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Sample records for b-box protein involved

  1. LZF1/SALT TOLERANCE HOMOLOG3, an Arabidopsis B-Box Protein Involved in Light-Dependent Development and Gene Expression, Undergoes COP1-Mediated Ubiquitination[W

    PubMed Central

    Datta, Sourav; Johansson, Henrik; Hettiarachchi, Chamari; Irigoyen, María Luisa; Desai, Mintu; Rubio, Vicente; Holm, Magnus

    2008-01-01

    B-box containing proteins play an important role in light signaling in plants. Here, we identify LIGHT-REGULATED ZINC FINGER1/SALT TOLERANCE HOMOLOG3 (STH3), a B-box encoding gene that genetically interacts with two key regulators of light signaling, ELONGATED HYPOCOTYL5 (HY5) and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). STH3 physically interacts with HY5 in vivo and shows a COP1-dependent localization to nuclear speckles when coexpressed with COP1 in plant cells. A T-DNA insertion mutant, sth3, is hyposensitive to high fluence blue, red, and far-red light and has elongated hypocotyls under short days. Analyses of double mutants between sth3, sth2, and hy5 suggest that they have partially overlapping functions. Interestingly, functional assays in protoplasts suggest that STH3 can activate transcription both independently and together with STH2 through the G-box promoter element. Furthermore, sth3 suppresses the cop1 hypocotyl phenotype in the dark as well as the anthocyanin accumulation in the light. Finally, COP1 ubiquitinates STH3 in vitro, suggesting that STH3 is regulated by COP1. In conclusion, we have identified STH3 as a positive regulator of photomorphogenesis acting in concert with STH2 and HY5, while also being a target of COP1-mediated ubiquitination. PMID:18796637

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

    PubMed Central

    2016-01-01

    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

  3. Function of B-BOX under shade.

    PubMed

    Crocco, Carlos D; Holm, Magnus; Yanovsky, Marcelo J; Botto, Javier F

    2011-01-01

    Plants are capable of perceiving changes in the light environment and finely adjust their growth and development. Reductions of red to far-red ratio (R:FR) generated by an increase of the plant canopy above the plant are sensed by the phytochrome system triggering the shade-avoidance syndrome (SAS) that includes elongation of vegetative structures, reduction of branching and acceleration of flowering. Albeit the SAS is a strategy of major adaptative significance in plant communities, involving massive changes in gene expression, our knowledge of the SAS signaling network is still fragmented. By a selection and characterization of a T-DNA mutant with a long hypocotyl under shade, we identified BBX21, a protein with two B-box domains involved in the SAS. BBX21 belongs to a small eight member family of B-box containing proteins with both opposite and additive functions in the SAS signaling. BBX21 down-regulates the gene expression of auxin, brassinosteroid and ethylene signaling pathway components under shade. Furthermore BBX21 is a transcription factor that interacts genetically with COP1. We propose a model in which a dynamic balance of positive and negative B-box transcriptional regulators acts as a gas-and-brake mechanism into the COP1 signaling to regulate the expression of SAS.

  4. BZS1, a B-box Protein, Promotes Photomorphogenesis Downstream of Both Brassinosteroid and Light Signaling Pathways

    PubMed Central

    Fan, Xi-Ying; Sun, Yu; Cao, Dong-Mei; Bai, Ming-Yi; Luo, Xiao-Min; Yang, Hong-Juan; Wei, Chuang-Qi; Zhu, Sheng-Wei; Sun, Ying; Wang, Zhi-Yong

    2012-01-01

    Photomorphogenesis is controlled by multiple signaling pathways, including the light and brassinosteroid (BR) pathways. BR signaling activates the BZR1 transcription factor, which is required for suppressing photomorphogenesis in the dark. We identified a suppressor of the BR hypersensitive mutant bzr1–1D and named it bzr1–1D suppressor1-Dominant (bzs1–D). The bzs1–D mutation was caused by overexpression of a B-box zinc finger protein BZS1, which is transcriptionally repressed by BZR1. Overexpression of BZS1 causes de-etiolation in the dark, short hypocotyls in the light, reduced sensitivity to BR treatment, and repression of many BR-activated genes. Knockdown of BZS1 by co-suppression partly suppressed the short hypocotyl phenotypes of BR-deficient or insensitive mutants. These results support that BZS1 is a negative regulator of BR response. BZS1 overexpressors are hypersensitive to different wavelengths of light and loss of function of BZS1 reduces plant sensitivity to light and partly suppresses the constitutive photomorphogenesis 1 (cop1) mutant in the dark, suggesting a positive role in light response. BZS1 protein accumulates at an increased level after light treatment of dark-grown BZS1–OX plants and in the cop1 mutants, and BZS1 interacts with COP1 in vitro, suggesting that light regulates BZS1 through COP1-mediated ubiquitination and proteasomal degradation. These results demonstrate that BZS1 mediates the crosstalk between BR and light pathways. PMID:22535582

  5. Genome-wide survey of B-box proteins in potato (Solanum tuberosum)-Identification, characterization and expression patterns during diurnal cycle, etiolation and de-etiolation.

    PubMed

    Talar, Urszula; Kiełbowicz-Matuk, Agnieszka; Czarnecka, Jagoda; Rorat, Tadeusz

    2017-01-01

    Plant B-box domain proteins (BBX) mediate many light-influenced developmental processes including seedling photomorphogenesis, seed germination, shade avoidance and photoperiodic regulation of flowering. Despite the wide range of potential functions, the current knowledge regarding BBX proteins in major crop plants is scarce. In this study, we identify and characterize the StBBX gene family in potato, which is composed of 30 members, with regard to structural properties and expression profiles under diurnal cycle, etiolation and de-etiolations. Based on domain organization and phylogenetic relationships, StBBX genes have been classified into five groups. Using real-time quantitative PCR, we found that expression of most of them oscillates following a 24-h rhythm; however, large differences in expression profiles were observed between the genes regarding amplitude and position of the maximal and minimal expression levels in the day/night cycle. On the basis of the time-of-day/time-of-night, we distinguished three expression groups specifically expressed during the light and two during the dark phase. In addition, we showed that the expression of several StBBX genes is under the control of the circadian clock and that some others are specifically associated with the etiolation and de-etiolation conditions. Thus, we concluded that StBBX proteins are likely key players involved in the complex diurnal and circadian networks regulating plant development as a function of light conditions and day duration.

  6. SALT TOLERANCE HOMOLOG2, a B-box protein in Arabidopsis that activates transcription and positively regulates light-mediated development.

    PubMed

    Datta, Sourav; Hettiarachchi, Chamari; Johansson, Henrik; Holm, Magnus

    2007-10-01

    CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and ELONGATED HYPOCOTYL5 (HY5) are two major regulators of light signaling in plants. Here, we identify SALT TOLERANCE HOMOLOG2 (STH2) as a gene that interacts genetically with both of these key regulators. STH2 encodes a B-box-containing protein that interacts physically with HY5 in yeast and in plant cells. Whereas STH2 is uniformly nuclear by itself, it shows a COP1-dependent localization to speckles when coexpressed with COP1. We identified two independent T-DNA insertion lines in STH2. Both alleles are hyposensitive to blue, red, and far-red light. The sth2 mutant, like hy5, shows an enhanced number of lateral roots and accumulates less anthocyanin. Analysis of double mutants between sth2 and hy5 indicates that STH2 has both HY5-dependent and -independent functions. Furthermore, besides partially suppressing the hypocotyl phenotype of dark-grown cop1 alleles, sth2 also suppresses the reduced number of lateral roots and high anthocyanin levels in light-grown cop1 alleles. Interestingly, we found that STH2 can activate transcription. Transient transfection assays in protoplasts using a LUC reporter driven by the chalcone isomerase promoter show that the B-boxes in STH2 and a functional G-box element in the promoter are required for this activity. In conclusion, we have identified STH2, a B-box protein in Arabidopsis thaliana, as a positive regulator of photomorphogenesis and report that the B-box domain plays a direct role in activating transcription in plants.

  7. 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

    2016-06-13

    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.

  8. 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

  9. BBX32, an Arabidopsis B-Box Protein, Functions in Light Signaling by Suppressing HY5-Regulated Gene Expression and Interacting with STH2/BBX21[W][OA

    PubMed Central

    Holtan, Hans E.; Bandong, Simona; Marion, Colleen M.; Adam, Luc; Tiwari, Shiv; Shen, Yu; Maloof, Julin N.; Maszle, Don R.; Ohto, Masa-aki; Preuss, Sasha; Meister, Rob; Petracek, Marie; Repetti, Peter P.; Reuber, T. Lynne; Ratcliffe, Oliver J.; Khanna, Rajnish

    2011-01-01

    A B-box zinc finger protein, B-BOX32 (BBX32), was identified as playing a role in determining hypocotyl length during a large-scale functional genomics study in Arabidopsis (Arabidopsis thaliana). Further analysis revealed that seedlings overexpressing BBX32 display elongated hypocotyls in red, far-red, and blue light, along with reduced cotyledon expansion in red light. Through comparative analysis of mutant and overexpression line phenotypes, including global expression profiling and growth curve studies, we demonstrate that BBX32 acts antagonistically to ELONGATED HYPOCOTYL5 (HY5). We further show that BBX32 interacts with SALT TOLERANCE HOMOLOG2/BBX21, another B-box protein previously shown to interact with HY5. Based on these data, we propose that BBX32 functions downstream of multiple photoreceptors as a modulator of light responses. As such, BBX32 potentially has a native role in mediating gene repression to maintain dark adaptation. PMID:21632973

  10. The mei-P26 Gene Encodes a RING Finger B-box Coiled-Coil-NHL Protein That Regulates Seizure Susceptibility in Drosophilia

    PubMed Central

    Glasscock, Edward; Singhania, Ayush; Tanouye, Mark A.

    2005-01-01

    Seizure-suppressor mutations provide unique insight into the genes and mechanisms involved in regulating nervous system excitability. Drosophila bang-sensitive (BS) mutants present a useful tool for identifying seizure suppressors since they are a well-characterized epilepsy model. Here we describe the isolation and characterization of a new Drosophila seizure-suppressor mutant that results from disruption of the meiotic gene mei-P26, which belongs to the RBCC-NHL family of proteins. The mei-P26 mutation reduces seizures in easily shocked (eas) and slamdance (sda) epileptic flies following mechanical stimulation and electroconvulsive shock. In addition, mutant mei-P26 flies exhibit seizure thresholds at least threefold greater than those of wild type. The mei-P26 phenotypes appear to result from missense mutation of a critical residue in the NHL protein-protein interaction domain of the protein. These results reveal a surprising role for mei-P26 outside of the germline as a regulator of seizure susceptibility, possibly by affecting synaptic development as a ubiquitin ligase. PMID:15937125

  11. The mei-P26 gene encodes a RING finger B-box coiled-coil-NHL protein that regulates seizure susceptibility in Drosophilia.

    PubMed

    Glasscock, Edward; Singhania, Ayush; Tanouye, Mark A

    2005-08-01

    Seizure-suppressor mutations provide unique insight into the genes and mechanisms involved in regulating nervous system excitability. Drosophila bang-sensitive (BS) mutants present a useful tool for identifying seizure suppressors since they are a well-characterized epilepsy model. Here we describe the isolation and characterization of a new Drosophila seizure-suppressor mutant that results from disruption of the meiotic gene mei-P26, which belongs to the RBCC-NHL family of proteins. The mei-P26 mutation reduces seizures in easily shocked (eas) and slamdance (sda) epileptic flies following mechanical stimulation and electroconvulsive shock. In addition, mutant mei-P26 flies exhibit seizure thresholds at least threefold greater than those of wild type. The mei-P26 phenotypes appear to result from missense mutation of a critical residue in the NHL protein-protein interaction domain of the protein. These results reveal a surprising role for mei-P26 outside of the germline as a regulator of seizure susceptibility, possibly by affecting synaptic development as a ubiquitin ligase.

  12. Interaction between composite elements in the napA promoter: both the B-box ABA-responsive complex and the RY/G complex are necessary for seed-specific expression.

    PubMed

    Ezcurra, I; Ellerström, M; Wycliffe, P; Stålberg, K; Rask, L

    1999-07-01

    During seed maturation, the transcriptional activity of napin genes is regulated by developmental signals involving the transcriptional activator ABI3 and abscisic acid (ABA). To localize cis elements involved in the seed-specific activity of the napin napA promoter, a systematic analysis was performed focusing on two major element complexes, the B-box and RY/G. Substitution mutation analysis using promoter-reporter gene fusions in stable transgenic tobacco showed synergistic interactions between elements within these complexes. The distal part of the B-box shows similarities to abscisic acid response elements and the proximal portion contains a CA-rich element. In vitro studies involving Exonuclease III protection and electrophoretic mobility shift assays revealed binding by nuclear proteins to elements within the B-box. The distal and proximal parts of the B-box were found to bind distinct nuclear protein complexes. By gain-of-function analysis with a tetramer of the B-box fused to a truncated (-46) cauliflower mosaic virus (CaMV) 35S minimal promoter, it was demonstrated that the B-box mediates strong activity in seeds. Further, it was shown that the elements in the B-box constitute an ABA-responsive complex, since the B-box tetramer mediates ABA-responsiveness in vegetative tissues to a construct containing the CaMV virus 35S enhancer (-343 to -90). Thus, the seed-specific activity of the napA promoter relies on the combinatorial interaction between the RY/G complex and the B-box ABA-responsive complex during the ABA response in seed development.

  13. Going Outside the TonB Box: Identification of Novel FepA-TonB Interactions In Vivo.

    PubMed

    Gresock, Michael G; Postle, Kathleen

    2017-05-15

    In Gram-negative bacteria, the cytoplasmic membrane protein TonB transmits energy derived from proton motive force to energize transport of important nutrients through TonB-dependent transporters in the outer membrane. Each transporter consists of a beta barrel domain and a lumen-occluding cork domain containing an essential sequence called the TonB box. To date, the only identified site of transporter-TonB interaction is between the TonB box and residues ∼158 to 162 of TonB. While the mechanism of ligand transport is a mystery, a current model based on site-directed spin labeling and molecular dynamics simulations is that, following ligand binding, the otherwise-sequestered TonB box extends into the periplasm for recognition by TonB, which mediates transport by pulling or twisting the cork. In this study, we tested that hypothesis with the outer membrane transporter FepA using in vivo photo-cross-linking to explore interactions of its TonB box and determine whether additional FepA-TonB interaction sites exist. We found numerous specific sites of FepA interaction with TonB on the periplasmic face of the FepA cork in addition to the TonB box. Two residues, T32 and A33, might constitute a ligand-sensitive conformational switch. The facts that some interactions were enhanced in the absence of ligand and that other interactions did not require the TonB box argued against the current model and suggested that the transport process is more complex than originally conceived, with subtleties that might provide a mechanism for discrimination among ligand-loaded transporters. These results constitute the first study on the dynamics of TonB-gated transporter interaction with TonB in vivoIMPORTANCE The TonB system of Gram-negative bacteria has a noncanonical active transport mechanism involving signal transduction and proteins integral to both membranes. To achieve transport, the cytoplasmic membrane protein TonB physically contacts outer membrane transporters such as Fep

  14. The B-box module of CYLD is responsible for its intermolecular interaction and cytoplasmic localization.

    PubMed

    Xie, Songbo; Chen, Miao; Gao, Siqi; Zhong, Tao; Zhou, Peng; Li, Dengwen; Zhou, Jun; Gao, Jinmin; Liu, Min

    2017-02-07

    The tumor suppressor protein cylindromatosis (CYLD), as a microtubule-associated deubiquitinase, plays a pivotal role in a wide range of cellular activities, including innate immunity, cell division, and ciliogenesis. Structural characterization reveals a small zinc-binding B-box inserted within the ubiquitin specific protease (USP) domain of CYLD; however, the exact role for this module remains yet to be elucidated. Here we identify a critical role for the B-box in facilitating the intermolecular interaction and subcellular localization of CYLD. By co-immunoprecipitation assays we uncover that CYLD has the ability to form an intermolecular complex. Native gel electrophoresis analysis and pull down assays show that the USP domain of CYLD is essential for its intermolecular interaction. Further investigation reveals that deletion of the B-box from the USP domain disrupts the intermolecular interaction of CYLD. Importantly, although loss of the B-box has no obvious effect on the deubiquitinase activity of CYLD, it abolishes the USP domain-mediated retention of CYLD in the cytoplasm. Collectively, these data demonstrate an important role for the B-box module of CYLD in mediating its assembly and subcellular distribution, which might be related to the functions of CYLD in various biological processes.

  15. In vivo inhibition of TonB-dependent processes by a TonB box consensus pentapeptide.

    PubMed

    Tuckman, M; Osburne, M S

    1992-01-01

    The TonB box, a conserved pentapeptide sequence found in TonB-dependent colicins and receptors, is thought to interact physically with the TonB protein to facilitate TonB-dependent processes. Strains of Escherichia coli were treated in vivo with the synthetic TonB box pentapeptide Glu-Thr-Val-Ile-Val. The pentapeptide inhibited several TonB-dependent processes, including cell growth in low-iron medium, phi 80 infection, and killing by colicins B and Ia. Two unrelated control pentapeptides had no effect on TonB-dependent processes.

  16. Conserved DNA Motifs, Including the CENP-B Box-like, Are Possible Promoters of Satellite DNA Array Rearrangements in Nematodes

    PubMed Central

    Car, Ana; Castagnone-Sereno, Philippe; Abad, Pierre; Plohl, Miroslav

    2013-01-01

    Tandemly arrayed non-coding sequences or satellite DNAs (satDNAs) are rapidly evolving segments of eukaryotic genomes, including the centromere, and may raise a genetic barrier that leads to speciation. However, determinants and mechanisms of satDNA sequence dynamics are only partially understood. Sequence analyses of a library of five satDNAs common to the root-knot nematodes Meloidogyne chitwoodi and M. fallax together with a satDNA, which is specific for M. chitwoodi only revealed low sequence identity (32–64%) among them. However, despite sequence differences, two conserved motifs were recovered. One of them turned out to be highly similar to the CENP-B box of human alpha satDNA, identical in 10–12 out of 17 nucleotides. In addition, organization of nematode satDNAs was comparable to that found in alpha satDNA of human and primates, characterized by monomers concurrently arranged in simple and higher-order repeat (HOR) arrays. In contrast to alpha satDNA, phylogenetic clustering of nematode satDNA monomers extracted either from simple or from HOR array indicated frequent shuffling between these two organizational forms. Comparison of homogeneous simple arrays and complex HORs composed of different satDNAs, enabled, for the first time, the identification of conserved motifs as obligatory components of monomer junctions. This observation highlights the role of short motifs in rearrangements, even among highly divergent sequences. Two mechanisms are proposed to be involved in this process, i.e., putative transposition-related cut-and-paste insertions and/or illegitimate recombination. Possibility for involvement of the nematode CENP-B box-like sequence in the transposition-related mechanism and together with previously established similarity of the human CENP-B protein and pogo-like transposases implicate a novel role of the CENP-B box and related sequence motifs in addition to the known function in centromere protein binding. PMID:23826269

  17. Possible Role of MADS AFFECTING FLOWERING 3 and B-BOX DOMAIN PROTEIN 19 in Flowering Time Regulation of Arabidopsis Mutants with Defects in Nonsense-Mediated mRNA Decay

    PubMed Central

    Nasim, Zeeshan; Fahim, Muhammad; Ahn, Ji Hoon

    2017-01-01

    Eukaryotic cells use nonsense-mediated mRNA decay (NMD) to clear aberrant mRNAs from the cell, thus preventing the accumulation of truncated proteins. In Arabidopsis, two UP-Frameshift (UPF) proteins, UPF1 and UPF3, play a critical role in NMD. Although deficiency of UPF1 and UPF3 leads to various developmental defects, little is known about the mechanism underlying the regulation of flowering time by NMD. Here, we showed that the upf1-5 and upf3-1 mutants had a late-flowering phenotype under long-day conditions and the upf1-5 upf3-1 double mutants had an additive effect in delaying flowering time. RNA sequencing of the upf mutants revealed that UPF3 exerted a stronger effect than UPF1 in the UPF-mediated regulation of flowering time. Among genes known to regulate flowering time, FLOWERING LOCUS C (FLC) mRNA levels increased (up to 8-fold) in upf mutants, as confirmed by qPCR. The upf1-5, upf3-1, and upf1-5 upf3-1 mutants responded to vernalization, suggesting a role of FLC in delayed flowering of upf mutants. Consistent with the high FLC transcript levels and delayed flowering in upf mutants, levels of FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) mRNAs were reduced in the upf mutants. However, RNA-seq did not identify an aberrant FLC transcript containing a premature termination codon (PTC), suggesting that FLC is not a direct target in the regulation of flowering time by NMD. Among flowering time regulators that act in an FLC-dependent manner, we found that MAF3, NF-YA2, NF-YA5, and TAF14 showed increased transcript levels in upf mutants. We also found that BBX19 and ATC, which act in an FLC-independent manner, showed increased transcript levels in upf mutants. An aberrant transcript containing a PTC was identified from MAF3 and BBX19 and the levels of the aberrant transcripts increased in upf mutants. Taking these results together, we propose that the late-flowering phenotype of upf mutants is mediated by at least two different

  18. Possible Role of MADS AFFECTING FLOWERING 3 and B-BOX DOMAIN PROTEIN 19 in Flowering Time Regulation of Arabidopsis Mutants with Defects in Nonsense-Mediated mRNA Decay.

    PubMed

    Nasim, Zeeshan; Fahim, Muhammad; Ahn, Ji Hoon

    2017-01-01

    Eukaryotic cells use nonsense-mediated mRNA decay (NMD) to clear aberrant mRNAs from the cell, thus preventing the accumulation of truncated proteins. In Arabidopsis, two UP-Frameshift (UPF) proteins, UPF1 and UPF3, play a critical role in NMD. Although deficiency of UPF1 and UPF3 leads to various developmental defects, little is known about the mechanism underlying the regulation of flowering time by NMD. Here, we showed that the upf1-5 and upf3-1 mutants had a late-flowering phenotype under long-day conditions and the upf1-5 upf3-1 double mutants had an additive effect in delaying flowering time. RNA sequencing of the upf mutants revealed that UPF3 exerted a stronger effect than UPF1 in the UPF-mediated regulation of flowering time. Among genes known to regulate flowering time, FLOWERING LOCUS C (FLC) mRNA levels increased (up to 8-fold) in upf mutants, as confirmed by qPCR. The upf1-5, upf3-1, and upf1-5 upf3-1 mutants responded to vernalization, suggesting a role of FLC in delayed flowering of upf mutants. Consistent with the high FLC transcript levels and delayed flowering in upf mutants, levels of FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) mRNAs were reduced in the upf mutants. However, RNA-seq did not identify an aberrant FLC transcript containing a premature termination codon (PTC), suggesting that FLC is not a direct target in the regulation of flowering time by NMD. Among flowering time regulators that act in an FLC-dependent manner, we found that MAF3, NF-YA2, NF-YA5, and TAF14 showed increased transcript levels in upf mutants. We also found that BBX19 and ATC, which act in an FLC-independent manner, showed increased transcript levels in upf mutants. An aberrant transcript containing a PTC was identified from MAF3 and BBX19 and the levels of the aberrant transcripts increased in upf mutants. Taking these results together, we propose that the late-flowering phenotype of upf mutants is mediated by at least two different

  19. Arabidopsis B-BOX32 interacts with CONSTANS-LIKE3 to regulate flowering.

    PubMed

    Tripathi, Prateek; Carvallo, Marcela; Hamilton, Elizabeth E; Preuss, Sasha; Kay, Steve A

    2017-01-03

    Plants have the ability to respond to seasonal environmental variations by monitoring day length to initiate flowering. The transition from vegetative to the reproductive stage is the critical developmental switch in flowering plants to ensure optimal fitness and/or yield. It has been previously reported that B-BOX32 (BBX32) has the potential to increase grain yield when ectopically expressed in soybean. In the present study, we performed a detailed molecular characterization of the Arabidopsis B-box domain gene BBX32 We showed that the circadian clock in Arabidopsis regulates BBX32 and expressed in the early morning. To understand the molecular mechanism of BBX32 regulation, we performed a large-scale yeast two-hybrid screen and identified CONSTANS-LIKE 3 (COL3)/BBX4 as one of its interacting protein partners. Using different genetic and biochemical assays, we have validated this interaction and shown that COL3 targets FT in the presence of BBX32 to regulate the flowering pathway. Based on these findings, we hypothesized that this BBX32-COL3 module could be an additional regulatory mechanism affecting the reproductive development in Arabidopsis that could be translated to crops for increased agricultural productivity.

  20. Arabidopsis B-BOX32 interacts with CONSTANS-LIKE3 to regulate flowering

    PubMed Central

    Tripathi, Prateek; Carvallo, Marcela; Hamilton, Elizabeth E.; Preuss, Sasha

    2017-01-01

    Plants have the ability to respond to seasonal environmental variations by monitoring day length to initiate flowering. The transition from vegetative to the reproductive stage is the critical developmental switch in flowering plants to ensure optimal fitness and/or yield. It has been previously reported that B-BOX32 (BBX32) has the potential to increase grain yield when ectopically expressed in soybean. In the present study, we performed a detailed molecular characterization of the Arabidopsis B-box domain gene BBX32. We showed that the circadian clock in Arabidopsis regulates BBX32 and expressed in the early morning. To understand the molecular mechanism of BBX32 regulation, we performed a large-scale yeast two-hybrid screen and identified CONSTANS-LIKE 3 (COL3)/BBX4 as one of its interacting protein partners. Using different genetic and biochemical assays, we have validated this interaction and shown that COL3 targets FT in the presence of BBX32 to regulate the flowering pathway. Based on these findings, we hypothesized that this BBX32-COL3 module could be an additional regulatory mechanism affecting the reproductive development in Arabidopsis that could be translated to crops for increased agricultural productivity. PMID:27999181

  1. Mutational spectrum of CENP-B box and α-satellite DNA on chromosome 21 in Down syndrome children.

    PubMed

    Chen, Qian; Tan, Bin; He, Jun-Lin; Liu, Xue-Qing; Chen, Xue-Mei; Gao, Ru-Fei; Zhu, Jing; Wang, Ying-Xiong; Qi, Hong-Bo

    2017-04-01

    The centromere is responsible for the correct inheritance of eukaryotic chromosomes during cell division. Centromere protein B (CENP‑B) and its 17 base pair binding site (CENP‑B box), which appears at regular intervals in centromeric α-satellite DNA (α-satDNA), are important for the assembly of the centromere components. Therefore, it is conceivable that CENP-B box mutations may induce errors in cell division. However, the association between the deoxynucleotide alterations of the CENP‑B box and the extra chromosome 21 (Chr21) present in patients with Down syndrome (DS) remains to be elucidated. The mutational spectrum of the α‑satDNA, including 4 functional CENP‑B boxes in Chr21 from 127 DS and 100 healthy children were analyzed by direct sequencing. The de novo occurrences of mutations within CENP‑B boxes in patients with DS were excluded. The prevalence of 6 novel mutations (g.661delC, g.1035_1036insA, g.1076_1077insC, g.670T>G, g.1239A>T, g.1343T>C) and 3 single nucleotide polymorphisms (g.727C/T, g.863A/C, g.1264C/G) were not significantly different between DS and controls (P>0.05). However, g.525C/G (P=0.01), g.601T/C (P=0.00000002), g.1279A/G (P=0.002), g.1294C/T (P=0.0006) and g.1302 G/T (P=0.004) were significantly associated with the prevalence of DS (P<0.05). The results indicated that CENP‑B boxes are highly conserved in DS patients and may not be responsible for Chr21 nondisjunction events. However, α‑satDNA in Chr21 is variable and deoxynucleotide deletions, mutations and polymorphisms may act as potential molecular diagnostic markers of DS.

