highly expressed in leaf and reproductive tissues. We further screened an Arabidopsis cDNA library for proteins that interact with the RRM-protein and identified five interactors. These proteins are involved in numerous non-telomere-associated cellular activities. In plants, the RRM-protein, both alone and in a complex with its interactors, localizes to nuclear speckles. Transcriptional analyses in wild-type and rrm mutant plants, as well as transcriptional co-analyses, suggest that TERT, the RRM-protein, and the RRM-protein interactors may play important roles in non-telomeric cellular functions. PMID:26617625

  17. cDNA Library Screening Identifies Protein Interactors Potentially Involved in Non-Telomeric Roles of Arabidopsis Telomerase.

    PubMed

    Dokládal, Ladislav; Honys, David; Rana, Rajiv; Lee, Lan-Ying; Gelvin, Stanton B; Sýkorová, Eva

    2015-01-01

    Telomerase-reverse transcriptase (TERT) plays an essential catalytic role in maintaining telomeres. However, in animal systems telomerase plays additional non-telomeric functional roles. We previously screened an Arabidopsis cDNA library for proteins that interact with the C-terminal extension (CTE) TERT domain and identified a nuclear-localized protein that contains an RNA recognition motif (RRM). This RRM-protein forms homodimers in both plants and yeast. Mutation of the gene encoding the RRM-protein had no detectable effect on plant growth and development, nor did it affect telomerase activity or telomere length in vivo, suggesting a non-telomeric role for TERT/RRM-protein complexes. The gene encoding the RRM-protein is highly expressed in leaf and reproductive tissues. We further screened an Arabidopsis cDNA library for proteins that interact with the RRM-protein and identified five interactors. These proteins are involved in numerous non-telomere-associated cellular activities. In plants, the RRM-protein, both alone and in a complex with its interactors, localizes to nuclear speckles. Transcriptional analyses in wild-type and rrm mutant plants, as well as transcriptional co-analyses, suggest that TERT, the RRM-protein, and the RRM-protein interactors may play important roles in non-telomeric cellular functions. PMID:26617625

  18. Involvement of decreased neuroglobin protein level in cognitive dysfunction induced by 1-bromopropane in rats.

    PubMed

    Guo, Ying; Yuan, Hua; Jiang, Lulu; Yang, Junlin; Zeng, Tao; Xie, Keqin; Zhang, Cuili; Zhao, Xiulan

    2015-03-10

    1-Bromopropane (1-BP) is used as a substitute for ozone-depleting solvents (ODS) in industrial applications. 1-BP could display central nervous system (CNS) neurotoxicity manifested by cognitive dysfunction. Neuroglobin (Ngb) is an endogenous neuroprotectant and is predominantly expressed in the nervous system. The present study aimed to investigate Ngb involvement in CNS neurotoxicity induced by 1-BP in rats. Male Wistar rats were randomly divided into 5 groups (n=14) and treated with 0, 100, 200, 400 and 800 mg/kg bw 1-BP, respectively, by gavage for consecutive 12 days. Rats displayed cognitive dysfunction dose-dependently through Morris water maze (MWM) test. Significant neuron loss in layer 5 of the prelimbic cortex (PL) was observed. Moreover, 1-BP decreased Ngb protein level in cerebral cortex and Ngb decrease was significantly positively correlated with cognitive dysfunction. Glutathione (GSH) content, GSH/oxidized glutathione (GSSG) ratio and glutamate cysteine ligase (GCL) activity decreased in cerebral cortex, coupled with the increase in GSSG content. GSH and GSH/GSSG ratio decrease were significantly positively correlated with cortical Ngb decrease. Additionally, levels of N-epsilon-hexanoyl-lysine (HEL) and 4-hydroxy-2-nonenal (4-HNE) modified proteins in cerebral cortex of 1-BP-treated rats increased significantly. In conclusion, it was suggested that 1-BP resulted in decreased endogenous neuroprotectant Ngb in cerebral cortex, which might play an important role in CNS neurotoxicity induced by 1-BP and that 1-BP-induced oxidative stress in cerebral cortex might partly be responsible for Ngb decrease. PMID:25557405

  19. Development of Novel In Vivo Chemical Probes to Address CNS Protein Kinase Involvement in Synaptic Dysfunction

    PubMed Central

    Watterson, D. Martin; Grum-Tokars, Valerie L.; Roy, Saktimayee M.; Schavocky, James P.; Bradaric, Brinda Desai; Bachstetter, Adam D.; Xing, Bin; Dimayuga, Edgardo; Saeed, Faisal; Zhang, Hong; Staniszewski, Agnieszka; Pelletier, Jeffrey C.; Minasov, George; Anderson, Wayne F.; Arancio, Ottavio; Van Eldik, Linda J.

    2013-01-01

    Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will

  20. Signatures of nitrogen limitation in the elemental composition of the proteins involved in the metabolic apparatus.

    PubMed

    Acquisti, Claudia; Kumar, Sudhir; Elser, James J

    2009-07-22

    Nitrogen (N) is a fundamental component of nucleotides and amino acids and is often a limiting nutrient in natural ecosystems. Thus, study of the N content of biomolecules may establish important connections between ecology and genomics. However, while significant differences in the elemental composition of whole organisms are well documented, how the flux of nutrients in the cell has shaped the evolution of different cellular processes remains poorly understood. By examining the elemental composition of major functional classes of proteins in four multicellular eukaryotic model organisms, we find that the catabolic machinery shows substantially lower N content than the anabolic machinery and the rest of the proteome. This pattern suggests that ecological selection for N conservation specifically targets cellular components that are highly expressed in response to nutrient limitation. We propose that the RNA component of the anabolic machineries is the mechanistic force driving the elemental imbalance we found, and that RNA functions as an intracellular nutrient reservoir that is degraded and recycled during starvation periods. A comparison of the elemental composition of the anabolic and catabolic machineries in species that have experienced different levels of N limitation in their evolutionary history (animals versus plants) suggests that selection for N conservation has preferentially targeted the catabolic machineries of plants, resulting in a lower N content of the proteins involved in their catabolic processes. These findings link the composition of major cellular components to the environmental factors that trigger the activation of those components, suggesting that resource availability has constrained the atomic composition and the molecular architecture of the biotic processes that enable cells to respond to reduced nutrient availability. PMID:19369262

  1. Heat shock protein 90 is involved in IL-17-mediated skin inflammation following thermal stimulation.

    PubMed

    Kim, Bo-Kyung; Park, Minhwa; Kim, Ji-Yon; Lee, Kyung-Ho; Woo, So-Youn

    2016-08-01

    The pathogenesis of inflammatory skin diseases involves interactions between immune cells and keratinocytes, including the T helper 17 (Th17)-mediated immune response. Several chemokines [chemokine (C-X-C motif) ligand (CXCL)1, CXCL5 and CXCL8] and antimicrobial peptides [β-defensin 1 (BD1), LL-37, S100A8 and S100A9] were transcriptionally upregulated in the keratinocyte cell line HaCaT upon stimulation with interleukin (IL)-17. Balneotherapy, the treatment of disease by bathing, is an alternative therapy that has frequently been used for the treatment of inflammatory skin diseases. Immersion in pools of thermal mineral water is often considered to have chemical, thermal, mechanical and immunomodulatory benefits. We examined the effect of thermal treatment on IL-17-mediated inflammation in a model of skin disease. As Act1 is required for IL-17 signaling and is a client protein of heat shock protein 90 (HSP90), we evaluated the effect of HSP90 inhibition on IL-17-mediated cytokine and antimicrobial peptide expression in keratinocytes following heat treatment. We found that after thermal stimulation, Act1 binding to HSP90α was significantly increased in the presence of IL-17 (100 ng/ml) and 17-N-allylamino-17-demethoxygeldanamycin (17-AAG, 1 µM). Antimicrobial peptide and chemokine expression generally increased after heat treatment; Act1 knockdown and 17‑AAG reversed this effect. These observations demonstrate the possible immunomodulatory effect of heat on keratinocytes during the progression of IL-17-mediated inflammatory skin diseases. PMID:27279135

  2. Involvement of protein phosphatases in the destabilization of methamphetamine-associated contextual memory.

    PubMed

    Yu, Yang-Jung; Huang, Chien-Hsuan; Chang, Chih-Hua; Gean, Po-Wu

    2016-09-01

    Destabilization refers to a memory that becomes unstable when reactivated and is susceptible to disruption by amnestic agents. Here we delineated the cellular mechanism underlying the destabilization of drug memory. Mice were conditioned with methamphetamine (MeAM) for 3 d, and drug memory was assessed with a conditioned place preference (CPP) protocol. Anisomycin (ANI) was administered 60 min after the CPP retrieval to disrupt reconsolidation. We found that destabilization of MeAM CPP after the application of ANI was blocked by the N-methyl-d-aspartate receptor (NMDAR) antagonist MK-801 and the NR2B antagonist ifenprodil (IFN) but not by the NR2A antagonist NVP-AAM077 (NVP). In addition, decrease in the phosphorylation of GluR1 at Serine845 (p-GluR1-Ser845), decrease in spine density, and a reduction in the AMPAR/NMDAR ratio in the basolateral amygdala (BLA) were reversed after the MK-801 treatment. The effect of ANI on destabilization was prevented by the protein phosphatase 2B (calcineurin, CaN) inhibitors cyclosporine A (CsA) and FK-506 and the protein phosphatase 1 (PP1) inhibitors calyculin A (CA) and okadaic acid (OA). These results suggest that memory destabilization involves the activation of NR2B-containing NMDARs, which in turn allows the influx of Ca(2+) Increased intracellular Ca(2+) stimulates CaN, leading to the dephosphorylation and inactivation of inhibitor 1 and the activation of PP1. PP1 then dephosphorylates p-GluR1-Ser845 to elicit AMPA receptor (AMPAR) endocytosis and destabilization of the drug memory. PMID:27531839

  3. Decreased activity of neutrophils in the presence of diferuloylmethane (curcumin) involves protein kinase C inhibition.

    PubMed

    Jancinová, Viera; Perecko, Tomás; Nosál, Radomír; Kostálová, Daniela; Bauerová, Katarína; Drábiková, Katarína

    2009-06-10

    Diferuloylmethane (curcumin) has been shown to act beneficially in arthritis, particularly through downregulated expression of proinflammatory cytokines and collagenase as well as through the modulated activities of T lymphocytes and macrophages. In this study its impact on activated neutrophils was investigated both in vitro and in experimental arthritis. Formation of reactive oxygen species in neutrophils was recorded on the basis of luminol- or isoluminol-enhanced chemiluminescence. Phosphorylation of neutrophil protein kinases C alpha and beta II was assessed by Western blotting, using phosphospecific antibodies. Adjuvant arthritis was induced in Lewis rats by heat-killed Mycobacterium butyricum. Diferuloylmethane or methotrexate was administered over a period of 28 days after arthritis induction. Under in vitro conditions, diferuloylmethane (1-100 microM) reduced dose-dependently oxidant formation both at extra- and intracellular level and it effectively reduced protein kinase C activation. Adjuvant arthritis was accompanied by an increased number of neutrophils in blood and by a more pronounced spontaneous as well as PMA (phorbol myristate acetate) stimulated chemiluminescence. Whereas the arthritis-related alterations in neutrophil count and in spontaneous chemiluminescence were not modified by diferuloylmethane, the increased reactivity of neutrophils to PMA was less evident in diferuloylmethane-treated animals. The effects of diferuloylmethane were comparable with those of methotrexate. Diferuloylmethane was found to be a potent inhibitor of neutrophil functions both in vitro and in experimental arthritis. As neutrophils are considered to be cells with the greatest capacity to inflict damage within diseased joints, the observed effects could represent a further mechanism involved in the antirheumatic activity of diferuloylmethane. PMID:19371737

  4. The effects of (-)-epicatechin on endothelial cells involve the G protein-coupled estrogen receptor (GPER).

    PubMed

    Moreno-Ulloa, Aldo; Mendez-Luna, David; Beltran-Partida, Ernesto; Castillo, Carmen; Guevara, Gustavo; Ramirez-Sanchez, Israel; Correa-Basurto, José; Ceballos, Guillermo; Villarreal, Francisco

    2015-10-01

    We have provided evidence that the stimulatory effects of (-)-epicatechin ((-)-EPI) on endothelial cell nitric oxide (NO) production may involve the participation of a cell-surface receptor. Thus far, such entity(ies) has not been fully elucidated. The G protein-coupled estrogen receptor (GPER) is a cell-surface receptor that has been linked to protective effects on the cardiovascular system and activation of intracellular signaling pathways (including NO production) similar to those reported with (-)-EPI. In bovine coronary artery endothelial cells (BCAEC) by the use of confocal imaging, we evidence the presence of GPER at the cell-surface and on F-actin filaments. Using in silico studies we document the favorable binding mode between (-)-EPI and GPER. Such binding is comparable to that of the GPER agonist, G1. By the use of selective blockers, we demonstrate that the activation of ERK 1/2 and CaMKII by (-)-EPI is dependent on the GPER/c-SRC/EGFR axis mimicking those effects noted with G1. We also evidence by the use of siRNA the role that GPER has on mediating ERK1/2 activation by (-)-EPI. GPER appears to be coupled to a non Gαi/o or Gαs, protein subtype. To extrapolate our findings to an ex vivo model, we employed phenylephrine pre-contracted aortic rings evidencing that (-)-EPI can mediate vasodilation through GPER activation. In conclusion, we provide evidence that suggests the GPER as a potential mediator of (-)-EPI effects and highlights the important role that GPER may have on cardiovascular system protection. PMID:26303816

  5. A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

    NASA Astrophysics Data System (ADS)

    Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

    2000-05-01

    RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

  6. Spermidine-induced improvement of reconsolidation of memory involves calcium-dependent protein kinase in rats.

    PubMed

    Girardi, Bruna Amanda; Ribeiro, Daniela Aymone; Signor, Cristiane; Muller, Michele; Gais, Mayara Ana; Mello, Carlos Fernando; Rubin, Maribel Antonello

    2016-01-01

    In this study, we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by the intrahippocampal administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4-mA footshock as an unconditioned stimulus. Twenty-four hours after training, animals were re-exposed to the apparatus in the absence of shock (reactivation session). Immediately after the reactivation session, spermidine (2-200 pmol/site), the PKC inhibitor 3-[1-(dimethylaminopropyl)indol-3-yl]-4-(indol-3-yl) maleimide hydrochloride (GF 109203X, 0.3-30 pg/site), the antagonist of the polyamine-binding site at the NMDA receptor, arcaine (0.2-200 pmol/site), or the PKC activator phorbol 12-myristate 13-acetate (PMA, 0.02-2 nmol/site) was injected. While the post-reactivation administration of spermidine (20 and 200 pmol/site) and PMA (2 nmol/site) improved memory reconsolidation, GF 109203X (1, 10, and 30 pg/site) and arcaine (200 pmol/site) impaired it. GF 109203X (0.3 pg/site) impaired memory reconsolidation in the presence of spermidine (200 pmol/site). PMA (0.2 nmol/site) prevented the arcaine (200 pmol/site)-induced impairment of memory reconsolidation. Anisomycin (2 µg/site) also impaired memory reconsolidation in the presence of spermidine (200 pmol/site). Drugs had no effect when they were administered in the absence of reactivation. These results suggest that the spermidine-induced enhancement of memory reconsolidation involves PKC activation. PMID:26670183

  7. Involvement of Cyclic Guanosine Monophosphate-Dependent Protein Kinase I in Renal Antifibrotic Effects of Serelaxin

    PubMed Central

    Wetzl, Veronika; Schinner, Elisabeth; Kees, Frieder; Hofmann, Franz; Faerber, Lothar; Schlossmann, Jens

    2016-01-01

    Introduction: Kidney fibrosis has shown to be ameliorated through the involvement of cyclic guanosine monophosphate (cGMP) and its dependent protein kinase I (cGKI). Serelaxin, the recombinant form of human relaxin-II, increases cGMP levels and has shown beneficial effects on kidney function in acute heart failure patients. Antifibrotic properties of serelaxin are supposed to be mediated via relaxin family peptide receptor 1 and subsequently enhanced nitric oxide/cGMP to inhibit transforming growth factor-β (TGF-β) signaling. This study examines the involvement of cGKI in the antifibrotic signaling of serelaxin. Methods and Results: Kidney fibrosis was induced by unilateral ureteral obstruction in wildtype (WT) and cGKI knock-out (KO) mice. After 7 days, renal antifibrotic effects of serelaxin were assessed. Serelaxin treatment for 7 days significantly increased cGMP in the kidney of WT and cGKI-KO. In WT, renal fibrosis was reduced through decreased accumulation of collagen1A1, total collagen, and fibronectin. The profibrotic connective tissue growth factor as well as myofibroblast differentiation were reduced and matrix metalloproteinases-2 and -9 were positively modulated after treatment. Moreover, Smad2 as well as extracellular signal-regulated kinase 1 (ERK1) phosphorylation were decreased, whereas phosphodiesterase (PDE) 5a phosphorylation was increased. However, these effects were not observed in cGKI-KO. Conclusion: Antifibrotic renal effects of serelaxin are mediated via cGMP/cGKI to inhibit Smad2- and ERK1-dependent TGF-β signaling and increased PDE5a phosphorylation. PMID:27462268

  8. Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly.

    PubMed

    Georgiadou, Michaella; Castagnini, Marta; Karimova, Gouzel; Ladant, Daniel; Pelicic, Vladimir

    2012-06-01

    The functionally versatile type IV pili (Tfp) are one of the most widespread virulence factors in bacteria. However, despite generating much research interest for decades, the molecular mechanisms underpinning the various aspects of Tfp biology remain poorly understood, mainly because of the complexity of the system. In the human pathogen Neisseria meningitidis for example, 23 proteins are dedicated to Tfp biology, 15 of which are essential for pilus biogenesis. One of the important gaps in our knowledge concerns the topology of this multiprotein machinery. Here we have used a bacterial two-hybrid system to identify and quantify the interactions between 11 Pil proteins from N. meningitidis. We identified 20 different binary interactions, many of which are novel. This represents the most complex interaction network between Pil proteins reported to date and indicates, among other things, that PilE, PilM, PilN and PilO, which are involved in pilus assembly, indeed interact. We focused our efforts on this subset of proteins and used a battery of assays to determine the membrane topology of PilN and PilO, map the interaction domains between PilE, PilM, PilN and PilO, and show that a widely conserved N-terminal motif in PilN is essential for both PilM-PilN interactions and pilus assembly. Finally, we show that PilP (another protein involved in pilus assembly) forms a complex with PilM, PilN and PilO. Taken together, these findings have numerous implications for understanding Tfp biology and provide a useful blueprint for future studies. PMID:22486968

  9. Identification of domains on the extrinsic 23 kDa protein possibly involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II.

    PubMed

    Tohri, Akihiko; Dohmae, Naoshi; Suzuki, Takehiro; Ohta, Hisataka; Inoue, Yasunori; Enami, Isao

    2004-03-01

    To elucidate the domains on the extrinsic 23 kDa protein involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II, we modified amino or carboxyl groups of the 23 kDa protein to uncharged methyl ester groups with N-succinimidyl propionate or glycine methyl ester in the presence of a water-soluble carbodiimide, respectively. The N-succinimidyl propionate-modified 23 kDa protein did not bind to the 33 kDa protein associated with PSII membranes, whereas the glycine methyl ester-modified 23 kDa protein completely bound. This indicates that positive charges on the 23 kDa protein are important for electrostatic interaction with the 33 kDa protein associated with the PSII membranes. Mapping of the N-succinimidyl propionate-modified sites of the 23 kDa protein was performed using Staphylococcus V8 protease digestion of the modified protein followed by determination of the mass of the resultant peptide fragments with MALDI-TOF MS. The results showed that six domains (Lys11-Lys14, Lys27-Lys38, Lys40, Lys90-Lys96, Lys143-Lys152, Lys166-Lys174) were modified with N-succinimidyl propionate. In these domains, Lys11, Lys13, Lys33, Lys38, Lys143, Lys166, Lys170 and Lys174 were wholly conserved in the 23 kDa protein from 12 species of higher plants. These positively charged lysyl residues on the 23 kDa protein may be involved in electrostatic interactions with the negatively charged carboxyl groups on the 33 kDa protein, the latter has been suggested to be important for the 23 kDa binding [Bricker, T.M. & Frankel, L.K. (2003) Biochemistry42, 2056-2061]. PMID:15009208

  10. Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake.

    PubMed

    Curie, C; Panaviene, Z; Loulergue, C; Dellaporta, S L; Briat, J F; Walker, E L

    2001-01-18

    Frequently, crop plants do not take up adequate amounts of iron from the soil, leading to chlorosis, poor yield and decreased nutritional quality. Extremely limited soil bioavailability of iron has led plants to evolve two distinct uptake strategies: chelation, which is used by the world's principal grain crops; and reduction, which is used by other plant groups. The chelation strategy involves extrusion of low-molecular-mass secondary amino acids (mugineic acids) known as 'phytosiderophores' which chelate sparingly soluble iron. The Fe(III)-phytosiderophore complex is then taken up by an unknown transporter at the root surface. The maize yellow stripe1 (ys1) mutant is deficient in Fe(III)-phytosiderophore uptake, therefore YS1 has been suggested to be the Fe(III)-phytosiderophore transporter. Here we show that ys1 is a membrane protein that mediates iron uptake. Expression of YS1 in a yeast iron uptake mutant restores growth specifically on Fe(III)-phytosiderophore media. Under iron-deficient conditions, ys1 messenger RNA levels increase in both roots and shoots. Cloning of ys1 is an important step in understanding iron uptake in grasses, and has implications for mechanisms controlling iron homeostasis in all plants. PMID:11201743

  11. The involvement of heat-shock proteins in the pathogenesis of autoimmune arthritis: a critical appraisal

    PubMed Central

    Huang, Min-Nung; Yu, Hua; Moudgil, Kamal D.

    2012-01-01

    Objectives To review the literature on the role of heat-shock proteins (HSPs) in the pathogenesis of autoimmune arthritis in animal models ans patients with rheumatoid arthritis (RA). Methods The published literature in Medline (PubMed), including our published work on the cell-mediated as well as humoral immune response to various HSPs was reviewed. Studies in both the pre-clinical animal models of arthritis as well as RA were examined critically and the data presented. Results In experimental arthritis, disease induction by different arthritogenic stimuli, including an adjuvant, led to immune response to mycobacterial HSP65 (BHSP65). However, attempts to induce arthritis by a purified HSP have not met with success. There are several reports of a significant immune response to HSP65 in RA patients. But, the issue of cause and effect is difficult to address. Nevertheless, several studies in animal models and a couple of clinical trials in RA patients have shown the beneficial effect of HSPs against autoimmune arthritis. Conclusions There is a clear association between immune response to HSPs, particularly HSP65, and the initiation and propagation of autoimmune arthritis in experimental models. The correlation is relatively less convincing in RA patients. In both cases, the ability of HSPs to modulate arthritis offers support, albeit an indirect one, for the involvement of these antigens in the disease process. PMID:19969325

  12. Are G-protein-coupled receptors involved in mediating larval settlement and metamorphosis of coral planulae?

    PubMed

    Tran, Cawa; Hadfield, Michael G

    2012-04-01

    Larvae of the scleractinian coral Pocillopora damicornis are induced to settle and metamorphose by the presence of marine bacterial biofilms, and the larvae of Montipora capitata respond to a combination of filamentous and crustose coralline algae. The primary goal of this study was to better understand metamorphosis of cnidarian larvae by determining what types of receptors and signal-transduction pathways are involved during stimulation of metamorphosis of P. damicornis and M. capitata. Evidence from studies on larvae of hydrozoans suggests that G-protein-coupled receptors (GPCRs) are good candidates. Settlement experiments were conducted in which competent larvae were exposed to neuropharmacological agents that affect GPCRs and their associated signal-transduction pathways, AC/cAMP and PI/DAG/PKC. On the basis of the results of these experiments, we conclude that GPCRs and these pathways do not mediate settlement and metamorphosis in either coral species. Two compounds that had an effect on both species, forskolin and phorbol-12-myristate-13-acetate (TPA), may be acting on other cellular processes not related to GPCRs. This study strengthens our understanding of the underlying physiological mechanisms that regulate metamorphosis in coral larvae. PMID:22589403

  13. Elevated expression of protein regulator of cytokinesis 1, involved in the growth of breast cancer cells.

    PubMed

    Shimo, Arata; Nishidate, Toshihiko; Ohta, Tomohiko; Fukuda, Mamoru; Nakamura, Yusuke; Katagiri, Toyomasa