  2. Genomic Organization, Phylogenetic and Expression Analysis of the B-BOX Gene Family in Tomato.

    PubMed

    Chu, Zhuannan; Wang, Xin; Li, Ying; Yu, Huiyang; Li, Jinhua; Lu, Yongen; Li, Hanxia; Ouyang, Bo

    2016-01-01

    The B-BOX (BBX) proteins encode a class of zinc-finger transcription factors possessing one or two B-BOX domains and in some cases an additional CCT (CO, CO-like and TOC1) motif, which play important roles in regulating plant growth, development and stress response. Nevertheless, no systematic study of BBX genes has undertaken in tomato (Solanum lycopersicum). Here we present the results of a genome-wide analysis of the 29 BBX genes in this important vegetable species. Their structures, conserved domains, phylogenetic relationships, subcellular localizations, and promoter cis-regulatory elements were analyzed; their tissue expression profiles and expression patterns under various hormones and stress treatments were also investigated in detail. Tomato BBX genes can be divided into five subfamilies, and twelve of them were found to be segmentally duplicated. Real-time quantitative PCR analysis showed that most BBX genes exhibited different temporal and spatial expression patterns. The expression of most BBX genes can be induced by drought, polyethylene glycol-6000 or heat stress. Some BBX genes were induced strongly by phytohormones such as abscisic acid, gibberellic acid, or ethephon. The majority of tomato BBX proteins was predicted to be located in nuclei, and the transient expression assay using Arabidopsis mesophyll protoplasts demonstrated that all the seven BBX members tested (SlBBX5, 7, 15, 17, 20, 22, and 24) were localized in nucleus. Our analysis of tomato BBX genes on the genome scale would provide valuable information for future functional characterization of specific genes in this family.

  3. Genomic Organization, Phylogenetic and Expression Analysis of the B-BOX Gene Family in Tomato

    PubMed Central

    Chu, Zhuannan; Wang, Xin; Li, Ying; Yu, Huiyang; Li, Jinhua; Lu, Yongen; Li, Hanxia; Ouyang, Bo

    2016-01-01

    The B-BOX (BBX) proteins encode a class of zinc-finger transcription factors possessing one or two B-BOX domains and in some cases an additional CCT (CO, CO-like and TOC1) motif, which play important roles in regulating plant growth, development and stress response. Nevertheless, no systematic study of BBX genes has undertaken in tomato (Solanum lycopersicum). Here we present the results of a genome-wide analysis of the 29 BBX genes in this important vegetable species. Their structures, conserved domains, phylogenetic relationships, subcellular localizations, and promoter cis-regulatory elements were analyzed; their tissue expression profiles and expression patterns under various hormones and stress treatments were also investigated in detail. Tomato BBX genes can be divided into five subfamilies, and twelve of them were found to be segmentally duplicated. Real-time quantitative PCR analysis showed that most BBX genes exhibited different temporal and spatial expression patterns. The expression of most BBX genes can be induced by drought, polyethylene glycol-6000 or heat stress. Some BBX genes were induced strongly by phytohormones such as abscisic acid, gibberellic acid, or ethephon. The majority of tomato BBX proteins was predicted to be located in nuclei, and the transient expression assay using Arabidopsis mesophyll protoplasts demonstrated that all the seven BBX members tested (SlBBX5, 7, 15, 17, 20, 22, and 24) were localized in nucleus. Our analysis of tomato BBX genes on the genome scale would provide valuable information for future functional characterization of specific genes in this family. PMID:27807440

  4. 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

  5. The rice B-box zinc finger gene family: genomic identification, characterization, expression profiling and diurnal analysis.

    PubMed

    Huang, Jianyan; Zhao, Xiaobo; Weng, Xiaoyu; Wang, Lei; Xie, Weibo

    2012-01-01

    The B-box (BBX) -containing proteins are a class of zinc finger proteins that contain one or two B-box domains and play important roles in plant growth and development. The Arabidopsis BBX gene family has recently been re-identified and renamed. However, there has not been a genome-wide survey of the rice BBX (OsBBX) gene family until now. In this study, we identified 30 rice BBX genes through a comprehensive bioinformatics analysis. Each gene was assigned a uniform nomenclature. We described the chromosome localizations, gene structures, protein domains, phylogenetic relationship, whole life-cycle expression profile and diurnal expression patterns of the OsBBX family members. Based on the phylogeny and domain constitution, the OsBBX gene family was classified into five subfamilies. The gene duplication analysis revealed that only chromosomal segmental duplication contributed to the expansion of the OsBBX gene family. The expression profile of the OsBBX genes was analyzed by Affymetrix GeneChip microarrays throughout the entire life-cycle of rice cultivar Zhenshan 97 (ZS97). In addition, microarray analysis was performed to obtain the expression patterns of these genes under light/dark conditions and after three phytohormone treatments. This analysis revealed that the expression patterns of the OsBBX genes could be classified into eight groups. Eight genes were regulated under the light/dark treatments, and eleven genes showed differential expression under at least one phytohormone treatment. Moreover, we verified the diurnal expression of the OsBBX genes using the data obtained from the Diurnal Project and qPCR analysis, and the results indicated that many of these genes had a diurnal expression pattern. The combination of the genome-wide identification and the expression and diurnal analysis of the OsBBX gene family should facilitate additional functional studies of the OsBBX genes.

  6. 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.

  7. Cytosolic events involved in chloroplast protein targeting.

    PubMed

    Lee, Dong Wook; Jung, Chanjin; Hwang, Inhwan

    2013-02-01

    Chloroplasts are unique organelles that are responsible for photosynthesis. Although chloroplasts contain their own genome, the majority of chloroplast proteins are encoded by the nuclear genome. These proteins are transported to the chloroplasts after translation in the cytosol. Chloroplasts contain three membrane systems (outer/inner envelope and thylakoid membranes) that subdivide the interior into three soluble compartments known as the intermembrane space, stroma, and thylakoid lumen. Several targeting mechanisms are required to deliver proteins to the correct chloroplast membrane or soluble compartment. These mechanisms have been extensively studied using purified chloroplasts in vitro. Prior to targeting these proteins to the various compartments of the chloroplast, they must be correctly sorted in the cytosol. To date, it is not clear how these proteins are sorted in the cytosol and then targeted to the chloroplasts. Recently, the cytosolic carrier protein AKR2 and its associated cofactor Hsp17.8 for outer envelope membrane proteins of chloroplasts were identified. Additionally, a mechanism for controlling unimported plastid precursors in the cytosol has been discovered. This review will mainly focus on recent findings concerning the possible cytosolic events that occur prior to protein targeting to the chloroplasts. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. 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.

  9. 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

  10. CENP-B box and pJalpha sequence distribution in human alpha satellite higher-order repeats (HOR).

    PubMed

    Rosandić, Marija; Paar, Vladimir; Basar, Ivan; Gluncić, Matko; Pavin, Nenad; Pilas, Ivan

    2006-01-01

    Using our Key String Algorithm (KSA) to analyze Build 35.1 assembly we determined consensus alpha satellite higher-order repeats (HOR) and consensus distributions of CENP-B box and pJalpha motif in human chromosomes 1, 4, 5, 7, 8, 10, 11, 17, 19, and X. We determined new suprachromosomal family (SF) assignments: SF5 for 13mer (2211 bp), SF5 for 13mer (2214 bp), SF2 for 11mer (1869 bp), SF1 for 18mer (3058 bp), SF3 for 12mer (2047 bp), SF3 for 14mer (2379 bp), and SF5 for 17mer (2896 bp) in chromosomes 4, 5, 8, 10, 11, 17, and 19, respectively. In chromosome 5 we identified SF5 13mer without any CENP-B box and pJalpha motif, highly homologous (96%) to 13mer in chromosome 19. Additionally, in chromosome 19 we identified new SF5 17mer with one CENP-B box and pJalpha motif, aligned to 13mer by deleting four monomers. In chromosome 11 we identified SF3 12mer, homologous to 12mer in chromosome X. In chromosome 10 we identified new SF1 18mer with eight CENP-B boxes in every other monomer (except one). In chromosome 4 we identified new SF5 13mer with CENP-B box in three consecutive monomers. We found four exceptions to the rule that CENP-B box belongs to type B and pJalpha motif to type A monomers.

  11. The solution structure of the C-terminal domain of TonB and interaction studies with TonB box peptides.

    PubMed

    Sean Peacock, R; Weljie, Aalim M; Peter Howard, S; Price, Feodor D; Vogel, Hans J

    2005-02-04

    The TonB protein transduces energy from the proton gradient across the cytoplasmic membrane of Gram-negative bacteria to TonB-dependent outer membrane receptors. It is a critically important protein in iron uptake, and deletion of this protein is known to decrease virulence of bacteria in animal models. This system has been used for Trojan horse antibiotic delivery. Here, we describe the high-resolution solution structure of Escherichia coli TonB residues 103-239 (TonB-CTD). TonB-CTD is monomeric with an unstructured N terminus (103-151) and a well structured C terminus (152-239). The structure contains a four-stranded antiparallel beta-sheet packed against two alpha-helices and an extended strand in a configuration homologous to the C-terminal domain of the TolA protein. Chemical shift perturbations to the TonB-CTD (1)H-(15)N HSCQ spectrum titrated with TonB box peptides modeled from the E.coli FhuA, FepA and BtuB proteins were all equivalent, indicating that all three peptides bind to the same region of TonB. Isothermal titration calorimetry measurements demonstrate that TonB-CTD interacts with the FhuA-derived peptide with a K(D)=36(+/-7) microM. On the basis of chemical shift data, the position of Gln160, and comparison to the TolA gp3 N1 complex crystal structure, we propose that the TonB box binds to TonB-CTD along the beta3-strand.

  12. Proteins involved in meiotic recombination: a role in male infertility?

    PubMed

    Sanderson, Matthew L; Hassold, Terry J; Carrell, Douglas T

    2008-01-01

    Meiotic recombination results in the formation of crossovers, by which genetic information is exchanged between homologous chromosomes during prophase I of meiosis. Recombination is a complex process involving many proteins. Alterations in the genes involved in recombination may result in infertility. Molecular studies have improved our understanding of the roles and mechanisms of the proteins and protein complexes involved in recombination, some of which have function in mitotic cells as well as meiotic cells. Human gene sequencing studies have been performed for some of these genes and have provided further information on the phenotypes observed in some infertile individuals. However, further studies are needed to help elucidate the particular role(s) of a given protein and to increase our understanding of these protein systems. This review will focus on our current understanding of proteins involved in meiotic recombination from a genomic perspective, summarizing our current understanding of known mutations and single nucleotide polymorphisms that may affect male fertility by altering meiotic recombination.

  13. Novel protein-protein interaction family proteins involved in chloroplast movement response.

    PubMed

    Kodama, Yutaka; Suetsugu, Noriyuki; Wada, Masamitsu

    2011-04-01

    To optimize photosynthetic activity, chloroplasts change their intracellular location in response to ambient light conditions; chloroplasts move toward low intensity light to maximize light capture, and away from high intensity light to avoid photodamage. Although several proteins have been reported to be involved in the chloroplast photorelocation movement response, any physical interaction among them was not found so far. We recently found a physical interaction between two plant-specific coiled-coil proteins, WEB1 (Weak Chloroplast Movement under Blue Light 1) and PMI2 (Plastid Movement Impaired 2), that were identified to regulate chloroplast movement velocity. Since the both coiled-coil regions of WEB1 and PMI2 were classified into an uncharacterized protein family having DUF827 (DUF: Domain of Unknown Function) domain, it was the first report that DUF827 proteins could mediate protein-protein interaction. In this mini-review article, we discuss regarding molecular function of WEB1 and PMI2, and also define a novel protein family composed of WEB1, PMI2 and WEB1/PMI2-like proteins for protein-protein interaction in land plants.

  14. Involvement of PCH family proteins in cytokinesis and actin distribution.

    PubMed

    Lippincott, J; Li, R

    2000-04-15

    Pombe Cdc15 homology (PCH) proteins constitute an extensive protein family whose members have been found in diverse eukaryotic organisms. These proteins are characterized by the presence of several conserved sequence and structural motifs. Recent studies in yeast and mammalian cultured cells have implicated these proteins in actin-based processes, in particular, cytokinesis. Here we review the recent findings on the in vivo localization, function, and binding partners of PCH family members. We also provide new microscopy data regarding the in vivo dynamics of a budding yeast PCH protein involved in cytokinesis.

  15. 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.

  16. Molecular Simulation Studies of Proteins Involved in Parkinson's Disease

    NASA Astrophysics Data System (ADS)

    Carloni, Paolo

    2007-12-01

    This contribution describes two recent computational studies related to proteins involved in Parkinson's Disease (PD). The first focuses on the interplay between dopamine and α-synuclein (AS), which plays a central role in PD (unpublished results). The second deals with the protein DJ-1, whose mutations are present in patients suffering from familiar PD [1]. Computational methods are used to investigate the relationship between such mutations and the protein oligomeric state, which may be important for the progression of the disease.

  17. 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

  18. Key proteins involved in insulin vesicle exocytosis and secretion

    PubMed Central

    Xiong, Qian-Yin; Yu, Cui; Zhang, Yao; Ling, Liefeng; Wang, Lizhuo; Gao, Jia-Lin

    2017-01-01

    In vivo insulin secretion is predominantly affected by blood glucose concentration, blood concentration of amino acids, gastrointestinal hormones and free nerve functional status, in addition to other factors. Insulin is one of the most important hormones in the body, and its secretion is precisely controlled by nutrients, neurotransmitters and hormones. The insulin exocytosis process is similar to the neurotransmitter release mechanism. There are various types of proteins and lipids that participate in the insulin secretory vesicle fusion process, such as soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, Ras-related proteins and vacuolar-type H+-ATPase (V-ATPase). Notably, the SNARE protein is the molecular basis of exocytotic activity. In the current review, the role of the vesicle membrane proteins (synaptobrevins, vesicle associated membrane proteins and target membrane proteins) and auxiliary proteins (Rab proteins and Munc-18 proteins) in vesicle fusion activity were summarized. A summary of these key proteins involved in insulin granule secretion will facilitate understanding of the pathogenesis of diabetes. PMID:28357064

  19. Exploiting genomic data to identify proteins involved in abalone reproduction.

    PubMed

    Mendoza-Porras, Omar; Botwright, Natasha A; McWilliam, Sean M; Cook, Mathew T; Harris, James O; Wijffels, Gene; Colgrave, Michelle L

    2014-08-28

    Aside from their critical role in reproduction, abalone gonads serve as an indicator of sexual maturity and energy balance, two key considerations for effective abalone culture. Temperate abalone farmers face issues with tank restocking with highly marketable abalone owing to inefficient spawning induction methods. The identification of key proteins in sexually mature abalone will serve as the foundation for a greater understanding of reproductive biology. Addressing this knowledge gap is the first step towards improving abalone aquaculture methods. Proteomic profiling of female and male gonads of greenlip abalone, Haliotis laevigata, was undertaken using liquid chromatography-mass spectrometry. Owing to the incomplete nature of abalone protein databases, in addition to searching against two publicly available databases, a custom database comprising genomic data was used. Overall, 162 and 110 proteins were identified in females and males respectively with 40 proteins common to both sexes. For proteins involved in sexual maturation, sperm and egg structure, motility, acrosomal reaction and fertilization, 23 were identified only in females, 18 only in males and 6 were common. Gene ontology analysis revealed clear differences between the female and male protein profiles reflecting a higher rate of protein synthesis in the ovary and higher metabolic activity in the testis. A comprehensive mass spectrometry-based analysis was performed to profile the abalone gonad proteome providing the foundation for future studies of reproduction in abalone. Key proteins involved in both reproduction and energy balance were identified. Genomic resources were utilised to build a database of molluscan proteins yielding >60% more protein identifications than in a standard workflow employing public protein databases. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Proteins involved in vesicular transport and membrane fusion.

    PubMed

    Waters, M G; Griff, I C; Rothman, J E

    1991-08-01

    In the past year, new information about proteins involved in vesicular transport has been plentiful. Particularly noteworthy are the complementary findings that Sec17p is required for vesicle consumption in endoplasmic reticulum-to-Golgi transport in yeast and that an analogous activity in mammalian cells, termed SNAP, is required for transport from the cis to the medial cisternae of the Golgi apparatus.

  1. 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

    2009-11-20

    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.

  2. Proteomic analysis of proteins involved in spermiogenesis in mouse.

    PubMed

    Guo, Xuejiang; Shen, Jian; Xia, Zhengrong; Zhang, Rui; Zhang, Ping; Zhao, Chun; Xing, Jun; Chen, Ling; Chen, Wen; Lin, Min; Huo, Ran; Su, Bing; Zhou, Zuomin; Sha, Jiahao

    2010-03-05

    Spermiogenesis is a unique process in mammals during which haploid round spermatids mature into spermatozoa in the testis. Its successful completion is necessary for fertilization and its malfunction is an important cause of male infertility. Here, we report the high-confidence identification of 2116 proteins in mouse haploid germ cells undergoing spermiogenesis: 299 of these were testis-specific and 155 were novel. Analysis of these proteins showed many proteins possibly functioning in unique processes of spermiogenesis. Of the 84 proteins annotated to be involved in vesicle-related events, VAMP4 was shown to be important for acrosome biogenesis by in vivo knockdown experiments. Knockdown of VAMP4 caused defects of acrosomal vesicle fusion and significantly increased head abnormalities in spermatids from testis and sperm from the cauda epididymis. Analysis of chromosomal distribution of the haploid genes showed underrepresentation on the X chromosome and overrepresentation on chromosome 11, which were due to meiotic sex chromosome inactivation and expansion of testis-expressed gene families, respectively. Comparison with transcriptional data showed translational regulation during spermiogenesis. This characterization of proteins involved in spermiogenesis provides an inventory of proteins useful for understanding the mechanisms of male infertility and may provide candidates for drug targets for male contraception and male infertility.

  3. Protein Phosphatases Involved in Regulating Mitosis: Facts and Hypotheses.

    PubMed

    Kim, Hyun-Soo; Fernandes, Gary; Lee, Chang-Woo

    2016-09-01

    Almost all eukaryotic proteins are subject to post-translational modifications during mitosis and cell cycle, and in particular, reversible phosphorylation being a key event. The recent use of high-throughput experimental analyses has revealed that more than 70% of all eukaryotic proteins are regulated by phosphorylation; however, the mechanism of dephosphorylation, counteracting phosphorylation, is relatively unknown. Recent discoveries have shown that many of the protein phosphatases are involved in the temporal and spatial control of mitotic events, such as mitotic entry, mitotic spindle assembly, chromosome architecture changes and cohesion, and mitotic exit. This implies that certain phosphatases are tightly regulated for timely dephosphorylation of key mitotic phosphoproteins and are essential for control of various mitotic processes. This review describes the physiological and pathological roles of mitotic phosphatases, as well as the versatile role of various protein phosphatases in several mitotic events.

  4. NEDD8 protein is involved in ubiquitinated inclusion bodies.

    PubMed

    Dil Kuazi, Afroz; Kito, Katsumi; Abe, Yasuhito; Shin, Ryong-Woon; Kamitani, Tetsu; Ueda, Norifumi

    2003-02-01

    Proteolysis by the ubiquitin-proteasome system is considered to play a pathological role in several degenerative diseases that involve ubiquitinated inclusion bodies. In recent years, several ubiquitin-like proteins have been isolated, but it is uncertain whether their roles are associated with protein degradation through the ubiquitin-proteasome system. NEDD8 (neural precursor cell-expressed and developmentally down-regulated gene), which consists of 81 amino acid residues, possesses the highest sequence similarity to ubiquitin. Recent studies have indicated that NEDD8 is covalently ligated to cullin family proteins, which are components of certain ubiquitin E3 ligases, by a pathway analogous to that of ubiquitin. Thus, by focusing on the structural and functional association between NEDD8 and ubiquitin, it would be of interest to know whether the NEDD8 system is involved in pathological disorders of the ubiquitin-proteasome system. This study has examined the immunohistochemical distribution of NEDD8 protein by using a highly purified antibody in normal tissues and in tissues known to contain ubiquitinated inclusions. NEDD8 protein expression was widely observed in most types of tissues. Furthermore, accumulation of the NEDD8 protein was commonly observed in ubiquitinated inclusion bodies, including Lewy bodies in Parkinson's disease, Mallory bodies in alcoholic liver disease, and Rosenthal fibres in astrocytoma. Two of ten cases of neurofibrillary tangles and senile plaques from patients with Alzheimer's disease showed intense staining for NEDD8 as well as for ubiquitin. These findings suggest the possibility that the NEDD8 system is involved in the metabolism of these inclusion bodies via the ubiquitin-proteasome system.

  5. 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.

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

    PubMed

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

    2014-10-17

    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.

  7. 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.

  8. Predicted structures of two proteins involved in human diseases.

    PubMed

    Zhou, H X; Wang, G

    2001-01-01

    Structures of 79 proteins involved in human diseases were predicted by sequence alignments with structural templates. The predicted structures for ALDP and CSA, proteins responsible for adrenoleukodystrophy and the Cockayne syndrome, respectively, were analyzed to elucidate the molecular basis of disease mutations. In particular we positioned residue P484 of ALDP in the homodimer interface. This positioning is consistent with a recent experimental finding that the mutation P484R significantly decreases the self-interaction of ALDP and suggests that the disease mechanism of this mutation lies in the impaired ALDP dimerization. We identified two new WD repeats in CSA and suggest that one of these forms part of the interaction surface with other proteins.

  9. Schizosaccharomyces U6 genes have a sequence within their introns that matches the B box consensus of tRNA internal promoters.

    PubMed

    Frendewey, D; Barta, I; Gillespie, M; Potashkin, J

    1990-04-25

    The gene for the U6 small nuclear RNA (snRNA) in the fission yeast Schizosaccharomyces pombe is interrupted by an intron whose structure is similar to those found in messenger RNA precursors (pre-mRNAs) (1). This is the only known example of a split snRNA gene from any organism--animal, plant, or yeast. To address the uniqueness of the S. pombe U6 gene, we have investigated the structures of the U6 genes from five Schizosaccharomyces strains and three other fungi. A fragment of the U6 coding sequence was amplified from the genomic DNA of each strain by the polymerase chain reaction (PCR). The sizes of the PCR products indicated that all of the fission yeast strains possess intron-containing U6 genes; whereas, the U6 genes from the other fungi appeared to be uninterrupted. The sequences of the Schizosaccharomyces U6 gene fragments revealed that each had an intron of approximately 50 base pairs in precisely the same position. In addition to the splice sites and putative branch point regions, a sequence immediately upstream of the branch point consensus was found to be conserved in all of the Schizosaccharomyces U6 genes. This sequence matches the consensus for the B box of eukaryotic tRNA promoters. These results raise the interesting possibility that synthesis of U6 RNA in fission yeast might involve the use of internal promoter elements similar to those found in other genes transcribed by RNA polymerase III.

  10. Bap, a Staphylococcus aureus Surface Protein Involved in Biofilm Formation

    PubMed Central

    Cucarella, Carme; Solano, Cristina; Valle, Jaione; Amorena, Beatriz; Lasa, Íñigo; Penadés, José R.

    2001-01-01

    Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing Staphylococcus aureus isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (bap [for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (Pseudomonas aeruginosa and Salmonella enterica serovar Typhi) and gram-positive (Enteroccocus faecalis) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids. bap was present in a small fraction of bovine mastitis isolates (5% of the 350 S. aureus isolates tested), but it was absent from the 75 clinical human S. aureus isolates analyzed. All staphylococcal isolates harboring bap were highly adherent and strong biofilm producers. In a mouse infection model bap was involved in pathogenesis, causing a persistent infection. PMID:11292810

  11. 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

  12. [Proteins of human milk involved in immunological processes].

    PubMed

    Lis, Jolanta; Orczyk-Pawiłowicz, Magdalena; Kątnik-Prastowska, Iwona

    2013-05-31

    Human milk contains a lot of components (i.e. proteins, carbohydrates, lipids, inorganic elements) which provide basic nutrients for infants during the first period of their lives. Qualitative composition of milk components of healthy mothers is similar, but their levels change during lactation stages. Colostrum is the fluid secreted during the first days postpartum by mammary epithelial cells. Colostrum is replaced by transitional milk during 5-15 days postpartum and from 15 days postpartum mature milk is produced. Human milk, apart from nutritional components, is a source of biologically active molecules, i.e. immunoglobulins, growth factors, cytokines, acute phase proteins, antiviral and antibacterial proteins. Such components of human milk are responsible for specific biological activities of human milk. This secretion plays an important role in growth and development of newborns. Bioactive molecules present in the milk support the immature immune system of the newborn and also protect against the development of infection. In this article we describe the pathways involved in the production and secretion of human milk, the state of knowledge on the proteome of human milk, and the contents of components of milk during lactation. Moreover, some growth factors and proteins involved in innate and specific immunity, intercellular communication, immunomodulation, and inflammatory processes have been characterized.

  13. 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.

  14. 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.

  15. Important amino acid residues involved in folding and binding of protein-protein complexes.

    PubMed

    Kulandaisamy, A; Lathi, V; ViswaPoorani, K; Yugandhar, K; Gromiha, M Michael

    2017-01-01

    Protein-protein interactions perform diverse functions in living organism. The integrative analysis of binding and stabilizing residues will provide insights on the functions of protein-protein complexes. In this work, we constructed a non-redundant dataset of 261 protein-protein complexes and identified binding site residues, stabilizing residues and common to both binding and stabilizing, termed as "key residues". We found that 6.1% of residues are involved in binding and 6.8% of residues are important for folding and stability. Among them, only 2% are involved in both folding and binding, which shows the importance and specific roles played by these residues. The key residues have been analyzed based on protein function, binding affinity, rigid and flexible complexes, amino acid preference and structure based parameters. We found that high affinity complexes have more key residues than low affinity complexes. In addition, key residues are enriched with the combination of specific hydrophobic and charged/polar residues. Atomic contacts between interacting proteins have distinct preferences of polar-polar, nonpolar-nonpolar and polar-nonpolar contacts in different functional classes of protein-protein complexes. Further, the influence of sequence and structural parameters such as surrounding hydrophobicity, solvent accessibility, secondary structure, long-range order and conservation score has been discussed. The analysis can be used to comprehend the interplay between stability and binding in protein-protein complexes. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2014-06-07

    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.