    2007-02-01

    To elucidate molecular mechanisms of mammary carcinogenesis and discover novel therapeutic targets for breast cancer, we previously carried out a genome-wide expression profile analysis of 81 breast cancer cases by means of a combination of cDNA microarray and laser microbeam microdissection. Among the upregulated genes, we focused on the functional significance of protein regulator of cytokinesis 1 (PRC1) in the development of breast cancer. Western blot analysis using breast cancer cell lines revealed a significant increase in endogenous PRC1 levels in G(2)/M phase. Treatment of breast cancer cells with small interfering RNA against PRC1 effectively suppressed its expression and inhibited the growth of breast cancer cell lines T47D and HBC5. Furthermore, we found an interaction between PRC1 and kinesin family member 2C/mitotic centromere-associated kinesin (KIF2C/MCAK) by coimmunoprecipitation and immunoblotting using COS-7 cells, in which these molecules were introduced exogenously. These findings suggest the involvement of a PRC1-KIF2C/MCAK complex in breast tumorigenesis, and this complex should be a promising target for the development of novel treatments for breast cancer. PMID:17233835

  14. Shrinkage activates a nonselective conductance: involvement of a Walker-motif protein and PKC.

    PubMed

    Nelson, D J; Tien, X Y; Xie, W; Brasitus, T A; Kaetzel, M A; Dedman, J R

    1996-01-01

    The ability of all cells to maintain their volume during an osmotic challenge is dependent on the regulated movement of salt and water across the plasma membrane. We demonstrate the phosphorylation-dependent gating of a nonselective conductance in Caco-2 cells during cellular shrinkage. Intracellular application of exogenous purified rat brain protein kinase C (PKC) resulted in the activation of a current similar to that activated during shrinkage with a Na(+)-to-Cl- permeability ratio of approximately 1.7:1. To prevent possible PKC- and/or shrinkage-dependent activation of cystic fibrosis transmembrane regulator (CFTR), which is expressed at high levels in Caco-2 cells, a functional anti-peptide antibody, anti-CFTR505-511, was introduced into the cells via the patch pipette. Anti-CFTR505-511, which is directed against the Walker motif in the first nucleotide binding fold of CFTR, prevented the PKC/shrink-age current activation. The peptide CFTR505-511 also induced current inhibition, suggesting the possible involvement of a regulatory element in close proximity to the channel that shares sequence homology with the first nucleotide binding fold of CFTR and whose binding to the channel is required for channel gating. PMID:8772443

  15. Protein binding sites involved in the assembly of the KplE1 prophage intasome.

    PubMed

    Panis, Gaël; Duverger, Yohann; Champ, Stéphanie; Ansaldi, Mireille

    2010-08-15

    The organization of the recombination regions of the KplE1 prophage in Escherichia coli K12 differs from that observed in the lambda prophage. Indeed, the binding sites characterized for the IntS integrase, the TorI recombination directionality factor (RDF) and the integration host factor (IHF) vary in number, spacing and orientation on the attL and attR regions. In this paper, we performed site-directed mutagenesis of the recombination sites to decipher if all sites are essential for the site-specific recombination reaction and how the KplE1 intasome is assembled. We also show that TorI and IntS form oligomers that are stabilized in the presence of their target DNA. Moreover, we found that IHF is the only nucleoid associated protein (NAP) involved in KplE1 recombination, although it is dispensable. This is consistent with the presence of only one functional IHF site on attR and none on attL. PMID:20494389

  16. The Fragile X Protein binds mRNAs involved in cancer progression and modulates metastasis formation

    PubMed Central

    Lucá, Rossella; Averna, Michele; Zalfa, Francesca; Vecchi, Manuela; Bianchi, Fabrizio; Fata, Giorgio La; Del Nonno, Franca; Nardacci, Roberta; Bianchi, Marco; Nuciforo, Paolo; Munck, Sebastian; Parrella, Paola; Moura, Rute; Signori, Emanuela; Alston, Robert; Kuchnio, Anna; Farace, Maria Giulia; Fazio, Vito Michele; Piacentini, Mauro; De Strooper, Bart; Achsel, Tilmann; Neri, Giovanni; Neven, Patrick; Evans, D Gareth; Carmeliet, Peter; Mazzone, Massimiliano; Bagni, Claudia

    2013-01-01

    The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression. PMID:24092663

  17. The fragile X protein binds mRNAs involved in cancer progression and modulates metastasis formation.

    PubMed

    Lucá, Rossella; Averna, Michele; Zalfa, Francesca; Vecchi, Manuela; Bianchi, Fabrizio; La Fata, Giorgio; Del Nonno, Franca; Nardacci, Roberta; Bianchi, Marco; Nuciforo, Paolo; Munck, Sebastian; Parrella, Paola; Moura, Rute; Signori, Emanuela; Alston, Robert; Kuchnio, Anna; Farace, Maria Giulia; Fazio, Vito Michele; Piacentini, Mauro; De Strooper, Bart; Achsel, Tilmann; Neri, Giovanni; Neven, Patrick; Evans, D Gareth; Carmeliet, Peter; Mazzone, Massimiliano; Bagni, Claudia

    2013-10-01

    The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression. PMID:24092663

  18. Direct Involvement of Retinoblastoma Family Proteins in DNA Repair by Non-homologous End-Joining

    PubMed Central

    Cook, Rebecca; Zoumpoulidou, Georgia; Luczynski, Maciej T.; Rieger, Simone; Moquet, Jayne; Spanswick, Victoria J.; Hartley, John A.; Rothkamm, Kai; Huang, Paul H.; Mittnacht, Sibylle

    2015-01-01

    Summary Deficiencies in DNA double-strand break (DSB) repair lead to genetic instability, a recognized cause of cancer initiation and evolution. We report that the retinoblastoma tumor suppressor protein (RB1) is required for DNA DSB repair by canonical non-homologous end-joining (cNHEJ). Support of cNHEJ involves a mechanism independent of RB1’s cell-cycle function and depends on its amino terminal domain with which it binds to NHEJ components XRCC5 and XRCC6. Cells with engineered loss of RB family function as well as cancer-derived cells with mutational RB1 loss show substantially reduced levels of cNHEJ. RB1 variants disabled for the interaction with XRCC5 and XRCC6, including a cancer-associated variant, are unable to support cNHEJ despite being able to confer cell-cycle control. Our data identify RB1 loss as a candidate driver of structural genomic instability and a causative factor for cancer somatic heterogeneity and evolution. PMID:25818292

  19. The methyltransferase adaptor protein Trm112 is involved in biogenesis of both ribosomal subunits

    PubMed Central

    Sardana, Richa; Johnson, Arlen W.

    2012-01-01

    We previously identified Bud23 as the methyltransferase that methylates G1575 of rRNA in the P-site of the small (40S) ribosomal subunit. In this paper, we show that Bud23 requires the methyltransferase adaptor protein Trm112 for stability in vivo. Deletion of Trm112 results in a bud23Δ-like mutant phenotype. Thus Trm112 is required for efficient small-subunit biogenesis. Genetic analysis suggests the slow growth of a trm112Δ mutant is due primarily to the loss of Bud23. Surprisingly, suppression of the bud23Δ-dependent 40S defect revealed a large (60S) biogenesis defect in a trm112Δ mutant. Using sucrose gradient sedimentation analysis and coimmunoprecipitation, we show that Trm112 is also involved in 60S subunit biogenesis. The 60S defect may be dependent on Nop2 and Rcm1, two additional Trm112 interactors that we identify. Our work extends the known range of Trm112 function from modification of tRNAs and translation factors to both ribosomal subunits, showing that its effects span all aspects of the translation machinery. Although Trm112 is required for Bud23 stability, our results suggest that Trm112 is not maintained in a stable complex with Bud23. We suggest that Trm112 stabilizes its free methyltransferase partners not engaged with substrate and/or helps to deliver its methyltransferase partners to their substrates. PMID:22956767

  20. Yeast Mitochondrial Interactosome Model: Metabolon Membrane Proteins Complex Involved in the Channeling of ADP/ATP

    PubMed Central

    Clémençon, Benjamin

    2012-01-01

    The existence of a mitochondrial interactosome (MI) has been currently well established in mammalian cells but the exact composition of this super-complex is not precisely known, and its organization seems to be different from that in yeast. One major difference is the absence of mitochondrial creatine kinase (MtCK) in yeast, unlike that described in the organization model of MI, especially in cardiac, skeletal muscle and brain cells. The aim of this review is to provide a detailed description of different partner proteins involved in the synergistic ADP/ATP transport across the mitochondrial membranes in the yeast Saccharomyces cerevisiae and to propose a new mitochondrial interactosome model. The ADP/ATP (Aacp) and inorganic phosphate (PiC) carriers as well as the VDAC (or mitochondrial porin) catalyze the import and export of ADP, ATP and Pi across the mitochondrial membranes. Aacp and PiC, which appear to be associated with the ATP synthase, consist of two nanomotors (F0, F1) under specific conditions and form ATP synthasome. Identification and characterization of such a complex were described for the first time by Pedersen and co-workers in 2003. PMID:22408429

  1. Increased abundance of proteins involved in phytosiderophore production in boron-tolerant barley.

    PubMed

    Patterson, John; Ford, Kris; Cassin, Andrew; Natera, Siria; Bacic, Antony

    2007-07-01

    Boron (B) phytotoxicity affects cereal-growing regions worldwide. Although B-tolerant barley (Hordeum vulgare) germplasm is available, molecules responsible for this tolerance mechanism have not been defined. We describe and use a new comparative proteomic technique, iTRAQ peptide tagging (iTRAQ), to compare the abundances of proteins from B-tolerant and -intolerant barley plants from a 'Clipper' x 'Sahara' doubled-haploid population selected on the basis of a presence or absence of two B-tolerance quantitative trait loci. iTRAQ was used to identify three enzymes involved in siderophore production (Iron Deficiency Sensitive2 [IDS2], IDS3, and a methylthio-ribose kinase) as being elevated in abundance in the B-tolerant plants. Following from this result, we report a potential link between iron, B, and the siderophore hydroxymugineic acid. We believe that this study highlights the potency of the iTRAQ approach to better understand mechanisms of abiotic stress tolerance in cereals, particularly when applied in conjunction with bulked segregant analysis. PMID:17478636

  2. Lincomycin Biosynthesis Involves a Tyrosine Hydroxylating Heme Protein of an Unusual Enzyme Family

    PubMed Central

    Novotna, Jitka; Olsovska, Jana; Novak, Petr; Mojzes, Peter; Chaloupkova, Radka; Kamenik, Zdenek; Spizek, Jaroslav; Kutejova, Eva; Mareckova, Marketa; Tichy, Pavel; Damborsky, Jiri; Janata, Jiri

    2013-01-01

    The gene lmbB2 of the lincomycin biosynthetic gene cluster of Streptomyces lincolnensis ATCC 25466 was shown to code for an unusual tyrosine hydroxylating enzyme involved in the biosynthetic pathway of this clinically important antibiotic. LmbB2 was expressed in Escherichia coli, purified near to homogeneity and shown to convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA). In contrast to the well-known tyrosine hydroxylases (EC 1.14.16.2) and tyrosinases (EC 1.14.18.1), LmbB2 was identified as a heme protein. Mass spectrometry and Soret band-excited Raman spectroscopy of LmbB2 showed that LmbB2 contains heme b as prosthetic group. The CO-reduced differential absorption spectra of LmbB2 showed that the coordination of Fe was different from that of cytochrome P450 enzymes. LmbB2 exhibits sequence similarity to Orf13 of the anthramycin biosynthetic gene cluster, which has recently been classified as a heme peroxidase. Tyrosine hydroxylating activity of LmbB2 yielding DOPA in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) was also observed. Reaction mechanism of this unique heme peroxidases family is discussed. Also, tyrosine hydroxylation was confirmed as the first step of the amino acid branch of the lincomycin biosynthesis. PMID:24324587

  3. NMR identification of the binding surfaces involved in the Salmonella and Shigella Type III secretion tip-translocon protein-protein interactions.

    PubMed

    McShan, Andrew C; Kaur, Kawaljit; Chatterjee, Srirupa; Knight, Kevin M; De Guzman, Roberto N

    2016-08-01

    The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein-protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N-terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled-coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N-terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip-translocon protein-protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097-1107. © 2016 Wiley Periodicals, Inc. PMID:27093649

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

    PubMed Central

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

    1992-01-01

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

  5. Identification of Protein Networks Involved in the Disease Course of Experimental Autoimmune Encephalomyelitis, an Animal Model of Multiple Sclerosis

    PubMed Central

    Plaisance, Stéphane; Baeten, Kurt; Hendriks, Jerome J. A.; Leprince, Pierre; Dumont, Debora; Robben, Johan; Brône, Bert; Stinissen, Piet; Noben, Jean-Paul; Hellings, Niels

    2012-01-01

    A more detailed insight into disease mechanisms of multiple sclerosis (MS) is crucial for the development of new and more effective therapies. MS is a chronic inflammatory autoimmune disease of the central nervous system. The aim of this study is to identify novel disease associated proteins involved in the development of inflammatory brain lesions, to help unravel underlying disease processes. Brainstem proteins were obtained from rats with MBP induced acute experimental autoimmune encephalomyelitis (EAE), a well characterized disease model of MS. Samples were collected at different time points: just before onset of symptoms, at the top of the disease and following recovery. To analyze changes in the brainstem proteome during the disease course, a quantitative proteomics study was performed using two-dimensional difference in-gel electrophoresis (2D-DIGE) followed by mass spectrometry. We identified 75 unique proteins in 92 spots with a significant abundance difference between the experimental groups. To find disease-related networks, these regulated proteins were mapped to existing biological networks by Ingenuity Pathway Analysis (IPA). The analysis revealed that 70% of these proteins have been described to take part in neurological disease. Furthermore, some focus networks were created by IPA. These networks suggest an integrated regulation of the identified proteins with the addition of some putative regulators. Post-synaptic density protein 95 (DLG4), a key player in neuronal signalling and calcium-activated potassium channel alpha 1 (KCNMA1), involved in neurotransmitter release, are 2 putative regulators connecting 64% of the identified proteins. Functional blocking of the KCNMA1 in macrophages was able to alter myelin phagocytosis, a disease mechanism highly involved in EAE and MS pathology. Quantitative analysis of differentially expressed brainstem proteins in an animal model of MS is a first step to identify disease-associated proteins and networks that

  6. Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells.

    PubMed

    Thomas, T; Shah, N; Klinge, C M; Faaland, C A; Adihkarakunnathu, S; Gallo, M A; Thomas, T J

    1999-04-01

    We investigated the effects of polyamine biosynthesis inhibition on the estrogenic signaling pathway of MCF-7 breast cancer cells using a protein-protein interaction system. Estrogen receptor (ER) linked to glutathione-S-transferase (GST) was used to examine the effects of two polyamine biosynthesis inhibitors, difluoromethylornithine (DFMO) and CGP 48664. ER was specifically associated with a 45 kDa protein in control cells. In cells treated with estradiol, nine proteins were associated with ER. Cells treated with polyamine biosynthesis inhibitors in the absence of estradiol retained the binding of their ER with a 45 kDa protein and the ER also showed low-affinity interactions with a number of cellular proteins; however, these associations were decreased by the presence of estradiol and the inhibitors. When samples from the estradiol+DFMO treatment group were incubated with spermidine prior to GST-ER pull down assay, an increased association of several proteins with ER was detected. The intensity of the ER-associated 45 kDa protein increased by 10-fold in the presence of 1000 microM spermidine. These results indicate a specific role for spermidine in ER association of proteins. Western blot analysis of samples eluted from GST-ER showed the presence of chicken ovalbumin upstream promoter-transcription factor, an orphan nuclear receptor, and the endogenous full-length ER. These results show that multiple proteins associate with ER and that the binding of some of these proteins is highly sensitive to intracellular polyamine concentrations. Overall, our results indicate the importance of the polyamine pathway in the gene regulatory function of estradiol in breast cancer cells. PMID:10194516

  7. Plastid ribosomal protein S5 is involved in photosynthesis, plant development, and cold stress tolerance in Arabidopsis.

    PubMed

    Zhang, Junxiang; Yuan, Hui; Yang, Yong; Fish, Tara; Lyi, Sangbom M; Thannhauser, Theodore W; Zhang, Lugang; Li, Li

    2016-04-01

    Plastid ribosomal proteins are essential components of protein synthesis machinery and have diverse roles in plant growth and development. Mutations in plastid ribosomal proteins lead to a range of developmental phenotypes in plants. However, how they regulate these processes is not fully understood, and the functions of some individual plastid ribosomal proteins remain unknown. To identify genes responsible for chloroplast development, we isolated and characterized a mutant that exhibited pale yellow inner leaves with a reduced growth rate in Arabidopsis. The mutant (rps5) contained a missense mutation of plastid ribosomal protein S5 (RPS5), which caused a dramatically reduced abundance of chloroplast 16S rRNA and seriously impaired 16S rRNA processing to affect ribosome function and plastid translation. Comparative proteomic analysis revealed that the rps5 mutation suppressed the expression of a large number of core components involved in photosystems I and II as well as many plastid ribosomal proteins. Unexpectedly, a number of proteins associated with cold stress responses were greatly decreased in rps5, and overexpression of the plastid RPS5 improved plant cold stress tolerance. Our results indicate that RPS5 is an important constituent of the plastid 30S subunit and affects proteins involved in photosynthesis and cold stress responses to mediate plant growth and development. PMID:27006483

  8. A New MAP Kinase Protein Involved in Estradiol-Stimulated Reproduction of the Helminth Parasite Taenia crassiceps

    PubMed Central

    Escobedo, Galileo; Soldevila, Gloria; Ortega-Pierres, Guadalupe; Chávez-Ríos, Jesús Ramsés; Nava, Karen; Fonseca-Liñán, Rocío; López-Griego, Lorena; Hallal-Calleros, Claudia; Ostoa-Saloma, Pedro; Morales-Montor, Jorge

    2010-01-01

    MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasite Taenia crassiceps. Our results show that 17β-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17β-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host and T. crassiceps, and may be considered as target for anti-helminth drugs design. PMID:20145710

  9. Plastid ribosomal protein S5 is involved in photosynthesis, plant development, and cold stress tolerance in Arabidopsis

    PubMed Central

    Zhang, Junxiang; Yuan, Hui; Yang, Yong; Fish, Tara; Lyi, Sangbom M.; Thannhauser, Theodore W; Zhang, Lugang; Li, Li

    2016-01-01

    Plastid ribosomal proteins are essential components of protein synthesis machinery and have diverse roles in plant growth and development. Mutations in plastid ribosomal proteins lead to a range of developmental phenotypes in plants. However, how they regulate these processes is not fully understood, and the functions of some individual plastid ribosomal proteins remain unknown. To identify genes responsible for chloroplast development, we isolated and characterized a mutant that exhibited pale yellow inner leaves with a reduced growth rate in Arabidopsis. The mutant (rps5) contained a missense mutation of plastid ribosomal protein S5 (RPS5), which caused a dramatically reduced abundance of chloroplast 16S rRNA and seriously impaired 16S rRNA processing to affect ribosome function and plastid translation. Comparative proteomic analysis revealed that the rps5 mutation suppressed the expression of a large number of core components involved in photosystems I and II as well as many plastid ribosomal proteins. Unexpectedly, a number of proteins associated with cold stress responses were greatly decreased in rps5, and overexpression of the plastid RPS5 improved plant cold stress tolerance. Our results indicate that RPS5 is an important constituent of the plastid 30S subunit and affects proteins involved in photosynthesis and cold stress responses to mediate plant growth and development. PMID:27006483

  10. A Proteomic Approach for the Identification of Up-Regulated Proteins Involved in the Metabolic Process of the Leiomyoma

    PubMed Central

    Ura, Blendi; Scrimin, Federica; Arrigoni, Giorgio; Franchin, Cinzia; Monasta, Lorenzo; Ricci, Giuseppe

    2016-01-01

    Uterine leiomyoma is the most common benign smooth muscle cell tumor of the uterus. Proteomics is a powerful tool for the analysis of complex mixtures of proteins. In our study, we focused on proteins that were upregulated in the leiomyoma compared to the myometrium. Paired samples of eight leiomyomas and adjacent myometrium were obtained and submitted to two-dimensional gel electrophoresis (2-DE) and mass spectrometry for protein identification and to Western blotting for 2-DE data validation. The comparison between the patterns revealed 24 significantly upregulated (p < 0.05) protein spots, 12 of which were found to be associated with the metabolic processes of the leiomyoma and not with the normal myometrium. The overexpression of seven proteins involved in the metabolic processes of the leiomyoma was further validated by Western blotting and 2D Western blotting. Four of these proteins have never been associated with the leiomyoma before. The 2-DE approach coupled with mass spectrometry, which is among the methods of choice for comparative proteomic studies, identified a number of proteins overexpressed in the leiomyoma and involved in several biological processes, including metabolic processes. A better understanding of the mechanism underlying the overexpression of these proteins may be important for therapeutic purposes. PMID:27070597

  11. Light-regulated stapled peptides to inhibit protein-protein interactions involved in clathrin-mediated endocytosis.

    PubMed

    Nevola, Laura; Martín-Quirós, Andrés; Eckelt, Kay; Camarero, Núria; Tosi, Sébastien; Llobet, Artur; Giralt, Ernest; Gorostiza, Pau

    2013-07-22

    Control of membrane traffic: Photoswitchable inhibitors of protein-protein interactions were applied to photoregulate clathrin-mediated endocytosis (CME) in living cells. Traffic light (TL) peptides acting as "stop" and "go" signals for membrane traffic can be used to dissect the role of CME in receptor internalization and in cell growth, division, and differentiation. PMID:23775788

  12. Acquisition of heme iron by Neisseria meningitidis does not involve meningococcal transferrin-binding proteins.

    PubMed

    Martel, N; Lee, B C

    1994-02-01

    Similarities in size between hemin-binding protein 1 (HmBP1) and transferrin-binding protein 1 (TBP1) of Neisseria meningitidis suggest that these proteins are functionally homologous. However, a meningococcal mutant lacking the transferrin-binding proteins retained the capacity to acquire iron from heme and hemoglobin. In immunoblots, hyperimmune polyclonal antiserum against TBP1 did not react with HmBP1. PMID:8300227

  13. TMTC1 and TMTC2 Are Novel Endoplasmic Reticulum Tetratricopeptide Repeat-containing Adapter Proteins Involved in Calcium Homeostasis*

    PubMed Central

    Sunryd, Johan C.; Cheon, Banyoon; Graham, Jill B.; Giorda, Kristina M.; Fissore, Rafael A.; Hebert, Daniel N.