  17. Identification of an additional protein involved in mannan biosynthesis

    PubMed Central

    Wang, Yan; Mortimer, Jennifer C; Davis, Jonathan; Dupree, Paul; Keegstra, Kenneth

    2013-01-01

    Galactomannans comprise a β-1,4-mannan backbone substituted with α-1,6-galactosyl residues. Genes encoding the enzymes that are primarily responsible for backbone synthesis and side-chain addition of galactomannans were previously identified and characterized. To identify additional genes involved in galactomannan biosynthesis, we previously performed deep EST profiling of fenugreek (Trigonella foenum-graecum L.) seed endosperm, which accumulates large quantities of galactomannans as a reserve carbohydrate during seed development. One of the candidate genes encodes a protein that is likely to be a glycosyltransferase. Because this protein is involved in mannan biosynthesis, we named it ‘mannan synthesis-related’ (MSR). Here, we report the characterization of a fenugreek MSR gene (TfMSR) and its two Arabidopsis homologs, AtMSR1 and AtMSR2. TfMSR was highly and specifically expressed in the endosperm. TfMSR, AtMSR1 and AtMSR2 proteins were all determined to be localized to the Golgi by fluorescence confocal microscopy. The level of mannosyl residues in stem glucomannans decreased by approximately 40% for Arabidopsis msr1 single T-DNA insertion mutants and by more than 50% for msr1 msr2 double mutants, but remained unchanged for msr2 single mutants. In addition, in vitro mannan synthase activity from the stems of msr1 single and msr1 msr2 double mutants also decreased. Expression of AtMSR1 or AtMSR2 in the msr1 msr2 double mutant completely or partially restored mannosyl levels. From these results, we conclude that the MSR protein is important for mannan biosynthesis, and offer some ideas about its role. PMID:22966747

  18. Genes and proteins involved in bacterial magnetic particle formation.

    PubMed

    Matsunaga, Tadashi; Okamura, Yoshiko

    2003-11-01

    Magnetic bacteria synthesize intracellular magnetosomes that impart a cellular swimming behaviour referred to as magnetotaxis. The magnetic structures aligned in chains are postulated to function as biological compass needles allowing the bacterium to migrate along redox gradients through the Earth's geomagnetic field lines. Despite the discovery of this unique group of microorganisms 28 years ago, the mechanisms of magnetic crystal biomineralization have yet to be fully elucidated. This review describes the current knowledge of the genes and proteins involved in magnetite formation in magnetic bacteria and the biotechnological applications of biomagnetites in the interdisciplinary fields of nanobiotechnology, medicine and environmental management.

  19. Identification of target proteins involved in cochlear otosclerosis.

    PubMed

    Richard, Céline; Doherty, Joni K; Fayad, Jose N; Cordero, Ana; Linthicum, Fred H

    2015-06-01

    Investigation of differential protein expression will provide clues to pathophysiology in otosclerosis. Otosclerosis is a bone remodeling disorder limited to the endochondral layer of the otic capsule within the temporal bone. Some authors have suggested an inflammatory etiology for otosclerosis resulting from persistent measles virus infection involving the otic capsule. Despite numerous genetic studies, implication of candidate genes in the otosclerotic process remains elusive. We employed liquid chromatography-mass spectrometry (LC-MS) analysis on formalin-fixed celloidin-embedded temporal bone tissues for postmortem investigation of otosclerosis. Proteomic analysis was performed using human temporal bones from a patient with severe otosclerosis and a control temporal bone. Sections were dissected under microscopy to remove otosclerotic lesions and normal otic capsule for proteomic analysis. Tandem 2D chromatography mass spectrometry was employed. Data analysis and peptide matching to FASTA human databases was done using SEQUEST and proteome discoverer software. TGFβ1 was identified in otosclerosis but not in the normal control temporal bone specimen. Aside from TGFβ1, many proteins and predicted cDNA-encoded proteins were observed, with implications in cell death and/or proliferation pathways, suggesting a possible role in otosclerotic bone remodeling. Immunostaining using TGFβ1 monoclonal revealed marked staining of the spongiotic otosclerotic lesions. Mechanisms involved in cochlear extension of otosclerosis are still unclear, but the implication of TGFβ1 is supported by the present proteomic data and immunostaining results. The established role of TGFβ1 in the chondrogenesis process supports the theory of a reaction targeting the globulae interossei within the otic capsule.

  20. 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.

  1. Possible involvement of poly(A) in protein synthesis.

    PubMed Central

    Jacobson, A; Favreau, M

    1983-01-01

    The experiments of this paper have re-evaluated the possibility that poly(A) is involved in protein synthesis by testing whether purified poly(A) might competitively inhibit in vitro protein synthesis in rabbit reticulocyte extracts. We have found that poly(A) inhibits the rate of translation of many different poly(A)+ mRNAs and that comparable inhibition is not observed with other ribopolymers. Inhibition by poly(A) preferentially affects the translation of adenylated mRNAs and can be overcome by increased mRNA concentrations or by translating mRNPs instead of mRNA. The extent of inhibition is dependent on the size of the competitor poly(A) as well as on the translation activity which a lysate has for poly(A)+ RNA. In light of our results and numerous experiments in the literature, we propose that poly(A) has a function in protein synthesis and that any role in the determination of mRNA stability is indirect. Images PMID:6137807

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

    PubMed

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

    2016-04-28

    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.

  3. 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

  4. 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

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

    2014-04-01

    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. 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. 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. The interplay between circadian rhythm and osmotic constraints in the regulation of the expression of a Solanum DBB transcriptional regulator is demonstrated. It is proposed that stress-dependent, post

  5. 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

  6. Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa.

    PubMed

    Goichon, Alexis; Bertrand, Julien; Chan, Philippe; Lecleire, Stéphane; Coquard, Aude; Cailleux, Anne-Françoise; Vaudry, David; Déchelotte, Pierre; Coëffier, Moïse

    2015-08-01

    Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg⁻¹ · h⁻¹). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis-based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student's t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and β-actin) or in protein biosynthesis (glutamyl-prolyl-transfer RNA synthetase, glutaminyl-transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling. © 2015 American Society for Nutrition.

  7. Lupine protein hydrolysates inhibit enzymes involved in the inflammatory pathway.

    PubMed

    Millán-Linares, María del Carmen; Yust, María del Mar; Alcaide-Hidalgo, Juan María; Millán, Francisco; Pedroche, Justo

    2014-05-15

    Lupine protein hydrolysates (LPHs) were obtained from a lupine protein isolate (LPI) by enzymatic hydrolysis using two proteases, Izyme AL and Alcalase 2.4 L, and their potential anti-inflammatory capacities were studied by determining their in vitro inhibition of the following enzymes that are involved in the inflammatory process: phospholipase A2 (PLA2), cyclooxygenase 2 (COX-2), thrombin, and transglutaminase (TG). The strongest inhibitory activities toward PLA2 and TG were found in the hydrolysates obtained by hydrolysis with Izyme and subsequently with Alcalase, with more than 70% inhibition obtained in some cases. All of the hydrolysates tested inhibited more than 60% of the COX-2 activity. In no case did the percentage of thrombin activity inhibition exceed 40%. The best inhibitory activities were found in the LPH obtained after 15 min of hydrolysis with Alcalase and in the LPH obtained after 60 min of hydrolysis with Izyme followed by 15 min of hydrolysis with Alcalase. Enzyme kinetic analyses were conducted to determine the Km and Vmax parameters of these two hydrolysates using the Lineweaver-Burk equation. Both hydrolysates competitively inhibited the thrombin and PLA2 activities. In the case of COX-2 and TG, the inhibition appeared to be the mixed type. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. 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

  9. What induces pocket openings on protein surface patches involved in protein-protein interactions?

    NASA Astrophysics Data System (ADS)

    Eyrisch, Susanne; Helms, Volkhard

    2009-02-01

    We previously showed for the proteins BCL-XL, IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

  10. Host membrane proteins involved in the replication of tobamovirus RNA.

    PubMed

    Ishibashi, Kazuhiro; Miyashita, Shuhei; Katoh, Etsuko; Ishikawa, Masayuki

    2012-12-01

    Eukaryotic positive-strand RNA viruses replicate their genomes in membrane-bound replication complexes composed of viral replication proteins and negative-strand RNA templates. These replication proteins are programmed to exhibit RNA polymerase and other replication-related activities only in replication complexes to avoid inducing double-stranded RNA-mediated host defenses. Host membrane components (e.g. proteins and lipids) should play important roles in the activation of replication proteins. Two host membrane proteins are components of the replication complex and activate the replication proteins of tobamoviruses. Interaction analyses using deletion mutants constructed based on structural information suggest a conformational change in replication proteins during the formation of a protein complex with RNA 5'-capping activity.

  11. Intrinsic disorder in proteins involved in amyotrophic lateral sclerosis.

    PubMed

    Santamaria, Nikolas; Alhothali, Marwa; Alfonso, Maria Harreguy; Breydo, Leonid; Uversky, Vladimir N

    2017-04-01

    Five structurally and functionally different proteins, an enzyme superoxide dismutase 1 (SOD1), a TAR-DNA binding protein-43 (TDP-43), an RNA-binding protein FUS, a cofilin-binding protein C9orf72, and polypeptides generated as a result of its intronic hexanucleotide expansions, and to lesser degree actin-binding profilin-1 (PFN1), are considered to be the major drivers of amyotrophic lateral sclerosis. One of the features common to these proteins is the presence of significant levels of intrinsic disorder. The goal of this study is to consider these neurodegeneration-related proteins from the intrinsic disorder perspective. To this end, we employed a broad set of computational tools for intrinsic disorder analysis and conducted intensive literature search to gain information on the structural peculiarities of SOD1, TDP-43, FUS, C9orf72, and PFN1 and their intrinsic disorder predispositions, and the roles of intrinsic disorder in their normal and pathological functions.

  12. 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

  13. Novel identification of matrix proteins involved in calcitic biomineralization.

    PubMed

    Rose-Martel, Megan; Smiley, Sandy; Hincke, Maxwell T

    2015-02-26

    Calcitic biomineralization is essential for otoconia formation in vertebrates. This process is characterized by protein-crystal interactions that modulate crystal growth on an extracellular matrix. An excellent model for the study of calcitic biomineralization is the avian eggshell, the fastest known biomineralization process. The objective of this study is to identify and characterize matrix proteins associated with the eggshell mammillary cones, which are hypothesized to regulate the earliest stage of eggshell calcification. Mammillary cones were isolated from 2 models, fertilized and unfertilized, and the released proteins were identified by RP-nanoLC and ES-MS/MS proteomics. Proteomics analysis identified 49 proteins associated with the eggshell membrane fibers and, importantly, 18 mammillary cone-specific proteins with an additional 18 proteins identified as enriched in the mammillary cones. Among the most promising candidates for modulating protein-crystal interactions were extracellular matrix proteins, including ABI family member 3 (NESH) binding protein (ABI3BP), tiarin-like, hyaluronan and proteoglycan link protein 3 (HAPLN3), collagen alpha-1(X), collagen alpha-1(II) and fibronectin, in addition to the calcium binding proteins calumenin, EGF-like repeats and discoidin 1-like domains 3 (EDIL3), nucleobindin-2 and SPARC. In conclusion, we identified several cone-resident proteins that are candidates to regulate initiation of eggshell calcification. Further study of these proteins will determine their roles in modulating calcitic biomineralization and lead to insight into the process of otoconia formation/regeneration. Biomineralization is essential for the development of hard tissues in vertebrates, which includes both calcium phosphate and calcium carbonate structures. Calcitic mineralization by calcium carbonate is an important process in the formation of otoconia, which are gravity receptor organs located in the inner ear and are responsible for balance

  14. Involvement of the Transmembrane Protein p23 in Biosynthetic Protein Transport

    PubMed Central

    Rojo, Manuel; Pepperkok, Rainer; Emery, Gregory; Kellner, Roland; Stang, Espen; Parton, Robert G.; Gruenberg, Jean

    1997-01-01

    Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/μm2 membrane surface area, or ≈30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment . Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport. PMID:9382861

  15. 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.

  16. 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

  17. 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.

  18. RNA Binding Proteins Posttranscriptionally Regulate Genes Involved In Oncogenesis

    DTIC Science & Technology

    2010-06-01

    Cloning and characterization of HuR, a ubiquitously expressed Elav-like protein . J Biol Chem 1996, 271(14):8144-8151. 21. Meisner NC, Hackermuller J...Hauptmann S: Expression of the ELAV-like protein HuR is associated with higher tumor grade and increased cyclooxygenase-2 expression in human breast...SH3 domain, ankyrin repeat and pH domain 3 tumor microarray reveals 47 annotated genes up regulated in the HA-HuR overexpressing tumors as compared to

  19. Centrophilin: a novel mitotic spindle protein involved in microtubule nucleation

    PubMed Central

    1991-01-01

    A novel protein has been identified which may serve a key function in nucleating spindle microtubule growth in mitosis. This protein, called centrophilin, is sequentially relocated from the centromeres to the centrosomes to the midbody in a manner dependent on the mitotic phase. Centrophilin was initially detected by immunofluorescence with a monoclonal, primate-specific antibody (2D3) raised against kinetochore- enriched chromosome extract from HeLa cells (Valdivia, M. M., and B. R. Brinkley. 1985. J. Cell Biol. 101:1124-1134). Centrophilin forms prominent crescents at the poles of the metaphase spindle, gradually diminishes during anaphase, and bands the equatorial ends of midbody microtubules in telophase. The formation and breakdown of the spindle and midbody correlates in time and space with the aggregation and disaggregation of centrophilin foci. Immunogold EM reveals that centrophilin is a major component of pericentriolar material in metaphase. During recovery from microtubule inhibition, centrophilin foci act as nucleation sites for the assembly of spindle tubules. The 2D3 probe recognizes two high molecular mass polypeptides, 180 and 210 kD, on immunoblots of whole HeLa cell extract. Taken together, these data and the available literature on microtubule dynamics point inevitably to a singular model for control of spindle tubule turnover. PMID:1991791

  20. Characterization and Modulation of Proteins Involved in SM Vesication

    DTIC Science & Technology

    2005-05-01

    lines such as HaCaT involve a number of undefined changes that occur over time in culture. We also found that NcoI cells , KC immortalized with HPV16 E6/7... lines such as HaCaT cells , we immortalized cells with a defined and physiologically relevant agent 21, HPV16 E6/7 (above), and utilized at passage 30...the retroviral vector, LXSN (Fig. 2A; Clontech). Following transient transfection of an amphotropic retrovirus packaging cell line (φNX; Gary Nolan

  1. Involvement of Drosophila uncoupling protein 5 in metabolism and aging.

    PubMed

    Sánchez-Blanco, Adolfo; Fridell, Yih-Woei C; Helfand, Stephen L

    2006-03-01

    A novel uncoupling protein, UCP5, has recently been characterized as a functional mitochondrial uncoupler in Drosophila. Here we demonstrate that UCP5 knockout (UCP5KO) flies are highly sensitive to starvation stress, a phenotype that can be reversed by ectopic neuronal expression of UCP5. UCP5KO flies live longer than controls on low-calorie diets, have a decreased level of fertility, and gain less weight than controls on high-calorie diets. However, isolated mitochondria from UCP5KO flies display the same respiration patterns as controls. Furthermore, total ATP levels in both UCP5KO and control flies are comparable. UCP5KO flies have a lower body composition of sugars, and during starvation stress their triglyceride reserves are depleted more rapidly than controls. Taken together, these data indicate that UCP5 is important to maintain metabolic homeostasis in the fly. We hypothesize that UCP5 influences hormonal control of metabolism.

  2. Apolipoprotein A-IV: a protein intimately involved in metabolism

    PubMed Central

    Wang, Fei; Kohan, Alison B.; Lo, Chun-Min; Liu, Min; Howles, Philip; Tso, Patrick

    2015-01-01

    The purpose of this review is to summarize our current understanding of the physiological roles of apoA-IV in metabolism, and to underscore the potential for apoA-IV to be a focus for new therapies aimed at the treatment of diabetes and obesity-related disorders. ApoA-IV is primarily synthesized by the small intestine, attached to chylomicrons by enterocytes, and secreted into intestinal lymph during fat absorption. In circulation, apoA-IV is associated with HDL and chylomicron remnants, but a large portion is lipoprotein free. Due to its anti-oxidative and anti-inflammatory properties, and because it can mediate reverse-cholesterol transport, proposed functions of circulating apoA-IV have been related to protection from cardiovascular disease. This review, however, focuses primarily on several properties of apoA-IV that impact other metabolic functions related to food intake, obesity, and diabetes. In addition to participating in triglyceride absorption, apoA-IV can act as an acute satiation factor through both peripheral and central routes of action. It also modulates glucose homeostasis through incretin-like effects on insulin secretion, and by moderating hepatic glucose production. While apoA-IV receptors remain to be conclusively identified, the latter modes of action suggest that this protein holds therapeutic promise for treating metabolic disease. PMID:25640749

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

    PubMed Central

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

    2015-01-01

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

  4. 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

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

    PubMed

    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.

  6. Interfacial interactions involved in the biological assembly of Chandipura virus nucleocapsid protein.

    PubMed

    Sreejith, R; Gulati, Sahil; Gupta, Sanjay

    2013-06-01

    The biological assembly of Chandipura virus nucleocapsid (N) protein has been modeled and the amino acid residues involved in specific intermolecular interactions among N monomers during oligomerisation have been predicted.

  7. TRIM proteins as RING finger E3 ubiquitin ligases.

    PubMed

    Ikeda, Kazuhiro; Inoue, Satoshi

    2012-01-01

    The tripartite motif(TRIM) proteins harboring the RING finger, B-box and coiled-coil (RBCC) domain motifs form a large protein family. The members of this family are involved in various biological processes, including growth, differentiation, apoptosis and transcription and also in diseases and oncogenesis. Recent studies have revealed that TRIM proteins play key roles in innate antiviral immunity. An accumulating body of evidence has demonstrated that some TRIM proteins function as E3 ubiquitin ligases in specific ubiquitin-mediated protein degradation pathways; however, the precise mechanisms underlying this function have not been fully elucidated. In this chapter, we focus on the TRIM family of proteins specially with regard to E3 ligase.

  8. 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.

  9. 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

  10. 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.

  11. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis1

    PubMed Central

    Pesaresi, Paolo; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Rothbart, Maxi; Hedtke, Boris

    2016-01-01

    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

  12. 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.

  13. Self protein-protein interactions are involved in TPPP/p25 mediated microtubule bundling

    PubMed Central

    DeBonis, Salvatore; Neumann, Emmanuelle; Skoufias, Dimitrios A.

    2015-01-01

    TPPP/p25 is a microtubule-associated protein, detected in protein inclusions associated with various neurodegenerative diseases. Deletion analysis data show that TPPP/p25 has two microtubule binding sites, both located in intrinsically disordered domains, one at the N-terminal and the other in the C-terminal domain. In copolymerization assays the full-length protein exhibits microtubule stimulation and bundling activity. In contrast, at the same ratio relative to tubulin, truncated forms of TPPP/p25 exhibit either lower or no microtubule stimulation and no bundling activity, suggesting a cooperative phenomenon which is enhanced by the presence of the two binding sites. The binding characteristics of the N- and C-terminally truncated proteins to taxol-stabilized microtubules are similar to the full-length protein. However, the C-terminally truncated TPPP/p25 shows a lower Bmax for microtubule binding, suggesting that it may bind to a site of tubulin that is masked in microtubules. Bimolecular fluorescent complementation assays in cells expressing combinations of various TPPP/p25 fragments, but not that of the central folded domain, resulted in the generation of a fluorescence signal colocalized with perinuclear microtubule bundles insensitive to microtubule inhibitors. The data suggest that the central folded domain of TPPP/p25 following binding to microtubules can drive s homotypic protein-protein interactions leading to bundled microtubules. PMID:26289831

  14. Inherited diseases involving g proteins and g protein-coupled receptors.

    PubMed

    Spiegel, Allen M; Weinstein, Lee S

    2004-01-01

    Heterotrimeric G proteins couple seven-transmembrane receptors for diverse extracellular signals to effectors that generate intracellular signals altering cell function. Mutations in the gene encoding the alpha subunit of the G protein-coupling receptors to stimulation of adenylyl cyclase cause developmental abnormalities of bone, as well as hormone resistance (pseudohypoparathyroidism caused by loss-of-function mutations) and hormone hypersecretion (McCune-Albright syndrome caused by gain-of-function mutations). Loss- and gain-of-function mutations in genes encoding G protein-coupled receptors (GPCRs) have been identified as the cause of an increasing number of retinal, endocrine, metabolic, and developmental disorders. GPCRs comprise an evolutionarily conserved gene superfamily ( 1 ). By coupling to heterotrimeric G proteins, GPCRs transduce a wide variety of extracellular signals including monoamine, amino acid, and nucleoside neurotransmitters, as well as photons, chemical odorants, divalent cations, hormones, lipids, peptides and proteins. Following a brief overview of G protein-coupled signal transduction, we review the growing body of evidence that mutations in genes encoding GPCRs and G proteins are an important cause of human disease.

  15. Exocyst Sec10 is involved in basolateral protein translation and translocation in the endoplasmic reticulum.

    PubMed

    Choi, Soo Young; Fogelgren, Ben; Zuo, Xiaofeng; Huang, Liwei; McKenna, Sarah; Lingappa, Vishwanath R; Lipschutz, Joshua H

    2012-01-01

    Protein translation and translocation at the rough endoplasmic reticulum (RER) are the first steps in the secretory pathway. The translocon through which newly made proteins are translocated into or across the RER membrane consists of three main subunits: Sec61α, -β, and -γ. Sec61β facilitates translocation, and we and others have shown that the highly conserved eight-protein exocyst complex interacts with Sec61β. We have also shown that the exocyst is involved in basolateral, not apical, protein synthesis and delivery. Recently, however, exocyst involvement in apical protein delivery has been reported. Furthermore, we have shown that the exocyst is necessary for formation of primary cilia, organelles found on the apical surface. GST pulldown was performed on lysate of renal tubule cells to investigate biochemical interactions. Cell-free assays consisting of cell-free extracts from rabbit reticulocytes, pancreatic endoplasmic reticulum (ER) microsomal membranes, transcripts of cDNA from apical and basolateral proteins, ATP/GTP, amino acids, and (35)S-methionine for protein detection were used to investigate the role of the exocyst in synthesis of polarized proteins. P(32)-orthophosphate and immunoprecipitation with antibody against Sec61β was used to investigate Sec61β phosphorylation in exocyst Sec10-overexpressing cells. Sec10 biochemically interacts with Sec61β using GST pulldown. Using cell-free assays, there is enhanced exocyst recruitment to endoplasmic reticulum membranes following exocyst depletion and basolateral G protein of vesicular stomatitis virus protein translation, compared to apical hemagglutinin of influenza virus protein translation. Finally, Sec10 overexpression increases Sec61β phosphorylation. These data confirm that the exocyst is preferentially involved in basolateral protein translation and translocation, and may well act through the phosphorylation of Sec61β. Copyright © 2012 S. Karger AG, Basel.

  16. Neuron membrane trafficking and protein kinases involved in autism and ADHD.

    PubMed

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

    2015-01-30

    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.

  17. The presequence pathway is involved in protein sorting to the mitochondrial outer membrane

    PubMed Central

    Wenz, Lena-Sophie; Opaliński, Łukasz; Schuler, Max-Hinderk; Ellenrieder, Lars; Ieva, Raffaele; Böttinger, Lena; Qiu, Jian; van der Laan, Martin; Wiedemann, Nils; Guiard, Bernard; Pfanner, Nikolaus; Becker, Thomas

    2014-01-01

    The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane. PMID:24781695

  18. Defense-related proteins involved in sugarcane responses to biotic stress.

    PubMed

    Souza, Thais P; Dias, Renata O; Silva-Filho, Marcio C

    2017-02-20

    Sugarcane is one of the most important agricultural crops in the world. However, pathogen infection and herbivore attack cause constant losses in yield. Plants respond to pathogen infection by inducing the expression of several protein types, such as glucanases, chitinases, thaumatins, peptidase inhibitors, defensins, catalases and glycoproteins. Proteins induced by pathogenesis are directly or indirectly involved in plant defense, leading to pathogen death or inducing other plant defense responses. Several of these proteins are induced in sugarcane by different pathogens or insects and have antifungal or insecticidal activity. In this review, defense-related proteins in sugarcane are described, with their putative mechanisms of action, pathogen targets and biotechnological perspectives.

  19. Autophagy-linked FYVE protein (Alfy) promotes autophagic removal of misfolded proteins involved in amyotrophic lateral sclerosis (ALS).

    PubMed

    Han, Huihui; Wei, Wanyi; Duan, Weisong; Guo, Yansu; Li, Yi; Wang, Jie; Bi, Yue; Li, Chunyan

    2015-03-01

    Autophagy-linked FYVE (Alfy) is a protein implicated in the selective degradation of aggregated proteins. In our present study, we found that Alfy was recruited into the aggregated G93A-SOD1 in transgenic mice with amyotrophic lateral sclerosis (ALS). We demonstrated that Alfy overexpression could decrease the expression of mutant proteins via the autophagosome-lysosome pathway, and thereby, the toxicity of mutant proteins was reduced. The clearance of the mutant proteins in NSC34 cells was significantly inhibited in an Alfy knockdown cellular model. We therefore deduced that Alfy translocalization likely is involved in the pathogenesis of ALS. Alfy may be developed into a useful target for ALS therapy.

  20. Exocyst Sec10 is Involved in Basolateral Protein Translation and Translocation in the Endoplasmic Reticulum

    PubMed Central

    Choi, Soo Young; Fogelgren, Ben; Zuo, Xiaofeng; Huang, Liwei; McKenna, Sarah; Lingappa, Vishwanath R.; Lipschutz, Joshua H.