    2014-01-01

    The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis. PMID:24764305

  14. DNA Processing Proteins Involved in the UV-Induced Stress Response of Sulfolobales

    PubMed Central

    van Wolferen, Marleen; Ma, Xiaoqing

    2015-01-01

    ABSTRACT The ups operon of Sulfolobus species is highly induced upon UV stress. Previous studies showed that the pili encoded by this operon are involved in cellular aggregation, which is essential for subsequent DNA exchange between cells, resulting in homologous recombination. The presence of this pilus system increases the fitness of Sulfolobus cells under UV light-induced stress conditions, as the transfer of DNA takes place in order to repair UV-induced DNA lesions via homologous recombination. Four conserved genes (saci_1497 to saci_1500) which encode proteins with putative DNA processing functions are present downstream of the ups operon. In this study, we show that after UV treatment the cellular aggregation of strains with saci_1497, saci_1498, and saci_1500 deletions is similar to that of wild-type strains; their survival rates, however, were reduced and similar to or lower than those of the pilus deletion strains, which could not aggregate anymore. DNA recombination assays indicated that saci_1498, encoding a ParB-like protein, plays an important role in DNA transfer. Moreover, biochemical analysis showed that the endonuclease III encoded by saci_1497 nicks UV-damaged DNA. In addition, RecQ-like helicase Saci_1500 is able to unwind homologous recombination intermediates, such as Holliday junctions. Interestingly, a saci_1500 deletion mutant was more sensitive to UV light but not to the replication-stalling agents hydroxyurea and methyl methanesulfonate, suggesting that Saci_1500 functions specifically in the UV damage pathway. Together these results suggest a role of Saci_1497 to Saci_1500 in the repair or transfer of DNA that takes place after UV-induced damage to the genomic DNA of Sulfolobus acidocaldarius. IMPORTANCE Sulfolobales species increase their fitness after UV stress by a UV-inducible pilus system that enables high rates of DNA exchange between cells. Downstream of the pilus operon, three genes that seem to play a role in the repair or

  15. Expression of proteins involved in DNA damage response in familial and sporadic breast cancer patients.

    PubMed

    Partipilo, Giulia; Simone, Giovanni; Scattone, Anna; Scarpi, Emanuela; Azzariti, Amalia; Mangia, Anita

    2016-01-01

    Understanding the expression of proteins involved in DNA damage response could improve knowledge of the pathways that contribute to familial and sporadic breast cancer (BC). We aimed to assess the different roles of BRCA1, poly(ADP-ribose) polymerase-1 (PARP1), BRCT-repeat inhibitor of hTERT expression (BRIT1) and novel SWItch 5 (SWI5) expression in 130 sporadic and 73 familial BC samples, by immunohistochemistry. In the sporadic group, negative nuclear BRCA1 (nBRCA1) expression was associated with positive PgR (p = 0.037). Negative association was found between nBRCA1 expression and HER2 (p = 0.001). In the familial group, nBRCA1 expression was associated with ER (p = 0.002). Reduced nBRCA1 expression was associated with higher histological grade and positive Ki67 both in sporadic (p = 0.0010, p = 0.047) and familial groups (p < 0.001, p = 0.001). Nuclear PARP1 (nPARP1) expression was associated with histological grade (p = 0.035) and positive PgR (p = 0.047) in sporadic cases. High cytoplasmic and low nuclear BRIT1 (cBRIT1 and nBRIT1) expression were associated with high histological grade in the familial group (p = 0.013, p = 0.025). Various statistical associations between the protein expressions were observed in the sporadic group, while in familial group only few associations were found. Univariate analyses showed that nPARP1 expression is able to discriminate between sporadic and familial tumors (OR 2.80, p = 0.002). Multivariate analyses proved that its overexpression is an independent factor associated with a high risk of sporadic tumor (OR 2.96, p = 0.017). Our findings indicate that nPARP1 expression is an independent factor for sporadic BCs and PARP1 inhibitors could be a promising therapy for different phenotypes. PMID:26205471

  16. Heat-shock protein 70 is involved in hyperbaric oxygen preconditioning on decompression sickness in rats.

    PubMed

    Ni, Xiao-Xiao; Ni, Ming; Fan, Dan-Feng; Sun, Qiang; Kang, Zhi-Min; Cai, Zhi-Yu; Liu, Yun; Liu, Kan; Li, Run-Ping; Xu, Wei-Gang

    2013-01-01

    Decompression sickness (DCS) is a major concern in diving and space walk. Hyperbaric oxygen (HBO) preconditioning has been proved to enhance tolerance to DCS via nitric oxide. Heat-shock protein (HSP) 70 was also found to have protective effects against DCS. We hypothesized that the beneficial effects of HBO preconditioning on DCS was related to levels of elevated HSP70. HSPs (70, 27 and 90) expressed in tissues of spinal cord and lung in rats was detected at different time points following HBO exposure by Western blot. HSP27 and HSP90 showed a slight but not significant increase after HBO. HSP70 increased and reached highest at 18 h following exposure before decreasing. Then rats were exposed to HBO and subjected to simulated air dive and rapid decompression to induce DCS 18 h after HBO. The severity of DCS, along with levels of HSP70 expression, as well as the extent of oxidative and apoptotic parameters in the lung and spinal cord were compared among different groups of rats pretreated with HBO, HBO plus NG-nitro-l-arginine-methyl ester (l-NAME), HBO plus quercetin or normobaric air. HBO preconditioning significantly reduced the morbidity of DCS (from 66.7% to 36.7%), reduced levels of oxidation (malondialdehyde, 8-hydroxyguanine and hydrogen peroxide) and apoptosis (caspase-3 and -9 activities and the number of apoptotic cells). l-NAME or quercetin eliminated most of the beneficial effects of HBO on DCS, and counteracted the stimulation of HSP70 by HBO. Bubbles in pulmonary artery were detected using ultrasound imaging to observe the possible effect of HBO preconditioning on DCS bubble formation. The amounts of bubbles in rats pretreated with HBO or air showed no difference. These results suggest that HSP70 was involved in the beneficial effects of HBO on DCS in rats, suspected be by the antioxidation and antiapoptosis effects. PMID:23479759

  17. The acid tolerance response of Salmonella typhimurium involves transient synthesis of key acid shock proteins.

    PubMed Central

    Foster, J W

    1993-01-01

    Although Salmonella typhimurium prefers neutral-pH environments, it can adapt to survive conditions of severe low-pH stress (pH 3.3). The process, termed the acid tolerance response (ATR), includes two distinct stages. The first stage, called pre-acid shock, is induced at pH 5.8 and involves the production of an inducible pH homeostasis system functional at external pH values below 4.0. The second stage occurs following an acid shock shift to pH 4.5 or below and is called the post-acid shock stage. During this stage of the ATR, 43 acid shock proteins (ASPs) are synthesized. The present data reveal that several ASPs important for pH 3.3 acid tolerance are only transiently produced. Their disappearance after 30 to 40 min of pH 4.4 acid shock coincides with an inability to survive subsequent pH 3.3 acid challenge. Clearly, an essential feature of inducible acid tolerance is an ability to synthesize these key ASPs. The pre-acid shock stage, with its inducible pH homeostasis system, offers the cell an enhanced ability to synthesize ASPs following rapid shifts to conditions below pH 4.0, an external pH that normally prevents ASP synthesis. The data also address possible signals for ASP synthesis. The inducing signal for 22 ASPs appears to be internal acidification, while external pH serves to induce 13 others. Of the 14 transient ASPs, 10 are induced in response to changes in internal pH. Mutations in the fur (ferric uptake regulator) locus that produce an Atr- acid-sensitive phenotype also eliminate induction of six transiently induced ASPs. Images PMID:8458840

  18. AMP-activated protein kinase is involved in perfluorohexanesulfonate -induced apoptosis of neuronal cells.

    PubMed

    Lee, Youn Ju; Choi, So-Young; Yang, Jae-Ho

    2016-04-01

    Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds (PFCs), has been used in a variety of industrial and consumer applications and detected in serum in the general population. This raised a concern over its possible detrimental health effects, including neurotoxic effects. We have previously shown that PFHxS induced neuronal apoptosis via the NMDA receptor-mediated extracellular signal-regulated kinase (ERK) pathway. Recently, it has been reported that AMP-activated protein kinase (AMPK) acts as a key signal molecule in neuronal excitotoxicity as well as providing a neuroprotective function. In the present study, we have examined the involvement of AMPK in PFHxS-induced neuronal apoptosis using neuronal differentiated PC12 cells. PFHxS induced significant increases in intracellular [Ca(2+)] via the NMDA receptor and the L-type voltage-gated calcium channel (L-VGCC). The inhibition of Ca(2+) loading by the NMDA receptor antagonist, MK801 and the L-VGCC blockers, nifedipine and diltiazem significantly reduced PFHxS-induced apoptosis. PFHxS induced sustained activation of AMPK and the inhibition of AMPK activation by compound C and AMPK siRNA significantly reduced PFHxS-induced caspase-3 activity. These results indicate the pro-apoptotic role of AMPK. The activation of AMPK was attenuated by MK801, nifedipine and diltiazem. However, the activation of AMPK was not affected by the ERK inhibitor, PD98059. Likewise, ERK activation was not affected by compound C but was substantially reduced by MK801, nifedipine or diltiazem. This suggests that the activation of AMPK and ERK is regulated by intracellular Ca(2+) loading in distinct pathways. Taken together, PFHxS-induced neuronal apoptosis is mediated by AMPK and ERK pathways, which are distinctly regulated by increased intracellular Ca(2+) via the NMDA receptor and L-VGCC. PMID:26826296

  19. Thermoregulatory uncoupling in heart muscle mitochondria: involvement of the ATP/ADP antiporter and uncoupling protein.

    PubMed

    Simonyan, R A; Skulachev, V P

    1998-09-25

    Possible involvement of the ATP/ADP antiporter and uncoupling protein (UCP) in thermoregulatory uncoupling of oxidative phosphorylation in heart muscle has been studied. To this end, effects of carboxyatractylate (cAtr) and GDP, specific inhibitors of the antiporter and UCP, on the membrane potential of the oligomycin-treated mitochondria from cold-exposed (6 degrees C, 48 h) and control rats have been measured. It is found that cAtr increases the membrane potential level in both cold-exposed and non-exposed groups, the effect being strongly enhanced by cooling. As for GDP, it is effective only in mitochondria from the cold-exposed rats. In these mitochondria, the coupling effect of GDP is smaller than that of cAtr. CDP, which does not interact with UCP, is without any influence on membrane potential. The cold exposure is found to increase the uncoupling efficiency of added natural (palmitate) or artificial (SF6847) uncouplers, the increase being cAtr- and GDP-sensitive in the case of palmitate. The fatty acid-free bovine serum albumin enhances delta psi in both cold-exposed and control groups, the effect being much larger in the former case. It is concluded that in heart muscle mitochondria the ATP/ADP antiporter is responsible for the 'mild uncoupling' under normal conditions and for major portion of the thermoregulatory uncoupling in the cold whereas the rest of thermoregulatory uncoupling is served by UCP (presumably by UCP2 since the UCP2 mRNA level is shown to strongly increase in rat heart muscle under the cold exposure conditions used). PMID:9771898

  20. Proteomics displays cytoskeletal proteins and chaperones involvement in Hedyotis corymbosa-induced photokilling in skin cancer cells.

    PubMed

    You, Bang-Jau; Wu, Yang-Chang; Wu, Chi-Yu; Bao, Bo-Ying; Chen, Mei-Yu; Chang, Yu-Hao; Lee, Hong-Zin

    2011-08-01

    Photodynamic therapy was found to be an effective therapy for local malignant tumors. This study demonstrated that 80 μg/ml Hedyotis corymbosa extracts with 0.8 J/cm(2) fluence dose caused M21 skin cancer cell death. Photoactivated H. corymbosa-induced M21 cell death is a typical apoptosis that is accompanied by nuclear condensation, externalization of phosphatidylserine and the changes in protein expression of apoptosis-related proteins, such as Bcl-2 and caspase family members. This study applied 2D electrophoresis to analyse the proteins involved in the photoactivated H. corymbosa-induced M21 cell apoptosis. We found 12 proteins to be markedly changed. According to the results of protein sequence analysis of these altered protein spots, we identified that the expression of cytoskeletal proteins and chaperones were involved in the photoactivated H. corymbosa-induced M21 cell apoptosis. We further demonstrated that photoactivated H. corymbosa caused a significant effect on the cytoskeleton distribution and mitochondrial activity in M21 cells. Based on the above findings, this study characterized the effects and mechanisms of the photoactivated H. corymbosa-induced apoptosis in M21 skin cancer cells. PMID:21569101

  1. Proteomic analysis of differentially expressed proteins involved in ethylene-induced chilling tolerance in harvested banana fruit

    PubMed Central

    Li, Taotao; Yun, Ze; Zhang, Dandan; Yang, Chengwei; Zhu, Hong; Jiang, Yueming; Duan, Xuewu

    2015-01-01

    To better understand the mechanism involved in ethylene-induced chilling tolerance in harvested banana fruit, a gel-based proteomic study followed by MALDI-TOF-TOF MS was carried out. Banana fruit were treated with 500 ppm ethylene for 12 h and then stored at 6°C. During cold storage, the chilling tolerance was assessed and the proteins from the peel were extracted for proteomic analysis. It was observed that ethylene pretreatment significantly induced the chilling tolerance in harvested banana fruit, manifesting as increases in maximal chlorophyll fluorescence (Fv/Fm) and decreased electrolyte leakage. Sixty-four proteins spots with significant differences in abundance were identified, most of which were induced by ethylene pretreatment during cold storage. The up-regulated proteins induced by ethylene pretreatment were mainly related to energy metabolism, stress response and defense, methionine salvage cycle and protein metabolism. These proteins were involved in ATP synthesis, ROS scavenging, protective compounds synthesis, protein refolding and degradation, and polyamine biosynthesis. It is suggested that these up-regulated proteins might play a role in the ethylene-induced chilling tolerance in harvested banana fruit. PMID:26528309

  2. Ligand-induced association of surface immunoglobulin with the detergent insoluble cytoskeleton may involve an 89K protein

    SciTech Connect

    Gupta, S.K.; Woda, B.

    1986-03-01

    Membrane immunoglobulin of B-lymphocytes is thought to play an important role in antigen recognition and cellular activation. Binding of cross-linking ligands to surface immunoglobulin (SIg) on intact cells converts it to a detergent insoluble state, and this conversion is associated with the transmission of a mitogenic signal. Insolubilized membrane proteins may be solubilized by incubating the detergent insoluble cytoskeletons in buffers which convert F-actin to G-actin ((Buffer 1), 0.34M sucrose, 0.5mM ATP, 0.5mM Dithiothrietol and lmM EDTA). Immunoprecipitation of SIg from the detergent soluble fraction of /sup 35/S-methionine labeled non ligand treated rat B-cells results in the co-isolation of an 89K protein and a 44K protein, presumably actin. The 89K protein is not associated with the fraction of endogenous detergent insoluble SIg. On treatment of rat B cells with cross-linking ligand (anti-Ig) the 89K protein becomes detergent insoluble along with most of the SIg and co-isolates with SIg on immunoprecipitation of the detergent insoluble, buffer l solubilized fraction. The migration of the SIg-associated 89K protein from the detergent soluble fraction to the detergent insoluble fraction after ligand treatment, suggests that this protein might be involved in linking SIg to the underlying cytoskeleton and could be involved in the transmission of a mitogenic signal.

  3. Proteomic analysis of differentially expressed proteins involved in ethylene-induced chilling tolerance in harvested banana fruit.

    PubMed

    Li, Taotao; Yun, Ze; Zhang, Dandan; Yang, Chengwei; Zhu, Hong; Jiang, Yueming; Duan, Xuewu

    2015-01-01

    To better understand the mechanism involved in ethylene-induced chilling tolerance in harvested banana fruit, a gel-based proteomic study followed by MALDI-TOF-TOF MS was carried out. Banana fruit were treated with 500 ppm ethylene for 12 h and then stored at 6°C. During cold storage, the chilling tolerance was assessed and the proteins from the peel were extracted for proteomic analysis. It was observed that ethylene pretreatment significantly induced the chilling tolerance in harvested banana fruit, manifesting as increases in maximal chlorophyll fluorescence (Fv/Fm) and decreased electrolyte leakage. Sixty-four proteins spots with significant differences in abundance were identified, most of which were induced by ethylene pretreatment during cold storage. The up-regulated proteins induced by ethylene pretreatment were mainly related to energy metabolism, stress response and defense, methionine salvage cycle and protein metabolism. These proteins were involved in ATP synthesis, ROS scavenging, protective compounds synthesis, protein refolding and degradation, and polyamine biosynthesis. It is suggested that these up-regulated proteins might play a role in the ethylene-induced chilling tolerance in harvested banana fruit. PMID:26528309

  4. RNA-protein interactions: involvement of NS3, NS5, and 3' noncoding regions of Japanese encephalitis virus genomic RNA.

    PubMed Central

    Chen, C J; Kuo, M D; Chien, L J; Hsu, S L; Wang, Y M; Lin, J H

    1997-01-01

    The mechanism of replication of the flavivirus Japanese encephalitis virus (JEV) is not well known. The structures at the 3' end of the viral genome are highly conserved among divergent flaviviruses, suggesting that they may function as cis-acting signals for RNA replication and, as such, might specifically bind to cellular or viral proteins. UV cross-linking experiments were performed to identify the proteins that bind with the JEV plus-strand 3' noncoding region (NCR). Two proteins, p71 and p110, from JEV-infected but not from uninfected cell extracts were shown to bind specifically to the plus-strand 3' NCR. The quantities of these binding proteins increased during the course of JEV infection and correlated with the levels of JEV RNA synthesis in cell extracts. UV cross-linking coupled with Western blot and immunoprecipitation analysis showed that the p110 and p71 proteins were JEV NS5 and NS3, respectively, which are proposed as components of the RNA replicase. The putative stem-loop structure present within the plus-strand 3' NCR was required for the binding of these proteins. Furthermore, both proteins could interact with each other and form a protein-protein complex in vivo. These findings suggest that the 3' NCR of JEV genomic RNA may form a replication complex together with NS3 and NS5; this complex may be involved in JEV minus-strand RNA synthesis. PMID:9094618

  5. Proteomic characterization of HIV-modulated membrane receptors, kinases and signaling proteins involved in novel angiogenic pathways

    PubMed Central

    Rasheed, Suraiya; Yan, Jasper S; Hussain, Adil; Lai, Bruce

    2009-01-01

    Background Kaposi's sarcoma (KS), hemangioma, and other angioproliferative diseases are highly prevalent in HIV-infected individuals. While KS is etiologically linked to the human herpesvirus-8 (HHV8) infection, HIV-patients without HHV-8 and those infected with unrelated viruses also develop angiopathies. Further, HIV-Tat can activate protein-tyrosine-kinase (PTK-activity) of the vascular endothelial growth factor receptor involved in stimulating angiogenic processes. However, Tat by itself or HHV8-genes alone cannot induce angiogenesis in vivo unless specific proteins/enzymes are produced synchronously by different cell-types. We therefore tested a hypothesis that chronic HIV-replication in non-endothelial cells may produce novel factors that provoke angiogenic pathways. Methods Genome-wide proteins from HIV-infected and uninfected T-lymphocytes were tested by subtractive proteomics analyses at various stages of virus and cell growth in vitro over a period of two years. Several thousand differentially regulated proteins were identified by mass spectrometry (MS) and >200 proteins were confirmed in multiple gels. Each protein was scrutinized extensively by protein-interaction-pathways, bioinformatics, and statistical analyses. Results By functional categorization, 31 proteins were identified to be associated with various signaling events involved in angiogenesis. 88% proteins were located in the plasma membrane or extracellular matrix and >90% were found to be essential for regeneration, neovascularization and angiogenic processes during embryonic development. Conclusion Chronic HIV-infection of T-cells produces membrane receptor-PTKs, serine-threonine kinases, growth factors, adhesion molecules and many diffusible signaling proteins that have not been previously reported in HIV-infected cells. Each protein has been associated with endothelial cell-growth, morphogenesis, sprouting, microvessel-formation and other biological processes involved in angiogenesis (p

  6. Cybip, a starfish cyclin B-binding protein, is involved in meiotic M-phase exit.

    PubMed

    Offner, Nicolas; Derancourt, Jean; Lozano, Jean Claude; Schatt, Philippe; Picard, André; Peaucellier, Gérard

    2003-01-01

    We designed a screen to identify starfish oocyte proteins able to bind monomeric cyclin B by affinity chromatography on a cyclin B splice variant displaying low affinity for cdc2. We identified a 15kDa protein previously described as a cdk-binding protein [Biochim. Biophys. Acta Mol. Cell Res. 1589 (2002) 219-231]. Cybip is encoded by a single polymorphic gene and the native protein is matured by cleaving a signal peptide. We firmly establish the fact that it is a true cyclin B-binding protein, since the recombinant protein binds recombinant cyclin B in absence of any cdk. Finally, we show that the microinjection of GST-cybip, and of anti-cybip antibody, in maturing starfish oocytes, inhibits H1 kinase and MPF inactivation, and first polar body emission. PMID:12480530

  7. Biochemical localization of a protein involved in Gluconacetobacter hansenii cellulose synthesis

    SciTech Connect

    Iyer, Prashanti R; Catchmark, Jeffrey M; Brown, Nicole Robitaille; Tien, Ming

    2011-02-08

    Using subcellular fractionation and Western blot methods, we have shown that AcsD, one of the proteins encoded by the Acetobacter cellulose synthase (acs) operon, is localized in the periplasmic region of the cell. AcsD protein was heterologously expressed in Escherichia coli and purified using histidine tag affinity methods. The purified protein was used to obtain rabbit polyclonal antibodies. The purity of the subcellular fractions was assessed by marker enzyme assays.

  8. Two regions of mature periplasmic maltose-binding protein of Escherichia coli involved in secretion.

    PubMed

    Duplay, P; Hofnung, M

    1988-10-01

    Six mutations in malE, the structural gene for the periplasmic maltose-binding protein (MBP) from Escherichia coli, prevent growth on maltose as a carbon source, as well as release of the mutant proteins by the cold osmotic-shock procedure. These mutations correspond to insertion of an oligonucleotide linker, concomitant with a deletion. One of the mutations (malE127) affects the N-terminal extension (the signal peptide), whereas the five others lie within the mature protein. As expected, the export of protein MalE127 is blocked at an early stage. This protein is neither processed to maturity nor sensitive to proteinase K in spheroplasts. In contrast, in the five other mutants, the signal peptide is cleaved and the protein is accessible to proteinase K added to spheroplasts. This indicates that the five mutant proteins are, at least in part, exported through the inner membrane. We propose that the corresponding mutations define two regions of the mature protein (between residues 18 and 42 and between residues 280 and 306), which are important for release of the protein from the inner membrane into the periplasm. We discuss the results in terms of possible conformational changes at this late step of export to the periplasm. PMID:3049532

  9. Identification of domains of the Tomato spotted wilt virus NSm protein involved in tubule formation, movement and symptomatology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Deletion and alanine-substitution mutants of the Tomato spotted wilt virus NSm protein were generated to identify domains involved in tubule formation, movement and symptomatology, using a heterologous expression system derived from Tobacco mosaic virus. Two regions of NSm were required for both tub...

  10. Direct interaction between nucleosome assembly protein 1 and the papillomavirus E2 proteins involved in activation of transcription.

    PubMed

    Rehtanz, Manuela; Schmidt, Hanns-Martin; Warthorst, Ursula; Steger, Gertrud

    2004-03-01

    Using a yeast two-hybrid screen, we identified human nucleosome assembly protein 1 (hNAP-1) as a protein interacting with the activation domain of the transcriptional activator encoded by papillomaviruses (PVs), the E2 protein. We show that the interaction between E2 and hNAP-1 is direct and not merely mediated by the transcriptional coactivator p300, which is bound by both proteins. Coexpression of hNAP-1 strongly enhances activation by E2, indicating a functional interaction as well. E2 binds to at least two separate domains within hNAP-1, one within the C terminus and an internal domain. The binding of E2 to hNAP-1 is necessary for cooperativity between the factors. Moreover, the N-terminal 91 amino acids are crucial for the transcriptional activity of hNAP-1, since deletion mutants lacking this N-terminal portion fail to cooperate with E2. We provide evidence that hNAP-1, E2, and p300 can form a ternary complex efficient in the activation of transcription. We also show that p53 directly interacts with hNAP-1, indicating that transcriptional activators in addition to PV E2 interact with hNAP-1. These results suggest that the binding of sequence-specific DNA binding proteins to hNAP-1 may be an important step contributing to the activation of transcription. PMID:14966293

  11. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins Involved in a Posttranscriptional Iron Regulatory Mechanism

    PubMed Central

    Figueroa-Angulo, Elisa E.; Calla-Choque, Jaeson S.; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-01-01

    Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis. PMID:26703754

  12. Involvement of heat shock protein 47 in Schistosoma japonicum-induced hepatic fibrosis in mice.

    PubMed

    Huang, Jia-Quan; Tao, Ran; Li, Lan; Ma, Ke; Xu, Lei; Ai, Guo; Fan, Xiang-Xue; Jiao, Yun-Tao; Ning, Qin