    2013-01-01

    Background Protein translation and translocation at the rough endoplasmic reticulum (RER) are the first steps in the secretory pathway. The translocon through which newly-made proteins are translocated into or across the RER membrane, consists of three main subunits, Sec61α, β, and γ. Sec61β facilitates translocation, and we and others showed that the highly-conserved eight protein exocyst complex interacts with Sec61β. We also showed that the exocyst was involved in basolateral, and not apical, protein synthesis and delivery. Recently, however, exocyst involvement in apical protein delivery was reported. Furthermore, we showed that the exocyst was necessary for formation of primary cilia, organelles found on the apical surface. Methods GST pulldown was performed on lysate of renal tubule cells to investigate biochemical interactions. Cell-free assays consisting of cell-free extracts from rabbit reticulocytes, pancreatic ER microsomal membranes, transcripts of cDNA from apical and basolateral proteins, ATP/GTP, amino acids, and 35S-methionine for protein detection, were used to investigate the role of the exocyst in synthesis of polarized proteins. P32-orthophosphate and immunoprecipitation with antibody against Sec61β was used to investigate the Sec61β phosphorylation in exocyst Sec10-overexpressing cells. Results Sec10 biochemically interacts with Sec61β using GST pulldown. Using cell-free assays, there is enhanced recruitment to ER membranes following exocyst depletion and basolateral VSVG protein translation, compared to apical HA protein translation. Finally, Sec10 overexpression increases Sec61β phosphorylation. Conclusion These data confirm that the exocyst is preferentially involved in basolateral protein translation and translocation, and may well act through the phosphorylation of Sec61β. PMID:23037926

  1. Clotting protein - An extracellular matrix (ECM) protein involved in crustacean hematopoiesis.

    PubMed

    Junkunlo, Kingkamon; Söderhäll, Kenneth; Söderhäll, Irene

    2017-09-21

    Hematopoietic progenitor cells in crustaceans are organized in lobule-like structures surrounded by different types of cells and extracellular matrix (ECM) protein in a Hematopoietic tissue (HPT). Here we show that the clotting protein (CP) is part of the ECM in HPT and is secreted during HPT cell culture. The formation of a filamentous network of CP was observed in HPT cell culture. A high amount of CP protein was detected at the surfaces of undifferentiated cells (round-shaped) compared with migrating cells (spindle shaped). Co-localization of the CP protein and TGase activity was observed on the cell surface and filamentous network between cells. A role for CP together with collagen was revealed in a 3D culture in which a collagen-I matrix was immobilized with CP or supplemented with CP. The results showed possible functions of CP, collagen, TGase and cytokine Ast1 in the regulation of HPT progenitor cell behavior. This is the first study to provide insight into the role of CP, which probably not only participates in clot formation but also functions as an ECM component protein controlling hematopoietic stem cell behavior. Copyright © 2017. Published by Elsevier Ltd.

  2. FRG1P-mediated aggregation of proteins involved in pre-mRNA processing.

    PubMed

    van Koningsbruggen, Silvana; Straasheijm, Kirsten R; Sterrenburg, Ellen; de Graaf, Natascha; Dauwerse, Hans G; Frants, Rune R; van der Maarel, Silvère M

    2007-02-01

    FRG1 is considered a candidate gene for facioscapulohumeral muscular dystrophy (FSHD) based on its location at chromosome 4qter and its upregulation in FSHD muscle. The FRG1 protein (FRG1P) localizes to nucleoli, Cajal bodies (and speckles), and has been suggested to be a component of the human spliceosome but its exact function is unknown. Recently, transgenic mice overexpressing high levels of FRG1P in skeletal muscle were described to present with muscular dystrophy. Moreover, upregulation of FRG1P was demonstrated to correlate with missplicing of specific pre-mRNAs. In this study, we have combined colocalization studies with yeast two-hybrid screens to identify proteins that associate with FRG1P. We demonstrate that artificially induced nucleolar aggregates of VSV-FRG1P specifically sequester proteins involved in pre-mRNA processing. In addition, we have identified SMN, PABPN1, and FAM71B, a novel speckle and Cajal body protein, as binding partners of FRG1P. All these proteins are, or seem to be, involved in RNA biogenesis. Our data confirm the presence of FRG1P in protein complexes containing human spliceosomes and support a potential role of FRG1P in either splicing or another step in nuclear RNA biogenesis. Intriguingly, among FRG1P-associated proteins are SMN and PABPN1, both being involved in neuromuscular disorders, possibly through RNA biogenesis-related processes.

  3. Pressure-temperature folding landscape in proteins involved in neurodegenerative diseases and cancer.

    PubMed

    Cordeiro, Yraima; Foguel, Debora; Silva, Jerson L

    2013-12-15

    High hydrostatic pressure (HHP) is a valuable tool to study processes such as protein folding, protein hydration and protein-protein interactions. HHP is a nondestructive technique because it reversibly affects internal cavities excluded from the solvent present in the hydrophobic core of proteins. HHP allows the solvation of buried amino acid side chains, thus shifting the equilibrium towards states of the studied molecule or molecular ensemble that occupy smaller volumes. HHP has long been used to dissociate multimeric proteins and protein aggregates and allows investigation of intermediate folding states, some of which are formed by proteins involved in human degenerative diseases, such as spongiform encephalopathies and Parkinson's disease, as well as cancer. When coupled with nuclear magnetic resonance and spectroscopic methods such as infrared and fluorescence spectroscopy, HHP treatment facilitates the understanding of protein folding and misfolding processes; the latter is related to protein aggregation into amyloid or amorphous species. In this review, we will address how HHP provides information about intermediate folding states and the aggregation processes of p53, which is related to cancer, and prion proteins, transthyretin and α-synuclein, which are related to human degenerative diseases.

  4. Purification of recombinant BtpA and Ycf3, proteins involved in membrane protein biogenesis in Synechocystis PCC 6803.

    PubMed

    Schwabe, Tatjana M E; Gloddek, Kirsten; Schluesener, Daniela; Kruip, Jochen

    2003-03-25

    The gene products Ycf3 (hypothetical chloroplast open reading frame) and BtpA (biogenesis of thylakoid protein) are thought to be involved in the biogenesis of the membrane protein complex photosystem I (PSI) from Synechocystis PCC 6803. PSI consists of 12 different subunits and binds more than 100 cofactors, making it a model protein to study different aspects of membrane protein biogenesis. For a detailed biophysical characterization of Ycf3 and BtpA pure proteins must be available in sufficient quantities. Therefore we cloned the corresponding genes into expression vectors. To facilitate purification we created His-tagged versions of Ycf3 and BtpA in addition to the unmodified forms. Immobilized metal affinity chromatography (IMAC) yielded His-tagged proteins which were used for the production of antibodies. Purification strategies for non-tagged proteins could also be established: Ycf3 could be purified in soluble form using a two-step purification in which ammonium sulfate precipitation was combined with anion-exchange chromatography (IEC). BtpA had to be purified from inclusion bodies by two-consecutive IEC steps under denaturing conditions. An optimized refolding protocol was established that yielded pure BtpA. In all cases, MALDI-TOF peptide mass fingerprinting (PMF) was used to confirm protein identity. Initially, size exclusion chromatography and CD-spectroscopy were used for biophysical characterization of the proteins. Both Ycf3 and BtpA show homo-oligomerization in vitro. In summary, purification protocols for Ycf3 and BtpA have been designed that yield pure proteins which can be used to probe the molecular function of these proteins for membrane protein biogenesis.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-10-18

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

  7. Involvement of a small GTP binding protein in HIV-1 release

    PubMed Central

    Audoly, Gilles; Popoff, Michel R; Gluschankof, Pablo

    2005-01-01

    Background There is evidence suggesting that actin binding to HIV-1 encoded proteins, or even actin dynamics themselves, might play a key role in virus budding and/or release from the infected cell. A crucial step in the reorganisation of the actin cytoskeleton is the engagement of various different GTP binding proteins. We have thus studied the involvement of GTP-binding proteins in the final steps of the HIV-1 viral replication cycle. Results Our results demonstrate that virus production is abolished when cellular GTP binding proteins involved in actin polymerisation are inhibited with specific toxins. Conclusion We propose a new HIV budding working model whereby Gag interactions with pre-existing endosomal cellular tracks as well as with a yet non identified element of the actin polymerisation pathway are required in order to allow HIV-1 to be released from the infected cell. PMID:16080789

  8. 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.

  9. 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

  10. Proteins involved in motility and sperm-egg interaction evolve more rapidly in mouse spermatozoa.

    PubMed

    Vicens, Alberto; Lüke, Lena; Roldan, Eduardo R S

    2014-01-01

    Proteomic studies of spermatozoa have identified a large catalog of integral sperm proteins. Rapid evolution of these proteins may underlie adaptive changes of sperm traits involved in different events leading to fertilization, although the selective forces underlying such rapid evolution are not well understood. A variety of selective forces may differentially affect several steps ending in fertilization, thus resulting in a compartmentalized adaptation of sperm proteins. Here we analyzed the evolution of genes associated to various events in the sperm's life, from sperm formation to sperm-egg interaction. Evolutionary analyses were performed on gene sequences from 17 mouse strains whose genomes have been sequenced. Four of these are derived from wild Mus musculus, M. domesticus, M. castaneus and M. spretus. We found a higher proportion of genes exhibiting a signature of positive selection among those related to sperm motility and sperm-egg interaction. Furthermore, sperm proteins involved in sperm-egg interaction exhibited accelerated evolution in comparison to those involved in other events. Thus, we identified a large set of candidate proteins for future comparative analyses of genotype-phenotype associations in spermatozoa of species subjected to different sexual selection pressures. Adaptive evolution of proteins involved in motility could be driven by sperm competition, since this selective force is known to increase the proportion of motile sperm and their swimming velocity. On the other hand, sperm proteins involved in gamete interaction could be coevolving with their egg partners through episodes of sexual selection or sexual conflict resulting in species-specific sperm-egg interactions and barriers preventing interspecies fertilization.

  11. 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

  12. A human skeletal muscle interactome centered on proteins involved in muscular dystrophies: LGMD interactome

    PubMed Central

    2013-01-01

    Background The complexity of the skeletal muscle and the identification of numerous human disease-causing mutations in its constitutive proteins make it an interesting tissue for proteomic studies aimed at understanding functional relationships of interacting proteins in both health and diseases. Method We undertook a large-scale study using two-hybrid screens and a human skeletal-muscle cDNA library to establish a proteome-scale map of protein-protein interactions centered on proteins involved in limb-girdle muscular dystrophies (LGMD). LGMD is a group of more than 20 different neuromuscular disorders that principally affect the proximal pelvic and shoulder girdle muscles. Results and conclusion The interaction network we unraveled incorporates 1018 proteins connected by 1492 direct binary interactions and includes 1420 novel protein-protein interactions. Computational, experimental and literature-based analyses were performed to assess the overall quality of this network. Interestingly, LGMD proteins were shown to be highly interconnected, in particular indirectly through sarcomeric proteins. In-depth mining of the LGMD-centered interactome identified new candidate genes for orphan LGMDs and other neuromuscular disorders. The data also suggest the existence of functional links between LGMD2B/dysferlin and gene regulation, between LGMD2C/γ-sarcoglycan and energy control and between LGMD2G/telethonin and maintenance of genome integrity. This dataset represents a valuable resource for future functional investigations. PMID:23414517

  13. Pin1: Intimate involvement with the regulatory protein kinase networks in the global phosphorylation landscape.

    PubMed

    Litchfield, David W; Shilton, Brian H; Brandl, Christopher J; Gyenis, Laszlo

    2015-10-01

    Protein phosphorylation is a universal regulatory mechanism that involves an extensive network of protein kinases. The discovery of the phosphorylation-dependent peptidyl-prolyl isomerase Pin1 added an additional layer of complexity to these regulatory networks. We have evaluated interactions between Pin1 and the regulatory kinome and proline-dependent phosphoproteome taking into consideration findings from targeted studies as well as data that has emerged from systematic phosphoproteomic workflows and from curated protein interaction databases. The relationship between Pin1 and the regulatory protein kinase networks is not restricted simply to the recognition of proteins that are substrates for proline-directed kinases. In this respect, Pin1 itself is phosphorylated in cells by protein kinases that modulate its functional properties. Furthermore, the phosphorylation-dependent targets of Pin1 include a number of protein kinases as well as other enzymes such as phosphatases and regulatory subunits of kinases that modulate the actions of protein kinases. As a result of its interactions with numerous protein kinases and their substrates, as well as itself being a target for phosphorylation, Pin1 has an intricate relationship with the regulatory protein kinase and phosphoproteomic networks that orchestrate complex cellular processes and respond to environmental cues. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. 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.

  15. Multiple proteins of White spot syndrome virus involved in recognition of beta-integrin.

    PubMed

    Zhang, Jing-Yan; Liu, Qing-Hui; Huang, Jie

    2014-06-01

    The recognition and attachment of virus to its host cell surface is a critical step for viral infection. Recent research revealed that beta-integrin was involved in White spot syndrome virus (WSSV) infection. In this study, the interaction of beta-integrin with structure proteins of WSSV and motifs involved in WSSV infection was examined. The results showed that envelope proteins VP26, VP31, VP37, VP90 and nucleocapsid protein VP136 interacted with LvInt. RGD-, YGL- and LDV-related peptide functioned as motifs of WSSV proteins binding with beta-integrin. The beta-integrin ligand of RGDT had better blocking effect compared with that of YGL- and LDV-related peptides. In vivo assay indicated that RGD-, LDV- and YGL-related peptides could partially block WSSV infection. These data collectively indicate that multiple proteins were involved in recognition of beta-integrin. Identification of proteins in WSSV that are associated with beta-integrin will assist development of new agents for effective control of the white spot syndrome.

  16. Not all mitochondrial carrier proteins support permeability transition pore formation: no involvement of uncoupling protein 1.

    PubMed

    Crichton, Paul G; Parker, Nadeene; Vidal-Puig, Antonio J; Brand, Martin D

    2009-12-15

    The mPTP (mitochondrial permeability transition pore) is a non-specific channel that is formed in the mitochondrial inner membrane in response to several stimuli, including elevated levels of matrix calcium. The pore is proposed to be composed of the ANT (adenine nucleotide translocase), voltage-dependent anion channel and cyclophilin D. Knockout studies, however, have demonstrated that ANT is not essential for permeability transition, which has led to the proposal that other members of the mitochondrial carrier protein family may be able to play a similar function to ANT in pore formation. To investigate this possibility, we have studied the permeability transition properties of BAT (brown adipose tissue) mitochondria in which levels of the mitochondrial carrier protein, UCP1 (uncoupling protein 1), can exceed those of ANT. Using an improved spectroscopic assay, we have quantified mPTP formation in de-energized mitochondria from wild-type and Ucp1KO (Ucp1-knockout) mice and assessed the dependence of pore formation on UCP1. When correctly normalized for differences in mitochondrial morphology, we find that calcium-induced mPTP activity is the same in both types of mitochondria, with similar sensitivity to GDP (approximately 50% inhibited), although the portion sensitive to cyclosporin A is higher in mitochondria lacking UCP1 (approximately 80% inhibited, compared with approximately 60% in mitochondria containing UCP1). We conclude that UCP1 is not a component of the cyclosporin A-sensitive mPTP in BAT and that playing a role in mPTP formation is not a general characteristic of the mitochondrial carrier protein family but is, more likely, restricted to specific members including ANT.

  17. Protein-protein interactions involved in the recognition of p27 by E3 ubiquitin ligase.

    PubMed Central

    Xu, Kui; Belunis, Charles; Chu, Wei; Weber, David; Podlaski, Frank; Huang, Kuo-Sen; Reed, Steven I; Vassilev, Lyubomir T

    2003-01-01

    The p27(Kip1) protein is a potent cyclin-dependent kinase inhibitor, the level of which is decreased in many common human cancers as a result of enhanced ubiquitin-dependent degradation. The multiprotein complex SCF(Skp2) has been identified as the ubiquitin ligase that targets p27, but the functional interactions within this complex are not well understood. One component, the F-box protein Skp2, binds p27 when the latter is phosphorylated on Thr(187), thus providing substrate specificity for the ligase. Recently, we and others have shown that the small cell cycle regulatory protein Cks1 plays a critical role in p27 ubiquitination by increasing the binding affinity of Skp2 for p27. Here we report the development of a homogeneous time-resolved fluorescence assay that allows the quantification of the molecular interactions between human recombinant Skp2, Cks1 and a p27-derived peptide phosphorylated on Thr(187). Using this assay, we have determined the dissociation constant of the Skp2-Cks1 complex (K(d) 140 +/- 14 nM) and have shown that Skp2 binds phosphorylated p27 peptide with high affinity only in the presence of Cks1 (K(d) 37 +/- 2 nM). Cks1 does not bind directly to the p27 phosphopeptide or to Skp1, which confirms its suggested role as an allosteric effector of Skp2. PMID:12529174

  18. 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.

  19. 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.

  20. 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

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

    PubMed

    Zienkiewicz, Agnieszka; Zienkiewicz, Krzysztof; Rejón, Juan David; Alché, Juan de Dios; Castro, Antonio Jesús; 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.

  2. Characterization of a DNA binding protein of bacteriophage PRD1 involved in DNA replication.

    PubMed Central

    Pakula, T M; Caldentey, J; Serrano, M; Gutierrez, C; Hermoso, J M; Salas, M; Bamford, D H

    1990-01-01

    Escherichia coli phage PRD1 protein P12, involved in PRD1 DNA replication in vivo, has been highly purified from E. coli cells harbouring a gene XII-containing plasmid. Protein P12 binds to single-stranded DNA as shown by gel retardation assays and nuclease protection experiments. Binding of protein P12 to single-stranded DNA increases about 14% the contour length of the DNA as revealed by electron microscopy. Binding to single-stranded DNA seems to be cooperative, and it is not sequence specific. Protein P12 also binds to double-stranded DNA although with an affinity 10 times lower than to single-stranded DNA. Using the in vitro phage phi 29 DNA replication system, it is shown that protein P12 stimulates the overall phi 29 DNA replication. Images PMID:2251117

  3. 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

  4. A Cellulose Synthase-Like Protein Involved in Hyphal Tip Growth and Morphological Differentiation in Streptomyces▿

    PubMed Central

    Xu, Hongbin; Chater, Keith F.; Deng, Zixin; Tao, Meifeng

    2008-01-01

    Cellulose synthase and cellulose synthase-like proteins, responsible for synthesizing β-glucan-containing polysaccharides, play a fundamental role in cellular architectures, such as plant cell and tissue morphogenesis, bacterial biofilm formation, and fruiting-body development. However, the roles of the proteins involved in the developmental process are not well understood. Here, we report that a cellulose synthase-like protein (CslASc) in Streptomyces has a function in hyphal tip growth and morphological differentiation. The cslASc replacement mutant showed pleiotropic defects, including the severe delay of aerial-hyphal formation and altered cell wall morphology. Calcofluor white fluorescence analysis demonstrated that polysaccharide synthesis at hyphal tips was dependent on CslASc. cslASc was constitutively transcribed, and an enhanced green fluorescent protein-CslASc fusion protein was mostly located at the hyphal tips. An extract enriched in morphogenetic chaplin proteins promoted formation of aerial hyphae by the mutant. Furthermore, a two-hybrid experiment indicated that the glycosyltransferase domain of CslASc interacted with the tropomyosin-like polarity-determining DivIVA protein, suggesting that the tip-located DivIVA governed tip recruitment of the CslASc membrane protein. These results imply that the cellulose synthase-like protein couples extracellular and cytoskeletal components functioning in tip growth and cell development. PMID:18487344

  5. Involvement of a tissue-specific RNA recognition motif protein in Drosophila spermatogenesis.

    PubMed Central

    Haynes, S R; Cooper, M T; Pype, S; Stolow, D T

    1997-01-01

    RNA binding proteins mediate posttranscriptional regulation of gene expression via their roles in nuclear and cytoplasmic mRNA metabolism. Many of the proteins involved in these processes have a common RNA binding domain, the RNA recognition motif (RRM). We have characterized the Testis-specific RRM protein gene (Tsr), which plays an important role in spermatogenesis in Drosophila melanogaster. Disruption of Tsr led to a dramatic reduction in male fertility due to the production of spermatids with abnormalities in mitochondrial morphogenesis. Tsr is located on the third chromosome at 87F, adjacent to the nuclear pre-mRNA binding protein gene Hrb87F. A 1.7-kb Tsr transcript was expressed exclusively in the male germ line. It encoded a protein containing two RRMs similar to those found in HRB87F as well as a unique C-terminal domain. TSR protein was located in the cytoplasm of spermatocytes and young spermatids but was absent from mature sperm. The cellular proteins expressed in premeiotic primary spermatocytes from Tsr mutant and wild-type males were assessed by two-dimensional gel electrophoresis. Lack of TSR resulted in the premature expression of a few proteins prior to meiosis; this was abolished by a transgenic copy of Tsr. These data demonstrate that TSR negatively regulated the expression of some testis proteins and, in combination with its expression pattern and subcellular localization, suggest that TSR regulates the stability or translatability of some mRNAs during spermatogenesis. PMID:9111341

  6. Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map.

    PubMed

    Collins, Sean R; Miller, Kyle M; Maas, Nancy L; Roguev, Assen; Fillingham, Jeffrey; Chu, Clement S; Schuldiner, Maya; Gebbia, Marinella; Recht, Judith; Shales, Michael; Ding, Huiming; Xu, Hong; Han, Junhong; Ingvarsdottir, Kristin; Cheng, Benjamin; Andrews, Brenda; Boone, Charles; Berger, Shelley L; Hieter, Phil; Zhang, Zhiguo; Brown, Grant W; Ingles, C James; Emili, Andrew; Allis, C David; Toczyski, David P; Weissman, Jonathan S; Greenblatt, Jack F; Krogan, Nevan J

    2007-04-12

    Defining the functional relationships between proteins is critical for understanding virtually all aspects of cell biology. Large-scale identification of protein complexes has provided one important step towards this goal; however, even knowledge of the stoichiometry, affinity and lifetime of every protein-protein interaction would not reveal the functional relationships between and within such complexes. Genetic interactions can provide functional information that is largely invisible to protein-protein interaction data sets. Here we present an epistatic miniarray profile (E-MAP) consisting of quantitative pairwise measurements of the genetic interactions between 743 Saccharomyces cerevisiae genes involved in various aspects of chromosome biology (including DNA replication/repair, chromatid segregation and transcriptional regulation). This E-MAP reveals that physical interactions fall into two well-represented classes distinguished by whether or not the individual proteins act coherently to carry out a common function. Thus, genetic interaction data make it possible to dissect functionally multi-protein complexes, including Mediator, and to organize distinct protein complexes into pathways. In one pathway defined here, we show that Rtt109 is the founding member of a novel class of histone acetyltransferases responsible for Asf1-dependent acetylation of histone H3 on lysine 56. This modification, in turn, enables a ubiquitin ligase complex containing the cullin Rtt101 to ensure genomic integrity during DNA replication.

  7. 3DSwap: curated knowledgebase of proteins involved in 3D domain swapping.

    PubMed

    Shameer, Khader; Shingate, Prashant N; Manjunath, S C P; Karthika, M; Pugalenthi, Ganesan; Sowdhamini, Ramanathan

    2011-01-01

    Three-dimensional domain swapping is a unique protein structural phenomenon where two or more protein chains in a protein oligomer share a common structural segment between individual chains. This phenomenon is observed in an array of protein structures in oligomeric conformation. Protein structures in swapped conformations perform diverse functional roles and are also associated with deposition diseases in humans. We have performed in-depth literature curation and structural bioinformatics analyses to develop an integrated knowledgebase of proteins involved in 3D domain swapping. The hallmark of 3D domain swapping is the presence of distinct structural segments such as the hinge and swapped regions. We have curated the literature to delineate the boundaries of these regions. In addition, we have defined several new concepts like 'secondary major interface' to represent the interface properties arising as a result of 3D domain swapping, and a new quantitative measure for the 'extent of swapping' in structures. The catalog of proteins reported in 3DSwap knowledgebase has been generated using an integrated structural bioinformatics workflow of database searches, literature curation, by structure visualization and sequence-structure-function analyses. The current version of the 3DSwap knowledgebase reports 293 protein structures, the analysis of such a compendium of protein structures will further the understanding molecular factors driving 3D domain swapping.

  8. Fusions involving protein kinase C and membrane-associated proteins in benign fibrous histiocytoma.

    PubMed

    Płaszczyca, Anna; Nilsson, Jenny; Magnusson, Linda; Brosjö, Otte; Larsson, Olle; Vult von Steyern, Fredrik; Domanski, Henryk A; Lilljebjörn, Henrik; Fioretos, Thoas; Tayebwa, Johnbosco; Mandahl, Nils; Nord, Karolin H; Mertens, Fredrik

    2014-08-01

    Benign fibrous histiocytoma (BFH) is a mesenchymal tumor that most often occurs in the skin (so-called dermatofibroma), but may also appear in soft tissues (so-called deep BFH) and in the skeleton (so-called non-ossifying fibroma). The origin of BFH is unknown, and it has been questioned whether it is a true neoplasm. Chromosome banding, fluorescence in situ hybridization, single nucleotide polymorphism arrays, RNA sequencing, RT-PCR and quantitative real-time PCR were used to search for recurrent somatic mutations in a series of BFH. BFHs were found to harbor recurrent fusions of genes encoding membrane-associated proteins (podoplanin, CD63 and LAMTOR1) with genes encoding protein kinase C (PKC) isoforms PRKCB and PRKCD. PKCs are serine-threonine kinases that through their many phosphorylation targets are implicated in a variety of cellular processes, as well as tumor development. When inactive, the amino-terminal, regulatory domain of PKCs suppresses the activity of their catalytic domain. Upon activation, which requires several steps, they typically translocate to cell membranes, where they interact with different signaling pathways. The detected PDPN-PRKCB, CD63-PRKCD and LAMTOR1-PRKCD gene fusions are all predicted to result in chimeric proteins consisting of the membrane-binding part of PDPN, CD63 or LAMTOR1 and the entire catalytic domain of the PKC. This novel pathogenetic mechanism should result in constitutive kinase activity at an ectopic location. The results show that BFH indeed is a true neoplasm, and that distorted PKC activity is essential for tumorigenesis. The findings also provide means to differentiate BFH from other skin and soft tissue tumors. This article is part of a Directed Issue entitled: Rare cancers.

  9. 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.…

  10. 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.…

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

    USDA-ARS?s Scientific Manuscript database

    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...

  12. A novel family of mammalian transmembrane proteins involved in cholesterol transport.

    PubMed

    Méndez-Acevedo, Kevin M; Valdes, Victor Julián; Asanov, Alexander; Vaca, Luis

    2017-08-07

    Cholesterol is an essential compound in mammalian cells because it is involved in a wide range of functions, including as a key component of membranes, precursor of important molecules such as hormones, bile acids and vitamin D. The cholesterol transport across the circulatory system is a well-known process in contrast to the intracellular cholesterol transport, which is poorly understood. Recently in our laboratory, we identified a novel protein in C. elegans involved in dietary cholesterol uptake, which we have named ChUP-1. Insillicoanalysis identified two putative orthologue candidate proteins in mammals. The proteins SIDT1 and SIDT2 share identity and conserved cholesterol binding (CRAC) domains with C. elegans ChUP-1. Both mammalian proteins are annotated as RNA transporters in databases. In the present study, we show evidence indicating that SIDT1 and SIDT2 not only do not transport RNA, but they are involved in cholesterol transport. Furthermore, we show that single point mutations directed to disrupt the CRAC domains of both proteins prevent FRET between SIDT1 and SIDT2 and the cholesterol analogue dehydroergosterol (DHE) and alter cholesterol transport.

  13. Pdsg1 and Pdsg2, Novel Proteins Involved in Developmental Genome Remodelling in Paramecium

    PubMed Central

    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

  14. 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.

  15. 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.

  16. Differential impact of REM sleep deprivation on cytoskeletal proteins of brain regions involved in sleep regulation.

    PubMed

    Rodríguez-Vázquez, Jennifer; Camacho-Arroyo, Ignacio; Velázquez-Moctezuma, Javier

    2012-01-01

    Rapid eye movement (REM) sleep is involved in memory consolidation, which implies synaptic plasticity. This process requires protein synthesis and the reorganization of the neural cytoskeleton. REM sleep deprivation (REMSD) has an impact on some neuronal proteins involved in synaptic plasticity, such as glutamate receptors and postsynaptic density protein 95, but its effects on cytoskeletal proteins is unknown. In this study, the effects of REMSD on the content of the cytoskeletal proteins MAP2 and TAU were analyzed. Adult female rats were submitted to selective REMSD by using the multiple platform technique. After 24, 48 or 72 h of REMSD, rats were decapitated and the following brain areas were dissected: pons, preoptic area, hippocampus and frontal cortex. Protein extraction and Western blot were performed. Results showed an increase in TAU content in the pons, preoptic area and hippocampus after 24 h of REMSD, while in the frontal cortex a significant increase in TAU content was observed after 72 h of REMSD. A TAU content decrease was observed in the hippocampus after 48 h of REMSD. Interestingly, a marked increase in TAU content was observed after 72 h of REMSD. MAP2 content only increased in the preoptic area at 24 h, and in the frontal cortex after 24 and 72 h of REMSD, without significant changes in the pons and hippocampus. These results support the idea that REM sleep plays an important role in the organization of neural cytoskeleton, and that this effect is tissue-specific.

  17. Recognition of nontrivial remote homology relationships involving proteins of Helicobacter pylori: implications for function recognition.

    PubMed

    Tyagi, Nidhi; Srinivasan, Narayanaswamy

    2013-01-01

    This chapter explains techniques for recognition of nontrivial remote homology relationships involving proteins of Helicobacter pylori and their implications for function recognition. Using the remote homology detection method, employing multiple-profile representations for every protein domain family, remotely related domain family information has been assigned for the 122, 77, and 95 protein sequences of 26695, and J99, and HPAG1 strains of H. pylori, respectively. Relationships for some of the H. pylori protein sequences with Pfam domain families are reported for the first time. In publicly available domain databases such as Pfam, for some of the H. pylori protein sequences functional domain information is associated only with part(s) of the proteins. In the current study other parts of such proteins have been shown to be remotely related to known domain families, raising the possibility of identifying functions for parts of the proteins that do not yet have domains assigned. Further, homologues of enzymes that potentially catalyze step(s) in various metabolic processes in H. pylori have been identified for the first time.

  18. Proteomic Analysis Reveals Proteins Involved in Seed Imbibition under Salt Stress in Rice.

    PubMed

    Xu, Enshun; Chen, Mingming; He, Hui; Zhan, Chengfang; Cheng, Yanhao; Zhang, Hongsheng; Wang, Zhoufei

    2016-01-01

    Enhancement of salinity tolerance during seed germination is very important for direct seeding in rice. In this study, the salt-tolerant japonica landrace Jiucaiqing was used to determine the regulators that are involved in seed imbibition under salt stress. Briefly, the comparative proteomic analysis was conducted between dry (0 h) and imbibed (24 h) seeds with 150 mM NaCl. Under salt stress, the uptake of water increased rapidly before 24 h imbibition (Phase I), followed by a plateau of seed imbibition from 24 to 96 h imbibition (Phase II). We identified 14 proteins involved in seed imbibition, in which the majority of these proteins were involved in energy supply and storage protein. The early imbibition process was mediated by protein catabolism; the most of proteins were down-regulated after 24 h imbibition. Eleven genes in salt stress treated seeds were expressed early during the seed imbibition in comparison to control seeds. By comparison, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (BPM), glutelin (GLU2.2 and GLU2.3), glucose-1-phosphate adenylyltransferase large subunit (GAS8), and cupin domain containing protein (CDP3.1 and CDP3.2) were near the regions of quantitative trait loci (QTLs) for seed dormancy, seed reserve utilization, and seed germination in Jiucaiqing. In particular, CDP3.1 was co-located in the region of qIR-3 for imbibition rate, and qGP-3 for germination percentage. The role of CDP3.1 was verified in enhancing seed germination under salt stress using T-DNA mutant. The identified proteins might be applicable for the improvement of seed germination under salt stress in rice.

  19. Proteomic Analysis Reveals Proteins Involved in Seed Imbibition under Salt Stress in Rice

    PubMed Central

    Xu, Enshun; Chen, Mingming; He, Hui; Zhan, Chengfang; Cheng, Yanhao; Zhang, Hongsheng; Wang, Zhoufei

    2017-01-01

    Enhancement of salinity tolerance during seed germination is very important for direct seeding in rice. In this study, the salt-tolerant japonica landrace Jiucaiqing was used to determine the regulators that are involved in seed imbibition under salt stress. Briefly, the comparative proteomic analysis was conducted between dry (0 h) and imbibed (24 h) seeds with 150 mM NaCl. Under salt stress, the uptake of water increased rapidly before 24 h imbibition (Phase I), followed by a plateau of seed imbibition from 24 to 96 h imbibition (Phase II). We identified 14 proteins involved in seed imbibition, in which the majority of these proteins were involved in energy supply and storage protein. The early imbibition process was mediated by protein catabolism; the most of proteins were down-regulated after 24 h imbibition. Eleven genes in salt stress treated seeds were expressed early during the seed imbibition in comparison to control seeds. By comparison, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (BPM), glutelin (GLU2.2 and GLU2.3), glucose-1-phosphate adenylyltransferase large subunit (GAS8), and cupin domain containing protein (CDP3.1 and CDP3.2) were near the regions of quantitative trait loci (QTLs) for seed dormancy, seed reserve utilization, and seed germination in Jiucaiqing. In particular, CDP3.1 was co-located in the region of qIR-3 for imbibition rate, and qGP-3 for germination percentage. The role of CDP3.1 was verified in enhancing seed germination under salt stress using T-DNA mutant. The identified proteins might be applicable for the improvement of seed germination under salt stress in rice. PMID:28105039

  20. Paraoxonase 1 and dietary hyperhomocysteinemia modulate the expression of mouse proteins involved in liver homeostasis.

    PubMed

    Suszyńska-Zajczyk, Joanna; Jakubowski, Hieronim

    2014-01-01

    Homocysteine (Hcy), a product of methionine metabolism, is elevated by the consumption of a high-methionine diet that can cause fatty liver disease. Paraoxonase 1 (Pon1), a hydrolase expressed mainly in the liver and carried in the circulation on high-density lipoprotein, participates in Hcy metabolism. Low Pon1 activity is linked to fatty liver disease. We hypothesize that hyperhomocysteinemia and low Pon1 induce changes in gene expression that could impair liver homeostasis. To test this hypothesis, we analyzed the liver proteome of Pon1(-/-) and Pon1(+/+) mice fed a high methionine diet (1% methionine in the drinking water) for 8 weeks using 2D IEF/SDS-PAGE gel electrophoresis and MALDI-TOF mass spectrometry. We identified seven liver proteins whose expression was significantly altered in Pon1(-/-) mice. In animals fed with a control diet, the expression of three liver proteins involved in lipoprotein metabolism (ApoE), iron metabolism (Ftl), and regulation of nitric oxide generation (Ddah1) was up-regulated by the Pon1(-/-) genotype. In mice fed with a high-methionine diet, expression of four liver proteins was up-regulated and of three proteins was down-regulated by the Pon1(-/-) genotype. The up-regulated proteins are involved in lipoprotein metabolism (ApoE), energy metabolism (Atp5h), oxidative stress response (Prdx2), and nitric oxide regulation (Ddah1). The down-regulated proteins are involved in energy metabolism (Gamt), iron metabolism (Ftl), and catechol metabolism (Comt). Expression of one protein (Ftl) was up-regulated both by the Pon1(-/-) genotype and a high-methionine diet. Our findings suggest that Pon1 interacts with diverse cellular processes - from lipoprotein metabolism, nitric oxide regulation, and energy metabolism to iron transport and antioxidant defenses - that are essential for normal liver homeostasis and modulation of these interactions by a high-methionine diet may contribute to fatty liver disease.

  1. Translationally Controlled Tumor Protein, a Dual Functional Protein Involved in the Immune Response of the Silkworm, Bombyx mori

    PubMed Central

    Hua, Xiaoting; Song, Liang; Xia, Qingyou

    2013-01-01

    Insect gut immunity is the first line of defense against oral infection. Although a few immune-related molecules in insect intestine has been identified by genomics or proteomics approach with comparison to well-studied tissues, such as hemolymph or fat body, our knowledge about the molecular mechanism underlying the gut immunity which would involve a variety of unidentified molecules is still limited. To uncover additional molecules that might take part in pathogen recognition, signal transduction or immune regulation in insect intestine, a T7 phage display cDNA library of the silkworm midgut is constructed. By use of different ligands for biopanning, Translationally Controlled Tumor Protein (TCTP) has been selected. BmTCTP is produced in intestinal epithelial cells and released into the gut lumen. The protein level of BmTCTP increases at the early time points during oral microbial infection and declines afterwards. In vitro binding assay confirms its activity as a multi-ligand binding molecule and it can further function as an opsonin that promotes the phagocytosis of microorganisms. Moreover, it can induce the production of anti-microbial peptide via a signaling pathway in which ERK is required and a dynamic tyrosine phosphorylation of certain cytoplasmic membrane protein. Taken together, our results characterize BmTCTP as a dual-functional protein involved in both the cellular and the humoral immune response of the silkworm, Bombyx mori. PMID:23894441

  2. Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation

    PubMed Central

    Blondel, Marc; Soubigou, Flavie; Evrard, Justine; Nguyen, Phu hai; Hasin, Naushaba; Chédin, Stéphane; Gillet, Reynald; Contesse, Marie-Astrid; Friocourt, Gaëlle; Stahl, Guillaume; Jones, Gary W.; Voisset, Cécile

    2016-01-01

    6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI+] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI+]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI+] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases. PMID:27633137

  3. Silkmapin of Hyriopsis cumingii, a novel silk-like shell matrix protein involved in nacre formation.

    PubMed

    Liu, Xiaojun; Dong, Shaojian; Jin, Can; Bai, Zhiyi; Wang, Guiling; Li, Jiale

    2015-01-25

    Understanding the role of matrix proteins in nacre formation and biomineralization in mollusks is important for the pearl industry. In this study, the gene encoding the novel Hyriopsis cumingii shell matrix protein silkmapin was characterized. The gene encodes a protein of 30.89kDa in which Gly accounts for 34.41% of the amino acid content, and the C-terminal region binds Ca(2+). Secondary structure prediction indicated a predominantly β-fold and a structure typical of filamentous proteins. Real-time quantitative PCR and in situ hybridization showed that silkmapin was expressed in epithelial cells at the edge and pallial of mantle tissue, indicated that silkmapin play roles in the shell nacreous and prismatic layer formation. Further real-time PCR results indicated an involvement in pearl formation via nucleation of calcium carbonate prior to formation of the nacre.

  4. Preliminary Characterization of MEDLE-2, a Protein Potentially Involved in the Invasion of Cryptosporidium parvum

    PubMed Central

    Li, Baoling; Wu, Haizhen; Li, Na; Su, Jiayuan; Jia, Ruilian; Jiang, Jianlin; Feng, Yaoyu; Xiao, Lihua

    2017-01-01

    Cryptosporidium spp. are important causes of diarrhea in humans, ruminants, and other mammals. Comparative genomic analysis indicated that genetically related and host-adapted Cryptosporidium species have different numbers of subtelomeric genes encoding the Cryptosporidium-specific MEDLE family of secreted proteins, which could contribute to differences in host specificity. In this study, a Cryptosporidium parvum-specific member of the protein family MEDLE-2 encoded by cgd5_4590 was cloned and expressed in Escherichia coli. Immunofluorescent staining with antibodies generated from the recombinant protein showed the expression of the protein in sporozoites and development stages. In vitro neutralization assay with the antibodies partially blocked the invasion of sporozoites. These results support the potential involvement of MEDLE-2 in the invasion of host cells. PMID:28912761

  5. Drosophila Orb2 targets genes involved in neuronal growth, synapse formation, and protein turnover

    PubMed Central

    Mastushita-Sakai, Tomoko; White-Grindley, Erica; Samuelson, Jessica; Seidel, Chris; Si, Kausik

    2010-01-01

    In the study of long-term memory, how memory persists is a fundamental and unresolved question. What are the molecular components of the long-lasting memory trace? Previous studies in Aplysia and Drosophila have found that a neuronal variant of a RNA-binding protein with a self-perpetuating prion-like property, cytoplasmic polyadenylation element binding protein, is required for the persistence of long-term synaptic facilitation in the snail and long-term memory in the fly. In this study, we have identified the mRNA targets of the Drosophila neuronal cytoplasmic polyadenylation element binding protein, Orb2. These Orb2 targets include genes involved in neuronal growth, synapse formation, and intriguingly, protein turnover. These targets suggest that the persistent form of the memory trace might be comprised of molecules that maintain a sustained, permissive environment for synaptic growth in an activated synapse. PMID:20547833

  6. Identification and characterization of proteins involved in rice urea and arginine catabolism.

    PubMed

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

    2010-09-01

    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 (K(m) = 67 mm, k(cat) = 490 s(-1)). The activity depended on the presence of manganese (K(d) = 1.3 microm). 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.

  7. 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

  8. 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

  9. 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.

  10. Identification of glandular (preputial and clitoral) proteins in house rat (Rattus rattus) involved in pheromonal communication.

    PubMed

    Archunan, G; Kamalakkannan, S; Achiraman, S; Rajkumar, R

    2004-10-01

    Proteins (18-20 kDa) belonging to lipocalin family have been reported to act as carriers for ligands binding to pheromones in mouse urine, pig saliva, hamster vaginal fluid and human sweat, that are involved in pheromonal communication. As the preputial gland is a major pheromonal source, the present study was aimed to detect the specific protein bands (around 18-20 kDa) in the preputial and clitoral glands of the house rat, R. rattus. The amount of protein was higher in preputial gland of the male than that of female (clitoral) gland. A 20 kDa protein was noted in male and female glands; however, the intensity of the band was much higher in male than in female. In addition, 70, 60, 35 kDa bands, identified in male preputial gland, were absent in females. The presence of higher concentration of glandular proteins in the male preputial gland suggests that male rats may depend more on these glandular proteins for the maintenance of reproductive and dominance behaviours. The results further suggest that these glandular proteins (20 kDa) may act as a carrier for ligand binding.

  11. 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.

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

    PubMed

    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.

  13. Staphylococcus aureus surface proteins involved in adaptation to oxacillin identified using a novel cell shaving approach.

    PubMed

    Solis, Nestor; Parker, Benjamin L; Kwong, Stephen M; Robinson, Gareth; Firth, Neville; Cordwell, Stuart J

    2014-06-06

    Staphylococcus aureus is a Gram-positive pathogen responsible for a variety of infections, and some strains are resistant to virtually all classes of antibiotics. Cell shaving proteomics using a novel probability scoring algorithm to compare the surfaceomes of the methicillin-resistant, laboratory-adapted S. aureus COL strain with a COL strain in vitro adapted to high levels of oxacillin (APT). APT displayed altered cell morphology compared with COL and increased aggregation in biofilm assays. Increased resistance to β-lactam antibiotics was observed, but adaptation to oxacillin did not confer multidrug resistance. Analysis of the S. aureus COL and APT surfaceomes identified 150 proteins at a threshold determined by the scoring algorithm. Proteins unique to APT included the LytR-CpsA-Psr (LCP) domain-containing MsrR and SACOL2302. Quantitative RT-PCR showed increased expression of sacol2302 in APT grown with oxacillin (>6-fold compared with COL). Overexpression of sacol2302 in COL to levels consistent with APT (+ oxacillin) did not influence biofilm formation or β-lactam resistance. Proteomics using iTRAQ and LC-MS/MS identified 1323 proteins (∼50% of the theoretical S. aureus proteome), and cluster analysis demonstrated elevated APT abundances of LCP proteins, capsule and peptidoglycan biosynthesis proteins, and proteins involved in wall remodelling. Adaptation to oxacillin also induced urease proteins, which maintained culture pH compared to COL. These results show that S. aureus modifies surface architecture in response to antibiotic adaptation.

  14. Acute myeloid leukemia fusion proteins deregulate genes involved in stem cell maintenance and DNA repair

    PubMed Central

    Alcalay, Myriam; Meani, Natalia; Gelmetti, Vania; Fantozzi, Anna; Fagioli, Marta; Orleth, Annette; Riganelli, Daniela; Sebastiani, Carla; Cappelli, Enrico; Casciari, Cristina; Sciurpi, Maria Teresa; Mariano, Angela Rosa; Minardi, Simone Paolo; Luzi, Lucilla; Muller, Heiko; Di Fiore, Pier Paolo; Frosina, Guido; Pelicci, Pier Giuseppe

    2003-01-01

    Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins. PMID:14660751

  15. UBX domain-containing proteins are involved in lipid homeostasis and stress responses in Pichia pastoris.

    PubMed

    Zhang, Meng; Yu, Qilin; Liu, Zhe; Liang, Chen; Zhang, Biao; Li, Mingchun

    2017-09-01

    Ubiquitin regulatory X (UBX) domain-containing proteins constitute a family of proteins and are substrate adaptors of AAA ATPase Cdc48. UBX proteins can bind to the N-terminal region of Cdc48 to perform endoplasmic reticulum associated protein degradation (ERAD). In this study, we identified two UBX domain-containing proteins, Ubx1 and Ubx2, in Pichia pastoris and found that the two proteins could recover the growth defect of Saccharomyces cerevisiae in ubx2Δ. Our results revealed that Ubx1 and Ubx2 play critical roles in synthesis of unsaturated fatty acids by affecting Spt23. In addition, the results demonstrated that both Ubx1 and Ubx2 are involved in lipid droplet formation and protein degradation. Deletion of UBX1 led to increased sensitivity to oxidative stress and disruption of UBX2 impaired cell viability under osmotic stress. The phenotypes of ubx1Δ+UBX2, ubx2Δ+UBX1 and ubx1Δubx2Δ and RNA-seq data suggested that Ubx1 and Ubx2 play different roles in cell functions, and the roles of Ubx1 may be more numerous than Ubx2. In summary, our findings provide new insights into the relationship between lipid homeostasis and cell functions in the oil-producing organism P. pastoris. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Identification of Bovine Sperm Surface Proteins Involved in Carbohydrate-mediated Fertilization Interactions*

    PubMed Central

    2016-01-01

    Glycan-protein interactions play a key role in mammalian fertilization, but data on the composition and identities of protein complexes involved in fertilization events are scarce, with the added complication that the glycans in such interactions tend to differ among species. In this study we have used a bovine model to detect, characterize and identify sperm lectins relevant in fertilization. Given the complexity of the sperm-toward-egg journey, two important aspects of the process, both primarily mediated by protein-sugar interactions, have been addressed: (1) formation of the sperm reservoir in the oviductal epithelium, and (2) gamete recognition (oocyte-sperm interaction). Using whole sperm cells and a novel affinity capture method, several groups of proteins with different glycan specificities, including 58 hitherto unreported as lectins, have been identified in sperm surface, underscoring both the efficacy of our selective approach and the complex composition and function of sperm. Based on these results and previous data, we suggest that sperm surface proteins play significant roles in fertilization events such as membrane remodeling, transport, protection and function, thus supporting the hypothesis that rather than a simple lock-and-key model, mammalian fertilization relies on a complex interactome involving multiple ligands/receptors and recognition/binding events. PMID:27094474

  17. LanCL proteins are not Involved in Lanthionine Synthesis in Mammals

    PubMed Central

    He, Chang; Zeng, Min; Dutta, Debapriya; Koh, Tong Hee; Chen, Jie; van der Donk, Wilfred A.

    2017-01-01

    LanC-like (LanCL) proteins are mammalian homologs of bacterial LanC enzymes, which catalyze the addition of the thiol of Cys to dehydrated Ser residues during the biosynthesis of lanthipeptides, a class of natural products formed by post-translational modification of precursor peptides. The functions of LanCL proteins are currently unclear. A recent proposal suggested that LanCL1 catalyzes the addition of the Cys of glutathione to protein- or peptide-bound dehydroalanine (Dha) to form lanthionine, analogous to the reaction catalyzed by LanC in bacteria. Lanthionine has been detected in human brain as the downstream metabolite lanthionine ketimine (LK), which has been shown to have neuroprotective effects. In this study, we tested the proposal that LanCL1 is involved in lanthionine biosynthesis by constructing LanCL1 knock-out mice and measuring LK concentrations in their brains using a mass spectrometric detection method developed for this purpose. To investigate whether other LanCL proteins (LanCL2/3) may confer a compensatory effect, triple knock-out (TKO) mice were also generated and tested. Very similar concentrations of LK (0.5–2.5 nmol/g tissue) were found in LanCL1 knock-out, TKO and wild type (WT) mouse brains, suggesting that LanCL proteins are not involved in lanthionine biosynthesis. PMID:28106097

  18. Endogenous basic fibroblast growth factor isoforms involved in different intracellular protein complexes.

    PubMed Central

    Patry, V; Bugler, B; Maret, A; Potier, M; Prats, H

    1997-01-01

    Four forms of basic fibroblast growth factor (bFGF or FGF-2) result from an alternative initiation of translation involving one AUG (155-amino acid form) and three CUGs (210-, 201- and 196-amino acid forms). These different forms of bFGF show different intracellular biological activities. To identify their intracellular targets, the 210- and 155-amino acid forms of bFGF were independently transfected into CHO cells and their correct subcellular localizations were verified, the 155-amino acid bFGF form being essentially cytoplasmic whereas the 210-amino acid protein was nuclear. The radiation fragmentation method was used to determine the target size of the different bFGF isoforms in the transfected CHO cells and to show that the 210- and 155-amino acids bFGF isoforms were included in protein complexes of 320 and 130 kDa respectively. Similar results were obtained using the SK-Hep1 cell line, which naturally expressed all forms of bFGF. Co-immunoprecipitation assays using different chimaeric bFGF-chloramphenicol acetyltransferase proteins showed that different cellular proteins are associated with different parts of the bFGF molecule. We conclude that bFGF isoforms are involved in different molecular complexes in the cytosol and nucleus, which would reflect different functions for these proteins. PMID:9337877

  19. LanCL proteins are not Involved in Lanthionine Synthesis in Mammals.

    PubMed

    He, Chang; Zeng, Min; Dutta, Debapriya; Koh, Tong Hee; Chen, Jie; van der Donk, Wilfred A

    2017-01-20

    LanC-like (LanCL) proteins are mammalian homologs of bacterial LanC enzymes, which catalyze the addition of the thiol of Cys to dehydrated Ser residues during the biosynthesis of lanthipeptides, a class of natural products formed by post-translational modification of precursor peptides. The functions of LanCL proteins are currently unclear. A recent proposal suggested that LanCL1 catalyzes the addition of the Cys of glutathione to protein- or peptide-bound dehydroalanine (Dha) to form lanthionine, analogous to the reaction catalyzed by LanC in bacteria. Lanthionine has been detected in human brain as the downstream metabolite lanthionine ketimine (LK), which has been shown to have neuroprotective effects. In this study, we tested the proposal that LanCL1 is involved in lanthionine biosynthesis by constructing LanCL1 knock-out mice and measuring LK concentrations in their brains using a mass spectrometric detection method developed for this purpose. To investigate whether other LanCL proteins (LanCL2/3) may confer a compensatory effect, triple knock-out (TKO) mice were also generated and tested. Very similar concentrations of LK (0.5-2.5 nmol/g tissue) were found in LanCL1 knock-out, TKO and wild type (WT) mouse brains, suggesting that LanCL proteins are not involved in lanthionine biosynthesis.

  20. SCIMP, a transmembrane adaptor protein involved in major histocompatibility complex class II signaling.

    PubMed

    Draber, Peter; Vonkova, Ivana; Stepanek, Ondrej; Hrdinka, Matous; Kucova, Marketa; Skopcova, Tereza; Otahal, Pavel; Angelisova, Pavla; Horejsi, Vaclav; Yeung, Mandy; Weiss, Arthur; Brdicka, Tomas

    2011-11-01

    Formation of the immunological synapse between an antigen-presenting cell (APC) and a T cell leads to signal generation in both cells involved. In T cells, the lipid raft-associated transmembrane adaptor protein LAT plays a central role. Its phosphorylation is a crucial step in signal propagation, including the calcium response and mitogen-activated protein kinase activation, and largely depends on its association with the SLP76 adaptor protein. Here we report the discovery of a new palmitoylated transmembrane adaptor protein, termed SCIMP. SCIMP is expressed in B cells and other professional APCs and is localized in the immunological synapse due to its association with tetraspanin-enriched microdomains. In B cells, it is constitutively associated with Lyn kinase and becomes tyrosine phosphorylated after major histocompatibility complex type II (MHC-II) stimulation. When phosphorylated, SCIMP binds to the SLP65 adaptor protein and also to the inhibitory kinase Csk. While the association with SLP65 initiates the downstream signaling cascades, Csk binding functions as a negative regulatory loop. The results suggest that SCIMP is involved in signal transduction after MHC-II stimulation and therefore serves as a regulator of antigen presentation and other APC functions.

  1. Identification of genes involved in radioresistance of nasopharyngeal carcinoma by integrating gene ontology and protein-protein interaction networks.

    PubMed

    Guo, Ya; Zhu, Xiao-Dong; Qu, Song; Li, Ling; Su, Fang; Li, Ye; Huang, Shi-Ting; Li, Dan-Rong

    2012-01-01

    Radioresistance remains one of the important factors in relapse and metastasis of nasopharyngeal carcinoma. Thus, it is imperative to identify genes involved in radioresistance and explore the underlying biological processes in the development of radioresistance. In this study, we used cDNA microarrays to select differential genes between radioresistant CNE-2R and parental CNE-2 cell lines. One hundred and eighty-three significantly differentially expressed genes (p<0.05) were identified, of which 138 genes were upregulated and 45 genes were downregulated in CNE-2R. We further employed publicly available bioinformatics related software, such as GOEAST and STRING to examine the relationship among differentially expressed genes. The results show that these genes were involved in type I interferon-mediated signaling pathway biological processes; the nodes tended to have high connectivity with the EGFR pathway, IFN-related pathways, NF-κB. The node STAT1 has high connectivity with other nodes in the protein-protein interaction (PPI) networks. Finally, the reliability of microarray data was validated for selected genes by semi-quantitative RT-PCR and Western blotting. The results were consistent with the microarray data. Our study suggests that microarrays combined with gene ontology and protein interaction networks have great value in the identification of genes of radioresistance in nasopharyngeal carcinoma; genes involved in several biological processes and protein interaction networks may be relevant to NPC radioresistance; in particular, the verified genes CCL5, STAT1-α, STAT2 and GSTP1 may become potential biomarkers for predicting NPC response to radiotherapy.