    2014-01-01

    Chronic infection with the blood fluke Schistosoma japonicum is associated with both liver cirrhosis and liver cancer. Previously, heat shock protein 47, a collagen-specific molecular chaperone, was shown to play a critical role in the maturation of procollagen. However, less is known about the role of heat shock protein 47 in S. japonicum-induced hepatic fibrosis. We therefore investigated the expression of heat shock protein 47 in S. japonicum-induced liver fibrosis and attempted to determine whether inhibition of heat shock protein 47 could have beneficial effects on fibrosis in vitro and in vivo. In this study, we found that the expression of heat shock protein 47 was significantly increased in patients with Schistosoma-induced fibrosis, as well as in rodent models. Immunohistochemistry revealed heat shock protein 47-positive cells were found in the periphery of egg granulomas. Administration of heat shock protein 47-targeted short hairpin (sh)RNA remarkably reduced heat shock protein 47 expression and collagen deposition in NIH3T3 cells and liver tissue of S. japonicum-infected mice. Life-table analysis revealed a dose-dependent prolongation of survival rates with the treatment of heat shock protein 47-shRNA in murine fibrosis models. Moreover, serum alanine aminotransferase and aspartate transaminase activity, splenomegaly, spleen weight index and portal hypertension were also measured, which showed improvement with the anti-fibrosis treatment. The fibrosis-related parameters assessed were expressions of Col1a1, Col3a1, TGF-β1, CTGF, IL-13, IL-17, MMP-9, TIMP-1 and PAI-1 in the liver. This study demonstrated that heat shock protein 47-targeted shRNA directly reduced collagen production of mouse liver fibrosis associated with S. japonicum. We conclude that heat shock protein 47 plays an essential role in S. japonicum-induced hepatic fibrosis in mice and may be a potential target for ameliorating the hepatic fibrosis caused by this parasite. PMID:24295791

  13. Morphine-Induced Preconditioning: Involvement of Protein Kinase A and Mitochondrial Permeability Transition Pore

    PubMed Central

    Dorsch, Marianne; Behmenburg, Friederike; Raible, Miriam; Blase, Dominic; Grievink, Hilbert; Hollmann, Markus W.; Heinen, André; Huhn, Ragnar

    2016-01-01

    hearts STAT3 inhibitor Stattic completely abolished morphine-induced preconditioning. Administration of Stattic and mPTP inhibitor cyclosporine A reduced infarct size to 31±6% (Stat+CsA, P<0.05 vs. Con). Cyclosporine A alone reduced infarct size to 26±7% (CsA P<0.05 vs. Con). In cardiomyocytes, PKA activity was increased by morphine. Conclusion Our data suggest that morphine-induced cardioprotection is mediated by STAT3-activation and inhibition of mPTP, with STA3 located upstream of mPTP. There is some evidence that protein kinase A is involved within the signalling pathway. PMID:26968004

  14. Identification of Proteins Possibly Involved in Glucosinolate Metabolism in L. agilis R16 and E. coli VL8.

    PubMed

    Luang-In, Vijitra; Narbad, Arjan; Cebeci, Fatma; Bennett, Mark; Rossiter, John T

    2015-04-01

    This study was aimed to identify sinigrin-induced bacterial proteins potentially involved in the metabolism of glucosinolate in two glucosinolate-metabolising bacteria Lactobacillus agilis R16 and Escherichia coli VL8. Sinigrin (2 mM) was used to induce the proteins in both bacteria under anaerobic incubation for 8 h at 30 °C for L. agilis R16 and 37 °C for E. coli VL8 and the controls without sinigrin were performed. Allyl isothiocyanate and allyl nitrile as two degradation products of sinigrin were detected in sinigrin-induced cultures of L. agilis R16 (27% total products) and E. coli VL8 (38% total products) from a complete sinigrin degradation in 8 h for both bacteria. 2D gel electrophoresis was conducted to identify induced proteins with at least twofold increased abundance. Sinigrin-induced L. agilis R16 and the control produced 1561 and 1543 protein spots, respectively. For E. coli VL8, 1363 spots were detected in sinigrin-induced and 1354 spots in the control. A combination of distinct proteins and upregulated proteins of 32 and 35 spots in L. agilis R16 and E. coli VL8, respectively were detected upon sinigrin induction. Of these, 12 and 16 spots from each bacterium respectively were identified by LC-MS/MS. In both bacteria most of the identified proteins are involved in carbohydrate metabolism, oxidoreduction system and sugar transport while the minority belong to purine metabolism, hydrolysis, and proteolysis. This indicated that sinigrin induction led to the expressions of proteins with similar functions in both bacteria and these proteins may play a role in bacterial glucosinolate metabolism. PMID:25805049

  15. Systematic Phenotypic Screen of Arabidopsis Peroxisomal Mutants Identifies Proteins Involved in β-Oxidation1[W][OPEN

    PubMed Central

    Cassin-Ross, Gaëlle; Hu, Jianping

    2014-01-01

    Peroxisomes are highly dynamic and multifunctional organelles essential to development. Plant peroxisomes accommodate a multitude of metabolic reactions, many of which are related to the β-oxidation of fatty acids or fatty acid-related metabolites. Recently, several dozens of novel peroxisomal proteins have been identified from Arabidopsis (Arabidopsis thaliana) through in silico and experimental proteomic analyses followed by in vivo protein targeting validations. To determine the functions of these proteins, we interrogated their transfer DNA insertion mutants with a series of physiological, cytological, and biochemical assays to reveal peroxisomal deficiencies. Sugar dependence and 2,4-dichlorophenoxybutyric acid and 12-oxo-phytodienoic acid response assays uncovered statistically significant phenotypes in β-oxidation-related processes in mutants for 20 of 27 genes tested. Additional investigations uncovered a subset of these mutants with abnormal seed germination, accumulation of oil bodies, and delayed degradation of long-chain fatty acids during early seedling development. Mutants for seven genes exhibited deficiencies in multiple assays, strongly suggesting the involvement of their gene products in peroxisomal β-oxidation and initial seedling growth. Proteins identified included isoforms of enzymes related to β-oxidation, such as acyl-CoA thioesterase2, acyl-activating enzyme isoform1, and acyl-activating enzyme isoform5, and proteins with functions previously unknown to be associated with β-oxidation, such as Indigoidine synthase A, Senescence-associated protein/B12D-related protein1, Betaine aldehyde dehydrogenase, and Unknown protein5. This multipronged phenotypic screen allowed us to reveal β-oxidation proteins that have not been discovered by single assay-based mutant screens and enabled the functional dissection of different isoforms of multigene families involved in β-oxidation. PMID:25253886

  16. Leucine alleviates dexamethasone-induced suppression of muscle protein synthesis via synergy involvement of mTOR and AMPK pathways.

    PubMed

    Wang, Xiao J; Yang, Xin; Wang, Ru X; Jiao, Hong C; Zhao, Jing P; Song, Zhi G; Lin, Hai

    2016-07-01

    Glucocorticoids (GCs) are negative muscle protein regulators that contribute to the whole-body catabolic state during stress. Mammalian target of rapamycin (mTOR)-signalling pathway, which acts as a central regulator of protein metabolism, can be activated by branched-chain amino acids (BCAA). In the present study, the effect of leucine on the suppression of protein synthesis induced by GCs and the pathway involved were investigated. In vitro experiments were conducted using cultured C2C12 myoblasts to study the effect of GCs on protein synthesis, and the involvement of mTOR pathway was investigated as well. After exposure to dexamethasone (DEX, 100 μmol/l) for 24 h, protein synthesis in muscle cells was significantly suppressed (P<0.05), the phosphorylations of mTOR, ribosomal protein S6 protein kinase 1 (p70s6k1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) were significantly reduced (P<0.05). Leucine supplementation (5 mmol/l, 10 mmol/l and 15 mmol/l) for 1 h alleviated the suppression of protein synthesis induced by DEX (P<0.05) and was accompanied with the increased phosphorylation of mTOR and decreased phosphorylation of AMPK (P<0.05). Branched-chain amino transferase 2 (BCAT2) mRNA level was not influenced by DEX (P>0.05) but was increased by leucine supplementation at a dose of 5 mmol/l (P<0.05). PMID:27129299

  17. Involvement of the "A" isozyme of methyltransferase II and the 29-kilodalton corrinoid protein in methanogenesis from monomethylamine.

    PubMed Central

    Burke, S A; Krzycki, J A

    1995-01-01

    An assay which allowed detection of proteins involved in the trimethylamine- or monomethylamine (MMA)-dependent methylation of coenzyme M (CoM) was developed. The two activities could be separated by anion-exchange chromatography. The unresolved activity responsible for MMA:CoM methyl transfer eluted from a gel permeation column in the molecular mass range of 32 kDa. The activity was purified to two monomeric proteins of 40 and 29 kDa. The preparation contained protein-bound corrinoid in a mixture of Co(II) and Co(III) states, as well as methyl-B12:CoM methyltransferase (MT2) activity. N-terminal sequence analysis demonstrated that the polypeptides were two previously identified proteins of undefined physiological function. The smaller polypeptide was the monomeric 29-kDa corrinoid protein. The larger polypeptide was the "A" isozyme of MT2. Individually purified preparations of both proteins increased the rate of MMA-dependent CoM methylation by approximately 1.7 mumol/min/mg of purified protein above background activity in the extract of methanol-grown cells. These results indicate that the 29-kDa corrinoid protein and the "A" isozyme of MT2 function in methanogenesis from MMA. A likely mechanism is that the 29-kDa corrinoid is methylated by MMA and the methyl group is then transferred by the "A" isozyme of MT2 to CoM. PMID:7635826

  18. Identification of candidate effector proteins potentially involved in Fusarium graminearum-wheat interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pathogen-derived small secreted cysteine-rich proteins (SSCPs) are known to be a common source of fungal effectors that trigger resistance or susceptibility in specific host plants. This group of proteins has not been well studied in Fusarium graminearum, the primary cause of Fusarium head blight (...

  19. Classical Swine Fever Virus p7 protein is a viroporin involved in virulence in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The non-structural protein p7 of Classical Swine Fever Virus (CSFV) is a hydrophobic polypeptide with an apparent molecular mass of 7 kDa. The protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that are predicted to form transmembrane helices and a cytos...

  20. Quantitative Proteomic Analysis of Differentially Expressed Protein Profiles Involved in Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Kuo, Kung-Kai; Kuo, Chao-Jen; Chiu, Chiang-Yen; Liang, Shih-Shin; Huang, Chun-Hao; Chi, Shu-Wen; Tsai, Kun-Bow; Chen, Chiao-Yun; Hsi, Edward; Cheng, Kuang-Hung; Chiou, Shyh-Horng

    2016-01-01

    Objectives The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. Methods Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. Results Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ≥2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include α-enolase, α-catenin, 14-3-3 β, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. Conclusions The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC. PMID:26262590

  1. The role of residue stability in transient protein-protein interactions involved in enzymatic phosphate hydrolysis. A computational study.

    PubMed

    Bonet, Jaume; Caltabiano, Gianluigi; Khan, Abdul Kareem; Johnston, Michael A; Corbí, Carles; Gómez, Alex; Rovira, Xavier; Teyra, Joan; Villà-Freixa, Jordi

    2006-04-01

    Finding why protein-protein interactions (PPIs) are so specific can provide a valuable tool in a variety of fields. Statistical surveys of so-called transient complexes (like those relevant for signal transduction mechanisms) have shown a tendency of polar residues to participate in the interaction region. Following this scheme, residues in the unbound partners have to compete between interacting with water or interacting with other residues of the protein. On the other hand, several works have shown that the notion of active site electrostatic preorganization can be used to interpret the high efficiency in enzyme reactions. This preorganization can be related to the instability of the residues important for catalysis. In some enzymes, in addition, conformational changes upon binding to other proteins lead to an increase in the activity of the enzymatic partner. In this article the linear response approximation version of the semimacroscopic protein dipoles Langevin dipoles (PDLD/S-LRA) model is used to evaluate the stability of several residues in two phosphate hydrolysis enzymes upon complexation with their activating partners. In particular, the residues relevant for PPI and for phosphate hydrolysis in the CDK2/Cyclin A and Ras/GAP complexes are analyzed. We find that the evaluation of the stability of residues in these systems can be used to identify not only active site regions but it can also be used as a guide to locate "hot spots" for PPIs. We also show that conformational changes play a major role in positioning interfacing residues in a proper "energetic" orientation, ready to interact with the residues in the partner protein surface. Thus, we extend the preorganization theory to PPIs, extrapolating the results we obtained from the above-mentioned complexes to a more general case. We conclude that the correlation between stability of a residue in the surface and the likelihood that it participates in the interaction can be a general fact for transient

  2. Mapping the H(+) (V)-ATPase interactome: identification of proteins involved in trafficking, folding, assembly and phosphorylation.