  2. Protein Kinase C-{delta} mediates down-regulation of heterogeneous nuclear ribonucleoprotein K protein: involvement in apoptosis induction

    SciTech Connect

    Gao, Feng-Hou; Wu, Ying-Li; Zhao, Meng; Chen, Guo-Qiang

    2009-11-15

    We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase C delta ({Delta}PKC-{delta}). By subcellular proteome analysis, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was identified as being significantly down-regulated in NSC606985-treated leukemic NB4 cells. HnRNP K, a docking protein for DNA, RNA, and transcriptional or translational molecules, is implicated in a host of processes involving the regulation of gene expression. However, the molecular mechanisms of hnRNP K reduction and its roles during apoptosis are still not understood. In the present study, we found that, following the appearance of the {Delta}PKC-{delta}, hnRNP K protein was significantly down-regulated in NSC606985, doxorubicin, arsenic trioxide and ultraviolet-induced apoptosis. We further provided evidence that {Delta}PKC-{delta} mediated the down-regulation of hnRNP K protein during apoptosis: PKC-{delta} inhibitor could rescue the reduction of hnRNP K; hnRNP K failed to be decreased in PKC-{delta}-deficient apoptotic KG1a cells; conditional induction of {Delta}PKC-{delta} in U937T cells directly down-regulated hnRNP K protein. Moreover, the proteasome inhibitor also inhibited the down-regulation of hnRNP K protein by apoptosis inducer and the conditional expression of {Delta}PKC-{delta}. More intriguingly, the suppression of hnRNP K with siRNA transfection significantly induced apoptosis. To our knowledge, this is the first demonstration that proteolytically activated PKC-{delta} down-regulates hnRNP K protein in a proteasome-dependent manner, which plays an important role in apoptosis induction.

  3. African swine fever virus proteins involved in evading host defence systems.

    PubMed

    Dixon, Linda K; Abrams, Charles C; Bowick, Gavin; Goatley, Lynnette C; Kay-Jackson, Pen C; Chapman, Dave; Liverani, Elisabetta; Nix, Rebecca; Silk, Rhiannon; Zhang, Fuquan

    2004-08-01

    African swine fever virus (ASFV) can cause an acutely fatal haemorrhagic fever in domestic pigs although in its natural hosts, warthogs, bushpigs and the soft tick vector, Ornithodoros moubata, ASFV causes inapparent persistent infections. The virus is a large, cytoplasmic, double-stranded DNA virus which has a tropism for macrophages. As it is the only member of the Asfarviridae family, ASFV encodes many novel genes not encoded by other virus families. The ability of the virus to persist in its natural hosts and in domestic pigs, which recover from infection with less virulent isolates, shows that the virus has effective mechanisms to evade host defence systems. This review focuses on recent progress made in understanding the function of ASFV-encoded proteins, which are involved in modulating the host response to infection. Growing evidence suggests that a major strategy used by the virus is to modulate signalling pathways in infected macrophages, thus interfering with the expression of a large number of immunomodulatory genes. One potent immunomodulatory protein, A238L, inhibits both activation of the host NFkappaB transcription factor and inhibits calcineurin phosphatase activity. Calcineurin-dependent pathways, including activation of the NFAT transcription factor, are therefore inhibited. Another ASFV-encoded protein, CD2v, resembles the host CD2 protein, which is expressed on T cells and NK cells. This virus protein causes the adsorption of red blood cells around virus-infected cells and extracellular virus particles. Expression of the CD2v protein aids virus dissemination in pigs and the protein also has a role in impairing bystander lymphocyte function. This may be mediated either by a direct interaction of CD2v extracellular domain with ligands on lymphocytes or by an indirect mechanism involving interaction of the CD2v cytoplasmic tail with host proteins involved in signalling or trafficking pathways. Two ASFV proteins, an IAP and a Bcl2 homologue

  4. A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C.

    PubMed

    Luangwedchakarn, Voravich; Day, Noorbibi K; Hitchcock, Remi; Brown, Pam G; Lerner, Danica L; Rucker, Rajivi P; Cianciolo, George J; Good, Robert A; Haraguchi, Soichi

    2003-05-01

    CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.

  5. 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

  6. 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.

  7. Absence of Aquaporin-4 in Skeletal Muscle Alters Proteins Involved in Bioenergetic Pathways and Calcium Handling

    PubMed Central

    Basco, Davide; Nicchia, Grazia Paola; D'Alessandro, Angelo; Zolla, Lello; Svelto, Maria; Frigeri, Antonio

    2011-01-01

    Aquaporin-4 (AQP4) is a water channel expressed at the sarcolemma of fast-twitch skeletal muscle fibers, whose expression is altered in several forms of muscular dystrophies. However, little is known concerning the physiological role of AQP4 in skeletal muscle and its functional and structural interaction with skeletal muscle proteome. Using AQP4-null mice, we analyzed the effect of the absence of AQP4 on the morphology and protein composition of sarcolemma as well as on the whole skeletal muscle proteome. Immunofluorescence analysis showed that the absence of AQP4 did not perturb the expression and cellular localization of the dystrophin-glycoprotein complex proteins, aside from those belonging to the extracellular matrix, and no alteration was found in sarcolemma integrity by dye extravasation assay. With the use of a 2DE-approach (BN/SDS-PAGE), protein maps revealed that in quadriceps, out of 300 Coomassie-blue detected and matched spots, 19 proteins exhibited changed expression in AQP4−/− compared to WT mice. In particular, comparison of the protein profiles revealed 12 up- and 7 down-regulated protein spots in AQP4−/− muscle. Protein identification by MS revealed that the perturbed expression pattern belongs to proteins involved in energy metabolism (i.e. GAPDH, creatine kinase), as well as in Ca2+ handling (i.e. parvalbumin, SERCA1). Western blot analysis, performed on some significantly changed proteins, validated the 2D results. Together these findings suggest AQP4 as a novel determinant in the regulation of skeletal muscle metabolism and better define the role of this water channel in skeletal muscle physiology. PMID:21552523

  8. Absence of aquaporin-4 in skeletal muscle alters proteins involved in bioenergetic pathways and calcium handling.

    PubMed

    Basco, Davide; Nicchia, Grazia Paola; D'Alessandro, Angelo; Zolla, Lello; Svelto, Maria; Frigeri, Antonio

    2011-04-28

    Aquaporin-4 (AQP4) is a water channel expressed at the sarcolemma of fast-twitch skeletal muscle fibers, whose expression is altered in several forms of muscular dystrophies. However, little is known concerning the physiological role of AQP4 in skeletal muscle and its functional and structural interaction with skeletal muscle proteome. Using AQP4-null mice, we analyzed the effect of the absence of AQP4 on the morphology and protein composition of sarcolemma as well as on the whole skeletal muscle proteome. Immunofluorescence analysis showed that the absence of AQP4 did not perturb the expression and cellular localization of the dystrophin-glycoprotein complex proteins, aside from those belonging to the extracellular matrix, and no alteration was found in sarcolemma integrity by dye extravasation assay. With the use of a 2DE-approach (BN/SDS-PAGE), protein maps revealed that in quadriceps, out of 300 Coomassie-blue detected and matched spots, 19 proteins exhibited changed expression in AQP4(-/-) compared to WT mice. In particular, comparison of the protein profiles revealed 12 up- and 7 down-regulated protein spots in AQP4-/- muscle. Protein identification by MS revealed that the perturbed expression pattern belongs to proteins involved in energy metabolism (i.e. GAPDH, creatine kinase), as well as in Ca(2+) handling (i.e. parvalbumin, SERCA1). Western blot analysis, performed on some significantly changed proteins, validated the 2D results. Together these findings suggest AQP4 as a novel determinant in the regulation of skeletal muscle metabolism and better define the role of this water channel in skeletal muscle physiology.

  9. Lacrimal gland PKC isoforms are differentially involved in agonist-induced protein secretion.

    PubMed

    Zoukhri, D; Hodges, R R; Sergheraert, C; Toker, A; Dartt, D A

    1997-01-01

    In the present study, we have synthesized and N-myristoylated peptides derived from the pseudosubstrate sequences of protein kinase C (PKC)-alpha, -delta, and -epsilon [Myr-PKC-alpha-(15-28), Myr-PKC-delta-(142-153), and Myr-PKC-epsilon-(149-164)], three isoforms present in rat lacrimal gland, and a peptide derived from the sequence of the endogenous inhibitor of protein kinase A [Myr-PKI-(17-25)]. Lacrimal gland acini were preincubated for 60 min with the myristoylated peptides (10(-10) to 3 x 10(-7) M), then protein secretion was stimulated with a phorbol ester, phorbol 12,13-dibutyrate (10(-6) M); vasoactive intestinal peptide (10(-8) M); a cholinergic agonist, carbachol (10(-5) M); or an alpha 1-adrenergic agonist, phenylephrine (10(-4) M), for 20 min. In intact lacrimal gland acini, Myr-PKC-alpha-(15-28) inhibited phorbol 12,13-dibutyrate-induced protein secretion. This effect was not reproduced by the acetylated peptide or by the myristoylated PKI, which inhibited vasoactive intestinal peptide-induced protein secretion, a response mediated by protein kinase A. Carbachol-induced protein secretion was inhibited by all three peptides. In contrast, phenylephrine-induced protein secretion was inhibited only by Myr-PKC-epsilon-(149-164), whereas Myr-PKC-alpha-(15-28) and Myr-PKC-delta-(142-153) had a stimulatory effect. None of these myristoylated peptides affected the calcium increase evoked by cholinergic or alpha 1-adrenergic agonists. We concluded that phorbol ester- and receptor-induced protein secretion involve different PKC isoforms in lacrimal gland.

  10. Protein receptor for activated C kinase 1 is involved in morphine reward in mice.

    PubMed

    Wan, L; Su, L; Xie, Y; Liu, Y; Wang, Y; Wang, Z

    2009-07-07

    Opiate addiction is associated with upregulation of cAMP signaling in the brain. cAMP-responsive element binding protein (CREB), a nuclear transcription factor, is a downstream component of the extracellular signal-regulated protein kinase (ERK) pathway, which has been shown to regulate different physiological and psychological responses of drug addiction. RACK1, the protein receptor for activated C kinase 1, is a multifunctional scaffolding protein known to be a key regulator of various signaling cascades in the CNS. RACK1 functions specifically in integrin mediated activation of ERK cascade and targets active ERK. We examined if RACK1 is involved in the mechanism of drug addiction by regulating CREB in mouse hippocampus and prefrontal cortex. Several expressions were observed. Chronic administration of morphine made the expression of RACK1 and CREB mRNA increase in hippocampus and prefrontal cortex. The expression of RACK1 and CREB protein was strongly positive in CA1, CA3 and dentate gyrus (DG) of the hippocampus of morphine-treated mice brain, especially the pyramidal neurons in the DG of the hippocampus. Using the small interfering RNA technology, we determined that the expression of CREB mRNA was decreased in hippocampus and prefrontal cortex of morphine-treated mice. The expression of RACK1 and CREB protein was negative in CA1, CA3 and DG of hippocampus. These findings suggest that morphine reward can influence the expression of RACK1 in mouse hippocampus and prefrontal cortex through regulating CREB transcription.

  11. Proteomic analysis of male 4C germ cell proteins involved in mouse meiosis.

    PubMed

    Guo, Xuejiang; Zhang, Ping; Qi, Yujuan; Chen, Wen; Chen, Xiangxiang; Zhou, Zuomin; Sha, Jiahao

    2011-01-01

    Male meiosis is a specialized type of cell division that gives rise to sperm. Errors in this process can result in the generation of aneuploid gametes, which are associated with birth defects and infertility in humans. Until now, there has been a lack of a large-scale identification of proteins involved in male meiosis in mammals. In this study, we report the high-confidence identification of 3625 proteins in mouse male germ cells with 4C DNA content undergoing meiosis I. Of these, 397 were found to be testis specific. Bioinformatics analysis of the proteome led to the identification of 28 proteins known to be essential for male meiosis in mice. We also found 172 proteins that had yeast orthologs known to be essential for meiosis. Chromosome distribution analysis of the proteome showed underrepresentation of the identified proteins on the X chromosome, which may be due to meiotic sex chromosome inactivation. Characterization of the proteome of 4C germ cells from mouse testis provides an inventory of proteins, which is useful for understanding meiosis and the mechanisms of male infertility.

  12. Involvement of protein tyrosine phosphatases in adipogenesis: New anti-obesity targets?

    PubMed Central

    Bae, Kwang-Hee; Kim, Won Kon; Lee, Sang Chul

    2012-01-01

    Obesity is a worldwide epidemic as well as being a major risk factor for diabetes, cardiovascular diseases and several types of cancers. Obesity is mainly due to the overgrowth of adipose tissue arising from an imbalance between energy intake and energy expenditure. Adipose tissue, primarily composed of adipocytes, plays a key role in maintaining whole body energy homeostasis. In view of the treatment of obesity and obesity-related diseases, it is critical to understand the detailed signal transduction mechanisms of adipogenic differentiation. Adipogenic differentiation is tightly regulated by many key signal cascades, including insulin signaling. These signal cascades generally transfer or amplify the signal by using serial tyrosine phosphorylations. Thus, protein tyrosine kinases and protein tyrosine phosphatases are closely related to adipogenic differentiation. Compared to protein tyrosine kinases, protein tyrosine phosphatases have received little attention in adipogenic differentiation. This review aims to highlight the involvement of protein tyrosine phosphatases in adipogenic differentiation and the possibility of protein tyrosine phosphatases as drugs to target obesity. [BMB Reports 2012; 45(12): 700-706] PMID:23261055

  13. Involvement of protein tyrosine phosphatases in adipogenesis: new anti-obesity targets?

    PubMed

    Bae, Kwang-Hee; Kim, Won Kon; Lee, Sang Chul

    2012-12-01

    Obesity is a worldwide epidemic as well as being a major risk factor for diabetes, cardiovascular diseases and several types of cancers. Obesity is mainly due to the overgrowth of adipose tissue arising from an imbalance between energy intake and energy expenditure. Adipose tissue, primarily composed of adipocytes, plays a key role in maintaining whole body energy homeostasis. In view of the treatment of obesity and obesity-related diseases, it is critical to understand the detailed signal transduction mechanisms of adipogenic differentiation. Adipogenic differentiation is tightly regulated by many key signal cascades, including insulin signaling. These signal cascades generally transfer or amplify the signal by using serial tyrosine phosphorylations. Thus, protein tyrosine kinases and protein tyrosine phosphatases are closely related to adipogenic differentiation. Compared to protein tyrosine kinases, protein tyrosine phosphatases have received little attention in adipogenic differentiation. This review aims to highlight the involvement of protein tyrosine phosphatases in adipogenic differentiation and the possibility of protein tyrosine phosphatases as drugs to target obesity.

  14. The Abi Proteins and Their Involvement in Bacteriocin Self-Immunity ▿ †

    PubMed Central

    Kjos, Morten; Snipen, Lars; Salehian, Zhian; Nes, Ingolf F.; Diep, Dzung B.

    2010-01-01

    The Abi protein family consists of putative membrane-bound metalloproteases. While they are involved in membrane anchoring of proteins in eukaryotes, little is known about their function in prokaryotes. In some known bacteriocin loci, Abi genes have been found downstream of bacteriocin structural genes (e.g., pln locus from Lactobacillus plantarum and sag locus from Streptococcus pyogenes), where they probably are involved in self-immunity. By modifying the profile hidden Markov model used to select Abi proteins in the Pfam protein family database, we show that this family is larger than presently recognized. Using bacteriocin-associated Abi genes as a means to search for novel bacteriocins in sequenced genomes, seven new bacteriocin-like loci were identified in Gram-positive bacteria. One such locus, from Lactobacillus sakei 23K, was selected for further experimental study, and it was confirmed that the bacteriocin-like genes (skkAB) exhibited antimicrobial activity when expressed in a heterologous host and that the associated Abi gene (skkI) conferred immunity against the cognate bacteriocin. Similar investigation of the Abi gene plnI and the Abi-like gene plnL from L. plantarum also confirmed their involvement in immunity to their cognate bacteriocins (PlnEF and PlnJK, respectively). Interestingly, the immunity genes from these three systems conferred a high degree of cross-immunity against each other's bacteriocins, suggesting the recognition of a common receptor. Site-directed mutagenesis demonstrated that the conserved motifs constituting the putative proteolytic active site of the Abi proteins are essential for the immunity function of SkkI, and to our knowledge, this represents a new concept in self-immunity. PMID:20154137

  15. Hyperhomocysteinemia and bleomycin hydrolase modulate the expression of mouse brain proteins involved in neurodegeneration.

    PubMed

    Suszyńska-Zajczyk, Joanna; Luczak, Magdalena; Marczak, Lukasz; Jakubowski, Hieronim

    2014-01-01

    Homocysteine (Hcy) is a risk factor for Alzheimer's disease (AD). Bleomycin hydrolase (BLMH) participates in Hcy metabolism and is also linked to AD. The inactivation of the Blmh gene in mice causes accumulation of Hcy-thiolactone in the brain and increases susceptibility to Hcy-thiolactone-induced seizures. To gain insight into brain-related Blmh function, we used two-dimensional IEF/SDS-PAGE gel electrophoresis and MALDI-TOF/TOF mass spectrometry to examine brain proteomes of Blmh-/- mice and their Blmh+/+ littermates fed with a hyperhomocysteinemic high-Met or a control diet. We found that: (1) proteins involved in brain-specific function (Ncald, Nrgn, Stmn1, Stmn2), antioxidant defenses (Aop1), cell cycle (RhoGDI1, Ran), and cytoskeleton assembly (Tbcb, CapZa2) were differentially expressed in brains of Blmh-null mice; (2) hyperhomocysteinemia amplified effects of the Blmh-/- genotype on brain protein expression; (3) proteins involved in brain-specific function (Pebp1), antioxidant defenses (Sod1, Prdx2, DJ-1), energy metabolism (Atp5d, Ak1, Pgam-B), and iron metabolism (Fth) showed differential expression in Blmh-null brains only in hyperhomocysteinemic animals; (4) most proteins regulated by the Blmh-/- genotype were also regulated by high-Met diet, albeit in the opposite direction; and (5) the differentially expressed proteins play important roles in neural development, learning, plasticity, and aging and are linked to neurodegenerative diseases, including AD. Taken together, our findings suggest that Blmh interacts with diverse cellular processes from energy metabolism and anti-oxidative defenses to cell cycle, cytoskeleton dynamics, and synaptic plasticity essential for normal brain homeostasis and that modulation of these interactions by hyperhomocysteinemia underlies the involvement of Hcy in AD.

  16. Identification of proteins involved in Hg-Se antagonism in water hyacinth (Eichhornia crassipes).

    PubMed

    Pacheco, Pablo; Hanley, Traci; Figueroa, Julio A Landero

    2014-03-01

    Different studies have established the presence of a proteinaceus complex involved in Hg-Se agonism/antagonism in plants. In order to identify proteins involved in this mechanism, water hyacinth plants were divided into groups and supplemented with Hg, Se and a Hg-Se mixture. Proteins involved were identified through a screening separation by SEC-ICPMS followed by SAX-ICPMS and then peptide mapping of selected fractions by nanoLC-ESI-ITMS(2). Determination of total metal concentration showed that Se inhibits Hg translocation from roots to aerial compartments of the plant and that Se and Hg are antagonists to each other in terms of plant toxicity. In roots, stems and leaves Se was distributed mainly in two molecular mass fractions <670 kDa and ∼40 kDa, however, the proportion between these two fractions was inverted when Hg was co-administered. Hg throughout the plant was distributed in high and medium molecular mass compounds. Hg associated with molecules, ranging from <1.5 kDa to 15 kDa, was found in the root extract of Hg(ii) supplemented plants, but was absent in the root extract of Se(iv) and Hg(ii) supplemented plants. SAX showed that Hg and Se were mostly not associated with the same entity, since the complete overlapping of Hg and Se signals in all the peaks of SEC chromatograms was not observed. Changes in Se and Hg levels in water hyacinth were more evident in leaves in contrast to other compartments. Several proteins, possibly associated with either Se or Hg, were identified in roots, stems and leaves. Most of the identified proteins were associated with Hg and located in leaves, and these are associated specifically with chloroplast and mitochondria proteins, related to essential mechanisms in plants such as photosynthesis, carbon fixation and the electron transport chain.

  17. The abi proteins and their involvement in bacteriocin self-immunity.

    PubMed

    Kjos, Morten; Snipen, Lars; Salehian, Zhian; Nes, Ingolf F; Diep, Dzung B

    2010-04-01

    The Abi protein family consists of putative membrane-bound metalloproteases. While they are involved in membrane anchoring of proteins in eukaryotes, little is known about their function in prokaryotes. In some known bacteriocin loci, Abi genes have been found downstream of bacteriocin structural genes (e.g., pln locus from Lactobacillus plantarum and sag locus from Streptococcus pyogenes), where they probably are involved in self-immunity. By modifying the profile hidden Markov model used to select Abi proteins in the Pfam protein family database, we show that this family is larger than presently recognized. Using bacteriocin-associated Abi genes as a means to search for novel bacteriocins in sequenced genomes, seven new bacteriocin-like loci were identified in Gram-positive bacteria. One such locus, from Lactobacillus sakei 23K, was selected for further experimental study, and it was confirmed that the bacteriocin-like genes (skkAB) exhibited antimicrobial activity when expressed in a heterologous host and that the associated Abi gene (skkI) conferred immunity against the cognate bacteriocin. Similar investigation of the Abi gene plnI and the Abi-like gene plnL from L. plantarum also confirmed their involvement in immunity to their cognate bacteriocins (PlnEF and PlnJK, respectively). Interestingly, the immunity genes from these three systems conferred a high degree of cross-immunity against each other's bacteriocins, suggesting the recognition of a common receptor. Site-directed mutagenesis demonstrated that the conserved motifs constituting the putative proteolytic active site of the Abi proteins are essential for the immunity function of SkkI, and to our knowledge, this represents a new concept in self-immunity.

  18. The SERRATE protein is involved in alternative splicing in Arabidopsis thaliana

    PubMed Central

    Raczynska, Katarzyna Dorota; Stepien, Agata; Kierzkowski, Daniel; Kalak, Malgorzata; Bajczyk, Mateusz; McNicol, Jim; Simpson, Craig G.; Szweykowska-Kulinska, Zofia; Brown, John W. S.; Jarmolowski, Artur

    2014-01-01

    How alternative splicing (AS) is regulated in plants has not yet been elucidated. Previously, we have shown that the nuclear cap-binding protein complex (AtCBC) is involved in AS in Arabidopsis thaliana. Here we show that both subunits of AtCBC (AtCBP20 and AtCBP80) interact with SERRATE (AtSE), a protein involved in the microRNA biogenesis pathway. Moreover, using a high-resolution reverse transcriptase-polymerase chain reaction AS system we have found that AtSE influences AS in a similar way to the cap-binding complex (CBC), preferentially affecting selection of 5′ splice site of first introns. The AtSE protein acts in cooperation with AtCBC: many changes observed in the mutant lacking the correct SERRATE activity were common to those observed in the cbp mutants. Interestingly, significant changes in AS of some genes were also observed in other mutants of plant microRNA biogenesis pathway, hyl1-2 and dcl1-7, but a majority of them did not correspond to the changes observed in the se-1 mutant. Thus, the role of SERRATE in AS regulation is distinct from that of HYL1 and DCL1, and is similar to the regulation of AS in which CBC is involved. PMID:24137006

  19. Involvement of dietary bioactive proteins and peptides in autism spectrum disorders.

    PubMed

    Siniscalco, Dario; Antonucci, Nicola

    2013-12-01

    Autism and autism spectrum disorders (ASDs) are heterogeneous, severe neurodevelopmental pathologies. These enigmatic conditions have their origins in the interaction of multiple genes and environmental factors. Dysfunctions in social interactions and communication skills, restricted interests, repetitive and stereotypic verbal and non-verbal behaviours are the main core symptoms. Several biochemical processes are associated with ASDs: oxidative stress; endoplasmic reticulum stress; decreased methylation capacity; limited production of glutathione; mitochondrial dysfunction; intestinal impaired permeability and dysbiosis; increased toxic metal burden; immune dysregulation. Current available treatments for ASDs can be divided into behavioural, nutritional and medical approaches, although no defined standard approach exists. Dietary bioactive proteins and peptides show potential for application as health-promoting agents. Nowadays, increasing studies highlight a key role of bioactive proteins and peptides in ASDs. This review will focus on the state-of-the-art regarding the involvement of dietary bioactive proteins and peptides in ASDs. Identification of novel therapeutic targets for ASD management will be also discussed.

  20. Crystal structure of a bicupin protein HutD involved in histidine utilization in Pseudomonas.

    PubMed

    Gerth, M L; Liu, Y; Jiao, W; Zhang, X-X; Baker, E N; Lott, J S; Rainey, P B; Johnston, J M

    2017-08-01

    Cupins form one of the most functionally diverse superfamilies of proteins, with members performing a wide range of catalytic, non-catalytic, and regulatory functions. HutD is a predicted bicupin protein that is involved in histidine utilization (Hut) in Pseudomonas species. Previous genetic analyses have suggested that it limits the upper level of Hut pathway expression, but its mechanism of action is unknown. Here, we have determined the structure of PfluHutD at 1.74 Å resolution in several crystallization conditions, and identified N-formyl-l-glutamate (FG, a Hut pathway intermediate) as a potential ligand in vivo. Proteins 2017; 85:1580-1588. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  1. The ESCRT-II proteins are involved in shaping the sarcoplasmic reticulum in C. elegans.

    PubMed

    Lefebvre, Christophe; Largeau, Céline; Michelet, Xavier; Fourrage, Cécile; Maniere, Xavier; Matic, Ivan; Legouis, Renaud; Culetto, Emmanuel

    2016-04-01

    The sarcoplasmic reticulum is a network of tubules and cisternae localized in close association with the contractile apparatus, and regulates Ca(2+)dynamics within striated muscle cell. The sarcoplasmic reticulum maintains its shape and organization despite repeated muscle cell contractions, through mechanisms which are still under investigation. The ESCRT complexes are essential to organize membrane subdomains and modify membrane topology in multiple cellular processes. Here, we report for the first time that ESCRT-II proteins play a role in the maintenance of sarcoplasmic reticulum integrity inC. elegans ESCRT-II proteins colocalize with the sarcoplasmic reticulum marker ryanodine receptor UNC-68. The localization at the sarcoplasmic reticulum of ESCRT-II and UNC-68 are mutually dependent. Furthermore, the characterization of ESCRT-II mutants revealed a fragmentation of the sarcoplasmic reticulum network, associated with an alteration of Ca(2+)dynamics. Our data provide evidence that ESCRT-II proteins are involved in sarcoplasmic reticulum shaping.

  2. Two Neuronal G Proteins Are Involved in Chemosensation of the Caenorhabditis Elegans Dauer-Inducing Pheromone

    PubMed Central

    Zwaal, R. R.; Mendel, J. E.; Sternberg, P. W.; Plasterk, RHA.

    1997-01-01

    Caenorhabditis elegans uses chemosensation to determine its course of development. Young larvae can arrest as dauer larvae in response to increasing population density, which they measure by a nematode-excreted pheromone, and decreasing food supply. Dauer larvae can resume development in response to a decrease in pheromone and increase in food concentration. We show here that two novel G protein alpha subunits (GPA-2 and GPA-3) show promoter activity in subsets of chemosensory neurons and are involved in the decision to form dauer larvae primarily through the response to dauer pheromone. Dominant activating mutations in these G proteins result in constitutive, pheromone-independent dauer formation, whereas inactivation results in reduced sensitivity to pheromone, and, under certain conditions, an alteration in the response to food. Interactions between gpa-2, gpa-3 and other genes controlling dauer formation suggest that these G proteins may act in parallel to regulate the neuronal decision making that precedes dauer formation. PMID:9055081

  3. CUP-1 Is a Novel Protein Involved in Dietary Cholesterol Uptake in Caenorhabditis elegans

    PubMed Central

    Valdes, Victor J.; Athie, Alejandro; Salinas, Laura S.; Navarro, Rosa E.; Vaca, Luis

    2012-01-01

    Sterols transport and distribution are essential processes in all multicellular organisms. Survival of the nematode Caenorhabditis elegans depends on dietary absorption of sterols present in the environment. However the general mechanisms associated to sterol uptake in nematodes are poorly understood. In the present work we provide evidence showing that a previously uncharacterized transmembrane protein, designated Cholesterol Uptake Protein-1 (CUP-1), is involved in dietary cholesterol uptake in C. elegans. Animals lacking CUP-1 showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A CUP-1-GFP fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. Additionally, by FRET imaging, a direct interaction was found between the cholesterol analog DHE and the transmembrane “cholesterol recognition/interaction amino acid consensus” (CRAC) motif present in C. elegans CUP-1. In-silico analysis identified two mammalian homologues of CUP-1. Most interestingly, CRAC motifs are conserved in mammalian CUP-1 homologous. Our results suggest a role of CUP-1 in cholesterol uptake in C. elegans and open up the possibility for the existence of a new class of proteins involved in sterol absorption in mammals. PMID:22479487

  4. Mild copper deficiency alters gene expression of proteins involved in iron metabolism.

    PubMed

    Auclair, Sylvain; Feillet-Coudray, Christine; Coudray, Charles; Schneider, Susanne; Muckenthaler, Martina U; Mazur, Andrzej

    2006-01-01

    Iron and copper homeostasis share common proteins and are therefore closely linked to each other. For example, copper-containing proteins like ceruloplasmin and hephaestin oxidize Fe(2+) during cellular export processes for transport in the circulation bound to transferrin. Indeed, copper deficiency provokes iron metabolism disorders leading to anemia and liver iron accumulation. The aim of the present work was to understand the cross-talk between copper status and iron metabolism. For this purpose we have established dietary copper deficiency in C57BL6 male mice during twelve weeks. Hematological parameters, copper and iron status were evaluated. cDNA microarray studies were performed to investigate gene expression profiles of proteins involved in iron metabolism in the liver, duodenum and spleen. Our results showed that copper deficiency induces microcytic and hypochromic anemia as well as liver iron overload. Gene expression profiles, however, indicate that hepatic and intestinal mRNA expression neither compensates for hepatic iron overload nor the anemia observed in this mouse model. Instead, major modifications of gene expression occurred in the spleen. We observed increased mRNA levels of the transferrin receptors 1 and 2 and of several proteins involved in the heme biosynthesis pathway (ferrochelatase, UroD, UroS,...). These results suggest that copper-deficient mice respond to the deficiency induced anemia by an adaptation leading to an increase in erythrocyte synthesis.

  5. Propionibacterium freudenreichii Surface Protein SlpB Is Involved in Adhesion to Intestinal HT-29 Cells

    PubMed Central

    do Carmo, Fillipe L. R.; Rabah, Houem; Huang, Song; Gaucher, Floriane; Deplanche, Martine; Dutertre, Stéphanie; Jardin, Julien; Le Loir, Yves; Azevedo, Vasco; Jan, Gwénaël

    2017-01-01

    Propionibacterium freudenreichii is a beneficial bacterium traditionally used as a cheese ripening starter and more recently for its probiotic abilities based on the release of beneficial metabolites. In addition to these metabolites (short-chain fatty acids, vitamins, and bifidogenic factor), P. freudenreichii revealed an immunomodulatory effect confirmed in vivo by the ability to protect mice from induced acute colitis. This effect is, however, highly strain-dependent. Local action of metabolites and of immunomodulatory molecules is favored by the ability of probiotics to adhere to the host cells. This property depends on key surface compounds, still poorly characterized in propionibacteria. In the present study, we showed different adhesion rates to cultured human intestinal cells, among strains of P. freudenreichii. The most adhesive one was P. freudenreichii CIRM-BIA 129, which is known to expose surface-layer proteins. We evidenced here the involvement of these proteins in adhesion to cultured human colon cells. We then aimed at deciphering the mechanisms involved in adhesion. Adhesion was inhibited by antibodies raised against SlpB, one of the surface-layer proteins in P. freudenreichii CIRM-BIA 129. Inactivation of the corresponding gene suppressed adhesion, further evidencing the key role of slpB product in cell adhesion. This work confirms the various functions fulfilled by surface-layer proteins, including probiotic/host interactions. It opens new perspectives for the understanding of probiotic determinants in propionibacteria, and for the selection of the most efficient strains within the P. freudenreichii species. PMID:28642747

  6. Involvement of F-BOX proteins in progression and development of human malignancies.

    PubMed

    Uddin, Shahab; Bhat, Ajaz A; Krishnankutty, Roopesh; Mir, Fayaz; Kulinski, Michal; Mohammad, Ramzi M

    2016-02-01

    The Ubiquitin Proteasome System (UPS) is a core regulator with various protein components (ubiquitin-activating E1 enzymes, ubiquitin-conjugating E2 enzymes, ubiquitin-protein E3 ligases, and the 26S proteasome) which work together in a coordinated fashion to ensure the appropriate and efficient proteolysis of target substrates. E3 ubiquitin ligases are essential components of the UPS machinery, working with E1 and E2 enzymes to bind substrates and assist the transport of ubiquitin molecules onto the target protein. As the UPS controls the degradation of several oncogenes and tumor suppressors, dysregulation of this pathway leads to several human malignancies. A major category of E3 Ub ligases, the SCF (Skp-Cullin-F-box) complex, is composed of four principal components: Skp1, Cul1/Cdc53, Roc1/Rbx1/Hrt1, and an F-box protein (FBP). FBPs are the substrate recognition components of SCF complexes and function as adaptors that bring substrates into physical proximity with the rest of the SCF. Besides acting as a component of SCF complexes, FBPs are involved in DNA replication, transcription, cell differentiation and cell death. This review will highlight the recent literature on three well characterized FBPs SKP2, Fbw7, and beta-TRCP. In particular, we will focus on the involvement of these deregulated FBPs in the progression and development of various human cancers. We will also highlight some novel substrates recently identified for these FBPs.

  7. NAP-1, Nucleosome assembly protein 1, a histone chaperone involved in Drosophila telomeres.

    PubMed

    López-Panadès, Elisenda; Casacuberta, Elena

    2016-03-01

    Telomere elongation is a function that all eukaryote cells must accomplish in order to guarantee, first, the stability of the end of the chromosomes and second, to protect the genetic information from the inevitable terminal erosion. The targeted transposition of the telomere transposons HeT-A, TART and TAHRE perform this function in Drosophila, while the telomerase mechanism elongates the telomeres in most eukaryotes. In order to integrate telomere maintenance together with cell cycle and metabolism, different components of the cell interact, regulate, and control the proteins involved in telomere elongation. Different partners of the telomerase mechanism have already been described, but in contrast, very few proteins have been related with assisting the telomere transposons of Drosophila. Here, we describe for the first time, the implication of NAP-1 (Nucleosome assembly protein 1), a histone chaperone that has been involved in nuclear transport, transcription regulation, and chromatin remodeling, in telomere biology. We find that Nap-1 and HeT-A Gag, one of the major components of the Drosophila telomeres, are part of the same protein complex. We also demonstrate that their close interaction is necessary to guarantee telomere stability in dividing cells. We further show that NAP-1 regulates the transcription of the HeT-A retrotransposon, pointing to a positive regulatory role of NAP-1 in telomere expression. All these results facilitate the understanding of the transposon telomere maintenance mechanism, as well as the integration of telomere biology with the rest of the cell metabolism.

  8. An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer's disease

    PubMed Central

    Onuki, Reiko; Bando, Yoshio; Suyama, Eigo; Katayama, Taiichi; Kawasaki, Hiroaki; Baba, Tadashi; Tohyama, Masaya; Taira, Kazunari

    2004-01-01

    Various types of stress, such as disruption of calcium homeostasis, inhibition of protein glycosylation and reduction of disulfide bonds, result in accumulation of misfolded proteins in the endoplasmic reticulum (ER). The initial cellular response involves removal of such proteins by the ER, but excessive and/or long-term stress results in apoptosis. In this study, we used a randomized ribozyme library and ER stress-mediated apoptosis (tunicamycin-induced apoptosis) in SK-N-SH human neuroblastoma cells as a selective phenotype to identify factors involved in this process. We identified a double-stranded RNA-dependent protein kinase (PKR) as one of the participants in this process. The level of nuclear PKR was elevated, but the level of cytoplasmic PKR barely changed in tunicamycin-treated SK-N-SH cells. Furthermore, tunicamycin also raised levels of phosphorylated PKR in the nucleus. We also detected the accumulation of phosphorylated PKR in the nuclei of autopsied brain tissues in Alzheimer's disease. Thus, PKR might play a role in ER stress-induced apoptosis and in Alzheimer's disease. PMID:14765129

  9. Involvement of regucalcin as a suppressor protein in human carcinogenesis: insight into the gene therapy.

    PubMed

    Yamaguchi, Masayoshi

    2015-08-01

    Regucalcin, which its gene is located on the X chromosome, plays a multifunctional role as a suppressor protein in cell signal transduction in various types of cells and tissues. The suppression of regucalcin gene expression has been shown to involve in carcinogenesis. Regucalcin gene expression was uniquely downregulated in carcinogenesis of rat liver in vivo, although the expression of other many genes was upregulated, indicating that endogenous regucalcin plays a suppressive role in the development of hepatocarcinogenesis. Overexpression of endogenous regucalcin was found to suppress proliferation of rat cloned hepatoma cells in vitro. Moreover, the regucalcin gene and its protein levels were demonstrated specifically to downregulate in human hepatocellular carcinoma by analysis with multiple gene expression profiles and proteomics. Regucalcin gene expression was also found to suppress in human tumor tissues including kidney, lung, brain, breast and prostate, suggesting that repressed regucalcin gene expression leads to the development of carcinogenesis in various tissues. Regucalcin may play a role as a suppressor protein in carcinogenesis. Overexpression of endogenous regucalcin is suggested to reveal preventive and therapeutic effects on carcinogenesis. Delivery of the regucalcin gene may be a novel useful tool in the gene therapy of carcinogenesis. This review will discuss regarding to an involvement of regucalcin as a suppressor protein in human carcinogenesis in insight into the gene therapy.

  10. Involvement of the pepper antimicrobial protein CaAMP1 gene in broad spectrum disease resistance.

    PubMed

    Lee, Sung Chul; Hwang, In Sun; Choi, Hyong Woo; Hwang, Byung Kook

    2008-10-01

    Pathogen-inducible antimicrobial defense-related proteins have emerged as key antibiotic peptides and enzymes involved in disease resistance in plants. A novel antimicrobial protein gene, CaAMP1 (for Capsicum annuum ANTIMICROBIAL PROTEIN1), was isolated from pepper (C. annuum) leaves infected with Xanthomonas campestris pv vesicatoria. Expression of the CaAMP1 gene was strongly induced in pepper leaves not only during pathogen infection but also after exposure to abiotic elicitors. The purified recombinant CaAMP1 protein possessed broad-spectrum antimicrobial activity against phytopathogenic bacteria and fungi. CaAMP1:smGFP fusion protein was localized mainly in the external and intercellular regions of onion (Allium cepa) epidermal cells. The virus-induced gene silencing technique and gain-of-function transgenic plants were used to determine the CaAMP1 gene function in plant defense. Silencing of CaAMP1 led to enhanced susceptibility to X. campestris pv vesicatoria and Colletotrichum coccodes infection, accompanied by reduced PATHOGENESIS-RELATED (PR) gene expression. In contrast, overexpression of CaAMP1 in Arabidopsis (Arabidopsis thaliana) conferred broad-spectrum resistance to the hemibiotrophic bacterial pathogen Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora parasitica, and the fungal necrotrophic pathogens Fusarium oxysporum f. sp. matthiolae and Alternaria brassicicola. CaAMP1 overexpression induced the salicylic acid pathway-dependent genes PR1 and PR5 but not the jasmonic acid-dependent defense gene PDF1.2 during P. syringae pv tomato infection. Together, these results suggest that the antimicrobial CaAMP1 protein is involved in broad-spectrum resistance to bacterial and fungal pathogen infection.

  11. MATI, a Novel Protein Involved in the Regulation of Herbivore-Associated Signaling Pathways.

    PubMed

    Santamaría, M Estrella; Martinez, Manuel; Arnaiz, Ana; Ortego, Félix; Grbic, Vojislava; Diaz, Isabel

    2017-01-01

    The defense response of the plants against herbivores relies on a complex network of interconnected signaling pathways. In this work, we characterized a new key player in the response of Arabidopsis against the two-spotted spider mite Tetranychus urticae, the MATI (Mite Attack Triggered Immunity) gene. This gene was differentially induced in resistant Bla-2 strain relative to susceptible Kon Arabidopsis accessions after mite attack, suggesting a potential role in the control of spider mites. To study the MATI gene function, it has been performed a deep molecular characterization of the gene combined with feeding bioassays using modified Arabidopsis lines and phytophagous arthropods. The MATI gene belongs to a new gene family that had not been previously characterized. Biotic assays showed that it confers a high tolerance not only to T. urticae, but also to the chewing lepidopteran Spodoptera exigua. Biochemical analyses suggest that MATI encodes a protein involved in the accumulation of reducing agents upon herbivore attack to control plant redox homeostasis avoiding oxidative damage and cell death. Besides, molecular analyses demonstrated that MATI is involved in the modulation of different hormonal signaling pathways, affecting the expression of genes involved in biosynthesis and signaling of the jasmonic acid and salicylic acid hormones. The fact that MATI is also involved in defense through the modulation of the levels of photosynthetic pigments highlights the potential of MATI proteins to be exploited as biotechnological tools for pest control.

  12. MATI, a Novel Protein Involved in the Regulation of Herbivore-Associated Signaling Pathways

    PubMed Central

    Santamaría, M. Estrella; Martinez, Manuel; Arnaiz, Ana; Ortego, Félix; Grbic, Vojislava; Diaz, Isabel

    2017-01-01

    The defense response of the plants against herbivores relies on a complex network of interconnected signaling pathways. In this work, we characterized a new key player in the response of Arabidopsis against the two-spotted spider mite Tetranychus urticae, the MATI (Mite Attack Triggered Immunity) gene. This gene was differentially induced in resistant Bla-2 strain relative to susceptible Kon Arabidopsis accessions after mite attack, suggesting a potential role in the control of spider mites. To study the MATI gene function, it has been performed a deep molecular characterization of the gene combined with feeding bioassays using modified Arabidopsis lines and phytophagous arthropods. The MATI gene belongs to a new gene family that had not been previously characterized. Biotic assays showed that it confers a high tolerance not only to T. urticae, but also to the chewing lepidopteran Spodoptera exigua. Biochemical analyses suggest that MATI encodes a protein involved in the accumulation of reducing agents upon herbivore attack to control plant redox homeostasis avoiding oxidative damage and cell death. Besides, molecular analyses demonstrated that MATI is involved in the modulation of different hormonal signaling pathways, affecting the expression of genes involved in biosynthesis and signaling of the jasmonic acid and salicylic acid hormones. The fact that MATI is also involved in defense through the modulation of the levels of photosynthetic pigments highlights the potential of MATI proteins to be exploited as biotechnological tools for pest control. PMID:28649257

  13. Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia.

    PubMed

    Kissmehl, R; Treptau, T; Hofer, H W; Plattner, H

    1996-07-01

    In Paramecium tetraurelia cells synchronous exocytosis induced by aminoethyldextran (AED) is accompanied by an equally rapid dephosphorylation of a 63 kDa phosphoprotein (PP63) within 80 ms. In vivo, rephosphorylation occurs within a few seconds after AED triggering. In homogenates (P)P63 can be solubilized in all three phosphorylation states (phosphorylated, dephosphorylated and rephosphorylated) and thus tested in vitro. By using chelators of different divalent cations, de- and rephosphorylation of PP63 and P63 respectively can be achieved by an endogenous protein phosphatase/kinase system. Dephosphorylation occurs in the presence of EDTA, whereas in the presence of EGTA this was concealed by phosphorylation by endogenous kinase(s), thus indicating that phosphorylation of P63 is calcium-independent. Results obtained with protein phosphatase inhibitors (okadaic acid, calyculin A) allowed us to exclude a protein serine/threonine phosphatase of type I (with selective sensitivity in Paramecium). Protein phosphatase 2C is also less likely to be a candidate because of its requirement for high Mg2+ concentrations. According to previous evidence a protein serine/threonine phosphatase of type 2B (calcineurin; CaN) is possibly involved. We have now found that bovine brain CaN dephosphorylates PP63 in vitro. Taking into account the specific requirements of this phosphatase in vitro, with p-nitrophenyl phosphate as a substrate, we have isolated a cytosolic phosphatase of similar characteristics by combined preparative gel electrophoresis and affinity-column chromatography. In Paramecium this phosphatase also dephosphorylates PP63 in vitro (after 32P labelling in vivo). Using various combinations of ion exchange, affinity and hydrophobic interaction chromatography we have also isolated three different protein kinases from the soluble fraction, i.e. a cAMP-dependent protein kinase (PKA), a cGMP-dependent protein kinase (PKG) and a casein kinase. Among the kinases tested, PKA

  14. Oligophrenin-1 encodes a rhoGAP protein involved in X-linked mental retardation.

    PubMed

    Billuart, P; Bienvenu, T; Ronce, N; des Portes, V; Vinet, M C; Zemni, R; Roest Crollius, H; Carrié, A; Fauchereau, F; Cherry, M; Briault, S; Hamel, B; Fryns, J P; Beldjord, C; Kahn, A; Moraine, C; Chelly, J

    1998-04-30

    Primary or nonspecific X-linked mental retardation (MRX) is a heterogeneous condition in which affected patients do not have any distinctive clinical or biochemical features in common apart from cognitive impairment. Although it is present in approximately 0.15-0.3% of males, most of the genetic defects associated with MRX, which may involve more than ten different genes, remain unknown. Here we report the characterization of a new gene on the long arm of the X-chromosome (position Xq12) and the identification in unrelated individuals of different mutations that are predicted to cause a loss of function. This gene is highly expressed in fetal brain and encodes a protein of relative molecular mass 91K, named oligophrenin-1, which contains a domain typical of a Rho-GTPase-activating protein (rhoGAP). By enhancing their GTPase activity, GAP proteins inactivate small Rho and Ras proteins, so inactivation of rhoGAP proteins might cause constitutive activation of their GTPase targets. Such activation is known to affect cell migration and outgrowth of axons and dendrites in vivo. Our results demonstrate an association between cognitive impairment and a defect in a signalling pathway that depends on a Ras-like GTPase.

  15. A Novel RNA-Binding Protein Involves ABA Signaling by Post-transcriptionally Repressing ABI2

    PubMed Central

    Xu, Jianwen; Chen, Yihan; Qian, Luofeng; Mu, Rong; Yuan, Xi; Fang, Huimin; Huang, Xi; Xu, Enshun; Zhang, Hongsheng; Huang, Ji

    2017-01-01

    The Stress Associated RNA-binding protein 1 (SRP1) repressed by ABA, salt and cold encodes a C2C2-type zinc finger protein in Arabidopsis. The knock-out mutation in srp1 reduced the sensitivity of seed to ABA and salt stress during germination and post-germinative growth stages. In contrast, SRP1-overexpressing seedlings were more sensitive to ABA and salt compared to wild type plants. In the presence of ABA, the transcript levels of ABA signaling and germination-related genes including ABI3. ABI5. EM1 and EM6 were less induced in srp1 compared to WT. Interestingly, expression of ABI2 encoding a protein phosphatase 2C protein were significantly up-regulated in srp1 mutants. By in vitro analysis, SRP1 was identified as a novel RNA-binding protein directly binding to 3′UTR of ABI2 mRNA. Moreover, transient expression assay proved the function of SRP1 in reducing the activity of luciferase whose coding sequence was fused with the ABI2 3’UTR. Together, it is suggested that SRP1 is involved in the ABA signaling by post-transcriptionally repressing ABI2 expression in Arabidopsis. PMID:28174577

  16. New Proteins Involved in Sulfur Trafficking in the Cytoplasm of Allochromatium vinosum*

    PubMed Central

    Stockdreher, Yvonne; Sturm, Marga; Josten, Michaele; Sahl, Hans-Georg; Dobler, Nadine; Zigann, Renate; Dahl, Christiane

    2014-01-01

    The formation of periplasmic sulfur globules is an intermediate step during the oxidation of reduced sulfur compounds in various sulfur-oxidizing microorganisms. The mechanism of how this sulfur is activated and crosses the cytoplasmic membrane for further oxidation to sulfite by the dissimilatory reductase DsrAB is incompletely understood, but it has been well documented that the pathway involves sulfur trafficking mediated by sulfur-carrying proteins. So far sulfur transfer from DsrEFH to DsrC has been established. Persulfurated DsrC very probably serves as a direct substrate for DsrAB. Here, we introduce further important players in oxidative sulfur metabolism; the proteins Rhd_2599, TusA, and DsrE2 are strictly conserved in the Chromatiaceae, Chlorobiaceae, and Acidithiobacillaceae families of sulfur-oxidizing bacteria and are linked to genes encoding complexes involved in sulfur oxidation (Dsr or Hdr) in the latter two. Here we show via relative quantitative real-time PCR and microarray analysis an increase of mRNA levels under sulfur-oxidizing conditions for rhd_2599, tusA, and dsrE2 in Allochromatium vinosum. Transcriptomic patterns for the three genes match those of major genes for the sulfur-oxidizing machinery rather than those involved in biosynthesis of sulfur-containing biomolecules. TusA appears to be one of the major proteins in A. vinosum. A rhd_2599-tusA-dsrE2-deficient mutant strain, although not viable in liquid culture, was clearly sulfur oxidation negative upon growth on solid media containing sulfide. Rhd_2599, TusA, and DsrE2 bind sulfur atoms via conserved cysteine residues, and experimental evidence is provided for the transfer of sulfur between these proteins as well as to DsrEFH and DsrC. PMID:24648525

  17. New proteins involved in sulfur trafficking in the cytoplasm of Allochromatium vinosum.

    PubMed

    Stockdreher, Yvonne; Sturm, Marga; Josten, Michaele; Sahl, Hans-Georg; Dobler, Nadine; Zigann, Renate; Dahl, Christiane

    2014-05-02

    The formation of periplasmic sulfur globules is an intermediate step during the oxidation of reduced sulfur compounds in various sulfur-oxidizing microorganisms. The mechanism of how this sulfur is activated and crosses the cytoplasmic membrane for further oxidation to sulfite by the dissimilatory reductase DsrAB is incompletely understood, but it has been well documented that the pathway involves sulfur trafficking mediated by sulfur-carrying proteins. So far sulfur transfer from DsrEFH to DsrC has been established. Persulfurated DsrC very probably serves as a direct substrate for DsrAB. Here, we introduce further important players in oxidative sulfur metabolism; the proteins Rhd_2599, TusA, and DsrE2 are strictly conserved in the Chromatiaceae, Chlorobiaceae, and Acidithiobacillaceae families of sulfur-oxidizing bacteria and are linked to genes encoding complexes involved in sulfur oxidation (Dsr or Hdr) in the latter two. Here we show via relative quantitative real-time PCR and microarray analysis an increase of mRNA levels under sulfur-oxidizing conditions for rhd_2599, tusA, and dsrE2 in Allochromatium vinosum. Transcriptomic patterns for the three genes match those of major genes for the sulfur-oxidizing machinery rather than those involved in biosynthesis of sulfur-containing biomolecules. TusA appears to be one of the major proteins in A. vinosum. A rhd_2599-tusA-dsrE2-deficient mutant strain, although not viable in liquid culture, was clearly sulfur oxidation negative upon growth on solid media containing sulfide. Rhd_2599, TusA, and DsrE2 bind sulfur atoms via conserved cysteine residues, and experimental evidence is provided for the transfer of sulfur between these proteins as well as to DsrEFH and DsrC.

  18. 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

  19. CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity

    PubMed Central

    Karasawa, T; Wang, Q; David, L L; Steyger, P S

    2010-01-01

    Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin–agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63. PMID:21368867

  20. Involvement of HxuC Outer Membrane Protein in Utilization of Hemoglobin by Haemophilus influenzae

    PubMed Central

    Cope, Leslie D.; Love, Robert P.; Guinn, Sarah E.; Gilep, Andrei; Usanov, Sergei; Estabrook, Ronald W.; Hrkal, Zbynek; Hansen, Eric J.

    2001-01-01

    Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study from this laboratory indicated that nontypeable Haemophilus influenzae (NTHI) strain N182 expressed three outer membrane proteins, designated HgbA, HgbB, and HgbC, that bound hemoglobin or hemoglobin-haptoglobin and were encoded by open reading frames (ORFs) that contained a CCAA nucleotide repeat. Testing of mutants expressing the HgbA, HgbB, and HgbC proteins individually revealed that expression of any one of these proteins was sufficient to allow wild-type growth with hemoglobin. In contrast, mutants that expressed only HgbA or HgbC grew significantly better with hemoglobin-haptoglobin than did a mutant expressing only HgbB. Construction of an isogenic hgbA hgbB hgbC mutant revealed that the absence of these three gene products did not affect the ability of NTHI N182 to utilize hemoglobin as a source of heme, although this mutant was severely impaired in its ability to utilize hemoglobin-haptoglobin. The introduction of a tonB mutation into this triple mutant eliminated its ability to utilize hemoglobin, indicating that the pathway for hemoglobin utilization in the absence of HgbA, HgbB, and HgbC involved a TonB-dependent process. Inactivation in this triple mutant of the hxuC gene, which encodes a predicted TonB-dependent outer membrane protein previously shown to be involved in the utilization of free heme, resulted in loss of the ability to utilize hemoglobin. The results of this study reinforce the redundant nature of the heme acquisition systems expressed by H. influenzae. PMID:11254593

  1. Identification of ICIS-1, a new protein involved in cilia stability.

    PubMed

    Ponsard, Cecile; Skowron-Zwarg, Marie; Seltzer, Virginie; Perret, Eric; Gallinger, Julia; Fisch, Cathy; Dupuis-Williams, Pascale; Caruso, Nathalie; Middendorp, Sandrine; Tournier, Frederic

    2007-01-01

    Cilia are specialized organelles that exert critical functions in numerous organisms, including that of cell motility, fluid transport and protozoan locomotion. Ciliary architecture and function strictly depend on basal body formation, migration and axoneme elongation. Numerous ultrastructural studies have been undertaken in different species to elucidate the process of ciliogenesis. Recent analyses have led to identification of genes specifically expressed in ciliated organisms, but most proteins involved in ciliogenesis remain uncharacterized. Using human nasal epithelial cells capable of ciliary differentiation in vitro, differential display was carried out to identify new proteins associated with ciliogenesis. We isolated a new gene, ICIS-1 (Involved in CIlia Stability-1), upregulated during mucociliary differentiation. This gene is localized within the TGF-beta1 promoter and is ubiquitously expressed in human tissues. Functional analyses of gene expression inhibition by RNA interference in Paramecium tetraurelia indicated that the ICIS-1 homologue interfered with cilia stability or formation. These findings demonstrate that ICIS-1 is a new protein associated with ciliated cells and potentially related to cilia stability.