    PubMed

    Merkulova, Maria; Păunescu, Teodor G; Azroyan, Anie; Marshansky, Vladimir; Breton, Sylvie; Brown, Dennis

    2015-01-01

    V-ATPases (H(+) ATPases) are multisubunit, ATP-dependent proton pumps that regulate pH homeostasis in virtually all eukaryotes. They are involved in key cell biological processes including vesicle trafficking, endosomal pH sensing, membrane fusion and intracellular signaling. They also have critical systemic roles in renal acid excretion and blood pH balance, male fertility, bone remodeling, synaptic transmission, olfaction and hearing. Furthermore, V-ATPase dysfunction either results in or aggravates various other diseases, but little is known about the complex protein interactions that regulate these varied V-ATPase functions. Therefore, we performed a proteomic analysis to identify V-ATPase associated proteins and construct a V-ATPase interactome. Our analysis using kidney tissue revealed V-ATPase-associated protein clusters involved in protein quality control, complex assembly and intracellular trafficking. ARHGEF7, DMXL1, EZR, NCOA7, OXR1, RPS6KA3, SNX27 and 9 subunits of the chaperonin containing TCP1 complex (CCT) were found to interact with V-ATPase for the first time in this study. Knockdown of two interacting proteins, DMXL1 and WDR7, inhibited V-ATPase-mediated intracellular vesicle acidification in a kidney cell line, providing validation for the utility of our interactome as a screen for functionally important novel V-ATPase-regulating proteins. Our data, therefore, provide new insights and directions for the analysis of V-ATPase cell biology and (patho)physiology. PMID:26442671

  3. Mapping the H+ (V)-ATPase interactome: identification of proteins involved in trafficking, folding, assembly and phosphorylation

    PubMed Central

    Merkulova, Maria; Păunescu, Teodor G.; Azroyan, Anie; Marshansky, Vladimir; Breton, Sylvie; Brown, Dennis

    2015-01-01

    V-ATPases (H+ ATPases) are multisubunit, ATP-dependent proton pumps that regulate pH homeostasis in virtually all eukaryotes. They are involved in key cell biological processes including vesicle trafficking, endosomal pH sensing, membrane fusion and intracellular signaling. They also have critical systemic roles in renal acid excretion and blood pH balance, male fertility, bone remodeling, synaptic transmission, olfaction and hearing. Furthermore, V-ATPase dysfunction either results in or aggravates various other diseases, but little is known about the complex protein interactions that regulate these varied V-ATPase functions. Therefore, we performed a proteomic analysis to identify V-ATPase associated proteins and construct a V-ATPase interactome. Our analysis using kidney tissue revealed V-ATPase-associated protein clusters involved in protein quality control, complex assembly and intracellular trafficking. ARHGEF7, DMXL1, EZR, NCOA7, OXR1, RPS6KA3, SNX27 and 9 subunits of the chaperonin containing TCP1 complex (CCT) were found to interact with V-ATPase for the first time in this study. Knockdown of two interacting proteins, DMXL1 and WDR7, inhibited V-ATPase-mediated intracellular vesicle acidification in a kidney cell line, providing validation for the utility of our interactome as a screen for functionally important novel V-ATPase-regulating proteins. Our data, therefore, provide new insights and directions for the analysis of V-ATPase cell biology and (patho)physiology. PMID:26442671

  4. Cox17 Protein Is an Auxiliary Factor Involved in the Control of the Mitochondrial Contact Site and Cristae Organizing System*

    PubMed Central

    Chojnacka, Magdalena; Gornicka, Agnieszka; Oeljeklaus, Silke; Warscheid, Bettina; Chacinska, Agnieszka

    2015-01-01

    The mitochondrial contact site and cristae organizing system (MICOS) is a recently discovered protein complex that is crucial for establishing and maintaining the proper inner membrane architecture and contacts with the outer membrane of mitochondria. The ways in which the MICOS complex is assembled and its integrity is regulated remain elusive. Here, we report a direct link between Cox17, a protein involved in the assembly of cytochrome c oxidase, and the MICOS complex. Cox17 interacts with Mic60, thereby modulating MICOS complex integrity. This interaction does not involve Sco1, a partner of Cox17 in transferring copper ions to cytochrome c oxidase. However, the Cox17-MICOS interaction is regulated by copper ions. We propose that Cox17 is a newly identified factor involved in maintaining the architecture of the MICOS complex. PMID:25918166

  5. Evolution of an ancient protein function involved in organized multicellularity in animals.

    PubMed

    Anderson, Douglas P; Whitney, Dustin S; Hanson-Smith, Victor; Woznica, Arielle; Campodonico-Burnett, William; Volkman, Brian F; King, Nicole; Thornton, Joseph W; Prehoda, Kenneth E

    2016-01-01

    To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which - the evolution of GKPID's capacity to bind the cortical marker protein - can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals. PMID:26740169

  6. STP1, a gene involved in pre-tRNA processing, encodes a nuclear protein containing zinc finger motifs.

    PubMed Central

    Wang, S S; Stanford, D R; Silvers, C D; Hopper, A K

    1992-01-01

    STP1 is an unessential yeast gene involved in the removal of intervening sequences from some, but not all, families of intervening sequence-containing pre-tRNAs. Previously, we proposed that STP1 might encode a product that generates pre-tRNA conformations efficiently recognized by tRNA-splicing endonuclease. To test the predictions of this model, we have undertaken a molecular analysis of the STP1 gene and its products. The STP1 locus is located on chromosome IV close to at least two other genes involved in RNA splicing: PRP3 and SPP41. The STP1 open reading frame (ORF) could encode a peptide of 64,827 Da; however, inspection of putative transcriptional and translational regulatory signals and mapping of the 5' ends of mRNA provide evidence that translation of the STP1 ORF usually initiates at a second AUG to generate a protein of 58,081 Da. The STP1 ORF contains three putative zinc fingers. The first of these closely resembles both the DNA transcription factor consensus and the Xenopus laevis p43 RNA-binding protein consensus. The third motif more closely resembles the fingers found in spliceosomal proteins. Employing antisera to the endogenous STP1 protein and to STP1-LacZ fusion proteins, we show that the STP1 protein is localized to nuclei. The presence of zinc finger motifs and the nuclear location of the STP1 protein support the model that this gene product is involved directly in pre-tRNA splicing. Images PMID:1588961

  7. Surface proteins of C6/36 cells involved in dengue virus 4 binding and entry.

    PubMed

    Vega-Almeida, Tania Olivia; Salas-Benito, Mariana; De Nova-Ocampo, Mónica Ascensión; Del Angel, Rosa María; Salas-Benito, Juan Santiago

    2013-06-01

    Dengue virus (DENV) is the causative agent of the most important mosquito-borne viral disease, which is endemic to over 100 countries in tropical and subtropical areas of the world. It is transmitted to humans by Aedes mosquitoes. The first step in the viral infection of host cells is virion attachment to the plasma membrane, which is mediated by specific surface molecules. There are several molecules that participate in DENV infection of mosquitoes, but only a few have been identified. In this work, we co-purified 4 proteins from C6/36 cells using a recombinant DENV 4 E protein and identified them as 70 kDa Heat Shock and 70 kDa Heat Shock cognate proteins (HSP70/HSc70), Binding immunoglobulin protein (BiP), Thioredoxin/protein disulphide isomerase (PDI), and 44 kDa Endoplasmic reticulum resident protein (ERp44) via matrix-assisted laser desorption/ionisation time of flight (Maldi-ToF) analysis. Using immunofluorescence and flow cytometry assays, we observed re-localisation of HSP70/HSc70 and, to a lesser extent, BiP to the plasma membrane under stress conditions, such as during DENV infection. By performing binding and infection assays independently, we found that all 4 proteins participate in both processes, but to differing extents: HSP70/HSc70 is the most critical component, while ERp44 is less important. Viral infection was not inhibited when the cells were incubated with antibodies against all of the surface proteins after virus binding, which suggests that DENV entry to C6/36 cells is mediated by these proteins at the same step and not sequentially. PMID:23344777

  8. Identification of Vibrio harveyi proteins involved in the specific immune response of Senegalese sole (Solea senegalensis, Kaup).

    PubMed

    Medina, A; Mancera, J M; Martínez-Manzanares, E; Moriñigo, M A; Arijo, S

    2015-11-01

    Senegalese sole cultures are frequently affected by Vibrio harveyi disease outbreaks. Vaccines in aquaculture are one of the most successful methods of preventing fish pathologies; however, these vaccines are usually composed of inactivated whole cells containing a wide pool of antigens, and some do not induce any protection against pathogens. Thus, the aim of this study was to identify immunogenic proteins of V. harveyi involved in the specific antibody production by Senegalese sole. S. senegalensis specimens were immunized, by intraperitoneal injection, with V. harveyi bacterin supplemented with inactivated extracellular polymeric substances (ECP) and Freund incomplete adjuvant to obtain polyclonal antiserum. One month later, specimens were re-inoculated with the same antigens. Sera from immunized fish were collected two months post first immunization. Strong specific immune response to V. harveyi antigens was detected by ELISA using bacterin (limit dilutions of sera were 1:64000), ECP (1:4000) and outer membrane proteins (OMP) (1:4000) as antigens. Presence of immunogenic proteins in V. harveyi ECP and OMP were determined by 2D-PAGE. For Western Blot analysis some gels were transferred onto nitrocellulose membranes and incubated with sera from S. senegalensis specimens immunized against V. harveyi. 2D-PAGE and Western Blot showed at least five reactive proteins in the ECP and two in the OMP fraction. The spots that clearly reacted with the sole antiserum were excised from stained gel, and analyzed by mass spectrometry (MALDI/TOFTOF). A database search was then performed, using MASCOT as the search method. According to the results, the five ECP spots were identified as Maltoporine, protein homologous to Metal dependent phosphohydrolase, two porins isoforms of V. harveyi and a protein homologous to the cell division protein FtsH. Reactive proteins in the OMP fraction were identified as the protein 3-hydroxyisobutyrate dehydrogenase and a protein homologous to acid

  9. The Shwachman-Bodian-Diamond Syndrome Protein Family Is Involved in RNA Metabolism

    SciTech Connect

    Savchenko, A; Krogan, Nevan; Cort, John R.; Evdokimova, Elena; Lew, Jocelyne M.; Yee, Adelinda; Sanchez-Pulido, Luis; Andrade, Miguel; Bochkarev, Alexey; Watson, James D.; Kennedy, Michael A.; Greenblatt, Jack; Hughes, Timothy; Arrowsmith, Cheryl H.; Rommens, Johanna M.; Edwards, Aled M.

    2005-05-13

    A combination of structural, biochemical, and genetic studies in model organisms was used to infer a cellular role for the human protein (SBDS) responsible for Shwachman-Bodian-Diamond syndrome. The crystal structure of the SBDS homologue in Archaeoglobus fulgidus, AF0491, revealed a three domain protein. The N-terminal domain, which harbors the majority of disease-linked mutations, has a novel three-dimensional fold.

  10. Structural Insights into the MMACHC-MMADHC Protein Complex Involved in Vitamin B12 Trafficking.

    PubMed

    Froese, D Sean; Kopec, Jolanta; Fitzpatrick, Fiona; Schuller, Marion; McCorvie, Thomas J; Chalk, Rod; Plessl, Tanja; Fettelschoss, Victoria; Fowler, Brian; Baumgartner, Matthias R; Yue, Wyatt W

    2015-12-01

    Conversion of vitamin B12 (cobalamin, Cbl) into the cofactor forms methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl) is required for the function of two crucial enzymes, mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase, respectively. The intracellular proteins MMACHC and MMADHC play important roles in processing and targeting the Cbl cofactor to its destination enzymes, and recent evidence suggests that they may interact while performing these essential trafficking functions. To better understand the molecular basis of this interaction, we have mapped the crucial protein regions required, indicate that Cbl is likely processed by MMACHC prior to interaction with MMADHC, and identify patient mutations on both proteins that interfere with complex formation, via different mechanisms. We further report the crystal structure of the MMADHC C-terminal region at 2.2 Å resolution, revealing a modified nitroreductase fold with surprising homology to MMACHC despite their poor sequence conservation. Because MMADHC demonstrates no known enzymatic activity, we propose it as the first protein known to repurpose the nitroreductase fold solely for protein-protein interaction. Using small angle x-ray scattering, we reveal the MMACHC-MMADHC complex as a 1:1 heterodimer and provide a structural model of this interaction, where the interaction region overlaps with the MMACHC-Cbl binding site. Together, our findings provide novel structural evidence and mechanistic insight into an essential biological process, whereby an intracellular "trafficking chaperone" highly specific for a trace element cofactor functions via protein-protein interaction, which is disrupted by inherited disease mutations. PMID:26483544