  2. Raf kinase inhibitory protein suppresses a metastasis signalling cascade involving LIN28 and let-7

    PubMed Central

    Dangi-Garimella, Surabhi; Yun, Jieun; Eves, Eva M; Newman, Martin; Erkeland, Stefan J; Hammond, Scott M; Minn, Andy J; Rosner, Marsha Rich

    2009-01-01

    Raf kinase inhibitory protein (RKIP) negatively regulates the MAP kinase (MAPK), G protein-coupled receptor kinase-2, and NF-κB signalling cascades. RKIP has been implicated as a metastasis suppressor for prostate cancer, but the mechanism is not known. Here, we show that RKIP inhibits invasion by metastatic breast cancer cells and represses breast tumour cell intravasation and bone metastasis in an orthotopic murine model. The mechanism involves inhibition of MAPK, leading to decreased transcription of LIN28 by Myc. Suppression of LIN28 enables enhanced let-7 processing in breast cancer cells. Elevated let-7 expression inhibits HMGA2, a chromatin remodelling protein that activates pro-invasive and pro-metastatic genes, including Snail. LIN28 depletion and let-7 expression suppress bone metastasis, and LIN28 restores bone metastasis in mice bearing RKIP-expressing breast tumour cells. These results indicate that RKIP suppresses invasion and metastasis in part through a signalling cascade involving MAPK, Myc, LIN28, let-7, and downstream let-7 targets. RKIP regulation of two pluripotent stem cell genes, Myc and LIN28, highlights the importance of RKIP as a key metastasis suppressor and potential therapeutic agent. PMID:19153603

  3. 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.

  4. 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

  5. A Phytophthora sojae G-protein alpha subunit is involved in chemotaxis to soybean isoflavones.

    PubMed

    Hua, Chenlei; Wang, Yonglin; Zheng, Xiaobo; Dou, Daolong; Zhang, Zhengguang; Govers, Francine; Wang, Yuanchao

    2008-12-01

    For the soybean pathogen Phytophthora sojae, chemotaxis of zoospores to isoflavones is believed to be critical for recognition of the host and for initiating infection. However, the molecular mechanisms underlying this chemotaxis are largely unknown. To investigate the role of G-protein and calcium signaling in chemotaxis, we analyzed the expression of several genes known to be involved in these pathways and selected one that was specifically expressed in sporangia and zoospores but not in mycelium. This gene, named PsGPA1, is a single-copy gene in P. sojae and encodes a G-protein alpha subunit that shares 96% identity in amino acid sequence with that of Phytophthora infestans. To elucidate the function, expression of PsGPA1 was silenced by introducing antisense constructs into P. sojae. PsGPA1 silencing did not disturb hyphal growth or sporulation but severely affected zoospore behavior, including chemotaxis to the soybean isoflavone daidzein. Zoospore encystment and cyst germination were also altered, resulting in the inability of the PsGPA1-silenced mutants to infect soybean. In addition, the expressions of a calmodulin gene, PsCAM1, and two calcium- and calmodulin-dependent protein kinase genes, PsCMK3 and PsCMK4, were increased in the mutant zoospores, suggesting that PsGPA1 negatively regulates the calcium signaling pathways that are likely involved in zoospore chemotaxis.

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

    PubMed

    Ding, Fangrui; Tan, Aidi; 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

  7. The protein-retaining effects of growth hormone during fasting involve inhibition of muscle-protein breakdown.

    PubMed

    Nørrelund, H; Nair, K S; Jørgensen, J O; Christiansen, J S; Møller, N

    2001-01-01

    The metabolic response to fasting involves a series of hormonal and metabolic adaptations leading to protein conservation. An increase in the serum level of growth hormone (GH) during fasting has been well substantiated. The present study was designed to test the hypothesis that GH may be a principal mediator of protein conservation during fasting and to assess the underlying mechanisms. Eight normal subjects were examined on four occasions: 1) in the basal postabsorptive state (basal), 2) after 40 h of fasting (fast), 3) after 40 h of fasting with somatostatin suppression of GH (fast-GH), and 4) after 40 h of fasting with suppression of GH and exogenous GH replacement (fast+GH). The two somatostatin experiments were identical in terms of hormone replacement (except for GH), meaning that somatostatin, insulin, glucagon and GH were administered for 28 h; during the last 4 h, substrate metabolism was investigated. Compared with the GH administration protocol, IGF-I and free IGF-I decreased 35 and 70%, respectively, during fasting without GH. Urinary urea excretion and serum urea increased when participants fasted without GH (urea excretion: basal 392 +/- 44, fast 440 +/- 32, fast-GH 609 +/- 76, and fast+GH 408 +/- 36 mmol/24 h, P < 0.05; serum urea: basal 4.6 +/- 0.1, fast 6.2 +/- 0.1, fast-GH 7.0 +/- 0.2, and fast+GH 4.3 +/- 0.2 mmol/1, P < 0.01). There was a net release of phenylalanine across the forearm, and the negative phenylalanine balance was higher during fasting with GH suppression (balance: basal 9 +/- 3, fast 15 +/- 6, fast-GH 17 +/- 4, and fast+GH 11 +/- 5 nmol/min, P < 0.05). Muscle-protein breakdown was increased among participants who fasted without GH (phenylalanine rate of appearance: basal 17 +/- 4, fast 26 +/- 9, fast-GH 33 +/- 7, fast+GH 25 +/- 6 nmol/min, P < 0.05). Levels of free fatty acids and oxidation of lipid decreased during fasting without GH (P < 0.01). In summary, we find that suppression of GH during fasting leads to a 50% increase in

  8. Involvement of lipid peroxidation-derived aldehyde-protein adducts in autoimmunity mediated by trichloroethene.

    PubMed

    Wang, Gangduo; Ansari, G A S; Khan, M Firoze

    2007-12-01

    Lipid peroxidation, a major contributor to cellular damage, is also implicated in the pathogenesis of autoimmune diseases (AD). The focus of this study was to elucidate the role of lipid peroxidation-derived aldehydes in autoimmunity induced and/or exacerbated by chemical exposure. Previous studies showed that trichloroethene (TCE) is capable of inducing/accelerating autoimmunity. To test whether TCE-induced lipid peroxidation might be involved in the induction/exacerbation of autoimmune responses, groups of autoimmune-prone female MRL +/+ mice were treated with TCE (10 mmol/kg, i.p., every 4th day) for 6 or 12 wk. Significant increases of the formation of malondialdehyde (MDA)- and 4-hydroxynonenal (HNE)-protein adducts were found in the livers of TCE-treated mice at both 6 and 12 wk, but the response was greater at 12 wk. Further characterization of these adducts in liver microsomes showed increased formation of MDA-protein adducts with molecular masses of 86, 65, 56, 44, and 32 kD, and of HNE-protein adducts with molecular masses of 87, 79, 46, and 17 kD in TCE-treated mice. In addition, significant induction of anti-MDA- and anti-HNE-protein adduct-specific antibodies was observed in the sera of TCE-treated mice, and showed a pattern similar to MDA- or HNE-protein adducts. The increases in anti-MDA- and anti-HNE-protein adduct antibodies were associated with significant elevation in serum anti-nuclear-, anti-ssDNA- and anti-dsDNA-antibodies at 6 wk and, to a greater extent, at 12 wk. These studies suggest that TCE-induced lipid peroxidation is associated with induction/exacerbation of autoimmune response in MRL+/+ mice, and thus may play an important role in disease pathogenesis. Further interventional studies are needed to establish a causal relationship between lipid peroxidation and TCE-induced autoimmune response.

  9. Identification of phosphorylated proteins involved in the oncogenesis of prostate cancer via Pin1-proteomic analysis.

    PubMed

    Endoh, Kanji; Nishi, Mayuko; Ishiguro, Hitoshi; Uemura, Hiroji; Miyagi, Yohei; Aoki, Ichiro; Hirano, Hisashi; Kubota, Yoshinobu; Ryo, Akihide

    2012-05-01

    The peptidyl-prolyl isomerase Pin1 regulates a subset of phosphorylated proteins by catalyzing the cis-trans isomerization of their specific phosphorylated Ser/Thr-Pro motifs. Although Pin1 has been shown to be involved in cell transformation and the maintenance of the malignant phenotype in prostate cancer, its specific substrates during these processes have not yet been determined. Cancer-specific phosphorylated proteins were isolated from two human prostate cancer cell lines (PC-3, LNCaP) and the Dunning rat prostate cancer cell lines by GST-pull down analysis with recombinant GST-Pin1 protein. These proteins were then identified by the LC-MS/MS analysis using a Q-Tof micro mass spectrometer and processed for further functional analysis. We newly identified five prostate cancer-specific Pin1 binding proteins (PINBPs) in this screen. Among these, TRK-fused gene (TFG) was found to be preferentially up-regulated in prostate cancer cell lines and tissues. The targeted inhibition of TFG by specific siRNA resulted in the reduced cell proliferation and the induction of premature senescence in PC3 prostate cancer cells. We further found that TFG can facilitate the cell signaling mediated by NF-kappaB and androgen receptor (AR). Tissue micro-dissection based quantitative RT-PCR analysis of prostate cancer tissues following radical prostatectomy further revealed that TFG expression is closely associated with both a higher probability and shorter period of tumor recurrence following surgery. Pin1-based proteomics analysis is a useful tool for the identification of prostate cancer-specific phosphorylated proteins. TFG could be a potential diagnostic and/or prognostic marker and therapeutic target in prostate cancer. Copyright © 2011 Wiley Periodicals, Inc.

  10. Evolution and diversity of periplasmic proteins involved in copper homeostasis in gamma proteobacteria

    PubMed Central

    2012-01-01

    Background Different systems contributing to copper homeostasis in bacteria have been described in recent years involving periplasmic and transport proteins that provide resistance via metal efflux to the extracellular media (CopA/Cue, Cus, Cut, and Pco). The participation of these proteins in the assembly of membrane, periplasmic and secreted cuproproteins has also been postulated. The integration and interrelation of these systems and their apparent redundancies are less clear since they have been studied in alternative systems. Based on the idea that cellular copper is not free but rather it is transferred via protein-protein interactions, we hypothesized that systems would coevolve and be constituted by set numbers of essential components. Results By the use of a phylogenomic approach we identified the distribution of 14 proteins previously characterized as members of homeostasis systems in the genomes of 268 gamma proteobacteria. Only 3% of the genomes presented the complete systems and 5% of them, all intracellular parasites, lacked the 14 genes. Surprisingly, copper homeostatic pathways did not behave as evolutionary units with particular species assembling different combinations of basic functions. The most frequent functions, and probably because of its distribution the most vital, were copper extrusion from the cytoplasm to the periplasm performed by CopA and copper export from the cytoplasm to the extracellular space performed by CusC, which along with the remaining 12 proteins, assemble in nine different functional repertoires. Conclusions These observations suggest complex evolutionary dynamics and still unexplored interactions to achieve copper homeostasis, challenging some of the molecular transport mechanism proposed for these systems. PMID:23122209

  11. The Arabidopsis PLAT domain protein1 is critically involved in abiotic stress tolerance.

    PubMed

    Hyun, Tae Kyung; van der Graaff, Eric; Albacete, Alfonso; Eom, Seung Hee; Großkinsky, Dominik K; Böhm, Hannah; Janschek, Ursula; Rim, Yeonggil; Ali, Walid Wahid; Kim, Soo Young; Roitsch, Thomas

    2014-01-01

    Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty.

  12. Proteins involved in flor yeast carbon metabolism under biofilm formation conditions.

    PubMed

    Moreno-García, Jaime; García-Martínez, Teresa; Moreno, Juan; Mauricio, Juan Carlos

    2015-04-01

    A lack of sugars during the production of biologically aged wines after fermentation of grape must causes flor yeasts to metabolize other carbon molecules formed during fermentation (ethanol and glycerol, mainly). In this work, a proteome analysis involving OFFGEL fractionation prior to LC/MS detection was used to elucidate the carbon metabolism of a flor yeast strain under biofilm formation conditions (BFC). The results were compared with those obtained under non-biofilm formation conditions (NBFC). Proteins associated to processes such as non-fermentable carbon uptake, the glyoxylate and TCA cycles, cellular respiration and inositol metabolism were detected at higher concentrations under BFC than under the reference conditions (NBFC). This study constitutes the first attempt at identifying the flor yeast proteins responsible for the peculiar sensory profile of biologically aged wines. A better metabolic knowledge of flor yeasts might facilitate the development of effective strategies for improved production of these special wines.

  13. Sorcin, a calcium binding protein involved in the multidrug resistance mechanisms in cancer cells.

    PubMed

    Colotti, Gianni; Poser, Elena; Fiorillo, Annarita; Genovese, Ilaria; Chiarini, Valerio; Ilari, Andrea

    2014-09-05

    Sorcin is a penta-EF hand calcium binding protein, which participates in the regulation of calcium homeostasis in cells. Sorcin regulates calcium channels and exchangers located at the plasma membrane and at the endo/sarcoplasmic reticulum (ER/SR), and allows high levels of calcium in the ER to be maintained, preventing ER stress and possibly, the unfolded protein response. Sorcin is highly expressed in the heart and in the brain, and overexpressed in many cancer cells. Sorcin gene is in the same amplicon as other genes involved in the resistance to chemotherapeutics in cancer cells (multi-drug resistance, MDR) such as ABCB4 and ABCB1; its overexpression results in increased drug resistance to a number of chemotherapeutic agents, and inhibition of sorcin expression by sorcin-targeting RNA interference leads to reversal of drug resistance. Sorcin is increasingly considered a useful marker of MDR and may represent a therapeutic target for reversing tumor multidrug resistance.

  14. Involvement of Protein Kinase C-δ in Vascular Permeability in Acute Lung Injury.

    PubMed

    Ahn, Jong J; Jung, Jong P; Park, Soon E; Lee, Minhyun; Kwon, Byungsuk; Cho, Hong R

    2015-08-01

    Pulmonary edema is a major cause of mortality due to acute lung injury (ALI). The involvement of protein kinase C-δ (PKC-δ) in ALI has been a controversial topic. Here we investigated PKC-δ function in ALI using PKC-δ knockout (KO) mice and PKC inhibitors. Our results indicated that although the ability to produce proinflammatory mediators in response to LPS injury in PKC-δ KO mice was similar to that of control mice, they showed enhanced recruitment of neutrophils to the lung and more severe pulmonary edema. PKC-δ inhibition promoted barrier dysfunction in an endothelial cell layer in vitro, and administration of a PKC-δ-specific inhibitor significantly increased steady state vascular permeability. A neutrophil transmigration assay indicated that the PKC-δ inhibition increased neutrophil transmigration through an endothelial monolayer. This suggests that PKC-δ inhibition induces structural changes in endothelial cells, allowing extravasation of proteins and neutrophils.

  15. The H-NS protein is involved in the biogenesis of flagella in Escherichia coli.

    PubMed Central

    Bertin, P; Terao, E; Lee, E H; Lejeune, P; Colson, C; Danchin, A; Collatz, E

    1994-01-01

    The function of the flagellum-chemotaxis regulon requires the expression of many genes and is positively regulated by the cyclic AMP-catabolite activator protein (cAMP-CAP) complex. In this paper, we show that motile behavior was affected in Escherichia coli hns mutants. The loss of motility resulted from a complete lack of flagella. A decrease in the level of transcription of the flhD and fliA genes, which are both required for the synthesis of flagella, was observed in the presence of an hns mutation. Furthermore, the Fla- phenotype was not reversed to the wild type in the presence of a cfs mutation which renders the flagellum synthesis independent of the cAMP-CAP complex. These results suggest that the H-NS protein acts as a positive regulator of genes involved in the biogenesis of flagella by a mechanism independent of the cAMP-CAP pathway. Images PMID:8071234

  16. Vps41, a protein involved in lysosomal trafficking, interacts with caspase-8.

    PubMed

    Wang, Lu; Pan, Xiao; He, Liangqiang; Zhang, Rong; Chen, Wei; Zhang, Jing; Lu, Min; Hua, Zi-Chun

    2013-01-01

    Caspase-8 is a member of the cysteine-aspartic acid protease (caspase) family which plays a central role in apoptosis and development. We screened caspase-8 interacting proteins from mouse T-cell lymphoma and 7.5-day embryo cDNA libraries by yeast two-hybrid system and obtained eleven positive clones, including Vacuolar protein sorting 41 (Vps41), a protein involved in trafficking of proteins from the late Golgi to the vacuole. The interaction of Vps41 with caspase-8 was confirmed by co-immunoprecipitation (co-IP) and co-localization studies in HEK293T cells. Co-IP experiments also showed that Vps41 binds to the p18 subunit of caspase-8 through its WD40 region and RING-finger motif. Furthermore, we found that overexpression of Vps41 promotes Fas-induced apoptosis in A549 human lung adenocarcinoma cells. The cleavage of caspase-3, a caspase-8 downstream effector, was increased when cells were transfected with Vps41-overexpressing plasmid. Together, these results suggest a novel interaction of caspase-8 with Vps41 and provide a potential role of Vps41 beyond lysosomal trafficking.

  17. 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

  18. Analysis of nitrated proteins in Saccharomyces cerevisiae involved in mating signal transduction.

    PubMed

    Kang, Jeong Won; Lee, Na Young; Cho, Kyung-Cho; Lee, Min Young; Choi, Do-Young; Park, Sang-Hyun; Kim, Kwang Pyo

    2015-01-01

    Protein tyrosine nitration (PTN) is a PTM that regulates signal transduction and inflammatory responses, and is related to neurodegenerative and cardiovascular diseases. The cellular function of PTN remains unclear because the low stoichiometry of PTN limits the identification and quantification of nitrated peptides. Effective enrichment is an important aspect of PTN analysis. In this study, we analyzed the in vivo nitroproteome elicited by mating signal transduction in Saccharomyces cerevisiae using a novel chemical enrichment method followed by LC-MS/MS. Nitroproteome profiling successfully identified changes in the nitration states of 14 proteins during mating signal transduction in S. cerevisiae, making this the first reported in vivo nitroproteome in yeast. We investigated the biological functions of these nitroproteins and their relationships to mating signal transduction in S. cerevisiae using a protein-protein interaction network. Our results suggest that PTN and denitration may be involved in nonreactive nitrogen species-mediated signal transduction and can provide clues for understanding the functional roles of PTN in vivo.

  19. Analysis of outer membrane vesicle protein involved in biofilm formation of Helicobacter pylori.

    PubMed

    Yonezawa, Hideo; Osaki, Takako; Woo, Timothy; Kurata, Satoshi; Zaman, Cynthia; Hojo, Fuhito; Hanawa, Tomoko; Kato, Shuichi; Kamiya, Shigeru

    2011-12-01

    Helicobacter pylori is one of the most common causes of bacterial infection in humans. Infection with H. pylori is closely associated with gastritis and peptic ulcers and is a risk factor for gastric cancer and mucosa-associated lymphoid tissue lymphoma. H. pylori forms biofilms on glass surfaces at the air-liquid interface in in-vitro batch cultures. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. We also suggested the outer membrane vesicles (OMV) produced by strain TK1402 might be related to its biofilm forming ability. In the present study, we analyzed the protein profile of the OMV produced by strain TK1402 and found a unique 22-kDa protein in TK1402 OMV cultured for 2-3 days. In addition, this protein could not be detected in the OMVs produced by other H. pylori strains. These results suggest that the 22-kDa protein is involved in effective biofilm formation by strain TK1402. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. XAB2, a novel tetratricopeptide repeat protein involved in transcription-coupled DNA repair and transcription.

    PubMed

    Nakatsu, Y; Asahina, H; Citterio, E; Rademakers, S; Vermeulen, W; Kamiuchi, S; Yeo, J P; Khaw, M C; Saijo, M; Kodo, N; Matsuda, T; Hoeijmakers, J H; Tanaka, K

    2000-11-10

    Nucleotide excision repair is a highly versatile DNA repair system responsible for elimination of a wide variety of lesions from the genome. It is comprised of two subpathways: transcription-coupled repair that accomplishes efficient removal of damage blocking transcription and global genome repair. Recently, the basic mechanism of global genome repair has emerged from biochemical studies. However, little is known about transcription-coupled repair in eukaryotes. Here we report the identification of a novel protein designated XAB2 (XPA-binding protein 2) that was identified by virtue of its ability to interact with XPA, a factor central to both nucleotide excision repair subpathways. The XAB2 protein of 855 amino acids consists mainly of 15 tetratricopeptide repeats. In addition to interacting with XPA, immunoprecipitation experiments demonstrated that a fraction of XAB2 is able to interact with the transcription-coupled repair-specific proteins CSA and CSB as well as RNA polymerase II. Furthermore, antibodies against XAB2 inhibited both transcription-coupled repair and transcription in vivo but not global genome repair when microinjected into living fibroblasts. These results indicate that XAB2 is a novel component involved in transcription-coupled repair and transcription.

  1. Astroglial and neuronal proteins in cerebrospinal fluid as markers of CNS involvement in Lyme neuroborreliosis.

    PubMed

    Dotevall, L; Hagberg, L; Karlsson, J E; Rosengren, L E

    1999-03-01

    Is Lyme neuroborreliosis, even in its early phase, a parenchymatous disorder in the central nervous system (CNS), and not merely a meningitic process? We quantified cerebrospinal fluid (CSF) levels of four nerve and glial cell marker proteins in Lyme neuroborreliosis patients with pretreatment durations of 7-240 days. All 23 patients had meningoradiculitis, and six had objective signs of encephalopathy. Glial fibrillary acidic protein (GFAp) pretreatment levels in CSF, and the light subunit of neurofilament protein (NFL) levels were related to clinical outcome and declined significantly after treatment (P < 0.001 and P < 0.01, respectively). NFL was detectable in 11 out of 22 patients, and pre- and post-treatment NFL levels were associated with the duration of neurological symptoms within 100 days prior to treatment. Neuron-specific enolase (NSE) concentrations also decreased after therapy (P < 0.001), while CSF levels of glial S-100 protein remained unchanged. The pretreatment duration of disease was related to postinfectious sequelae. GFAp, NSE and NFL levels in CSF are unspecific indicators of astroglial and neuronal involvement in CNS disease. The findings in the present study are in agreement with the hypothesis that early and late stages of Lyme neuroborreliosis damage the CNS parenchyma. Copyright 1999 Lippincott Williams & Wilkins.

  2. 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

  3. The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division.

    PubMed

    Ambrose, J Christian; Shoji, Tsubasa; Kotzer, Amanda M; Pighin, Jamie A; Wasteneys, Geoffrey O

    2007-09-01

    Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.

  4. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Neuronal and microglial involvement in beta-amyloid protein deposition in Alzheimer's disease.

    PubMed Central

    Cras, P.; Kawai, M.; Siedlak, S.; Mulvihill, P.; Gambetti, P.; Lowery, D.; Gonzalez-DeWhitt, P.; Greenberg, B.; Perry, G.

    1990-01-01

    This study was undertaken to localize amyloid precursor protein (APP) and to determine how APP might be released and proteolyzed to yield the beta-amyloid protein deposits found in senile plaques in the brains of Alzheimer's disease patients. We found that antibodies to recombinantly expressed APP labeled many normal neurons and neurites. In addition, dystrophic neurites in different types of senile plaques and degenerating neurons in the temporal cortex and hippocampus of Alzheimer's disease patients were immunostained. We also detected small clusters of dystrophic APP immunoreactive neurites that were not associated with beta-amyloid protein deposits. Microglia was involved in different types of senile plaques and often were associated closely with APP immunoreactive neurites and neurons. The greatest concurrence of APP immunoreactivity and reactive microglia was seen in the subiculum and area CA1, regions with a high density of congophilic plaques and subject to intense Alzheimer's pathology. Our findings suggest that neuronally derived APP is the source for senile plaque beta-amyloid protein, while microglia may act as processing cells. Images Figure 1 Figure 2 PMID:2117395

  6. 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.

  7. The involvement of a protein kinase in phototaxis and gravitaxis of Euglena gracilis.

    PubMed

    Daiker, Viktor; Häder, Donat-P; Richter, Peter R; Lebert, Michael

    2011-05-01

    The unicellular flagellate Euglena gracilis shows positive phototaxis at low-light intensities (<10 W/m(2)) and a negative one at higher irradiances (>10 W/m(2)). Phototaxis is based on blue light-activated adenylyl cyclases, which produce cAMP upon irradiation. In the absence of light the cells swim upward in the water column (negative gravitaxis). The results of sounding rocket campaigns and of a large number of ground experiments led to the following model of signal perception and transduction in gravitaxis of E. gracilis: The body of the cell is heavier than the surrounding medium, sediments and thereby exerts a force onto the lower membrane. Upon deviation from a vertical swimming path mechano-sensitive ion channels are activated. Calcium is gated inwards which leads to an increase in the intracellular calcium concentration and causes a change of the membrane potential. After influx, calcium activates one of several calmodulins found in Euglena, which in turn activates an adenylyl cyclase (different from the one involved in phototaxis) to produce cAMP from ATP. One further element in the sensory transduction chain of both phototaxis and gravitaxis is a specific protein kinase A. We found five different protein kinases A in E. gracilis. The blockage of only one of these (PK.4, accession No. EU935859) by means of RNAi inhibited both phototaxis and gravitaxis, while inhibition of the other four affected neither phototaxis nor gravitaxis. It is assumed that cAMP directly activates this protein kinase A which may in turn phosphorylate a protein involved in the flagellar beating mechanism.

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

    PubMed

    Li, Junlin; Zhao, Guifang; Gao, Xiaocai

    2013-02-20

    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.

  9. 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

  10. 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.

  11. Involvement of protein kinase C in the delayed cytoprotection following sublethal ischaemia in rabbit myocardium.

    PubMed Central

    Baxter, G. F.; Goma, F. M.; Yellon, D. M.

    1995-01-01

    Rabbit hearts were preconditioned with four 5 min coronary artery occlusions 24 h before 30 min coronary occlusion with 120 min reperfusion. Preconditioning significantly reduced the percentage of myocardium infarcting within the risk zone from 49.1 +/- 4.3% to 31.8 +/- 3.5% (P < 0.05). When the protein kinase C (PKC) inhibitor, chelerythrine, was administered just before preconditioning, the delayed protection against infarction 24 h later was abolished. We conclude that the delayed cytoprotective response associated with ischaemic preconditioning of myocardium is likely to involve the early activation of one or more PKC subtypes. PMID:7545515

  12. Involvement of Retinoblastoma Protein and HBP1 in Histone H10 Gene Expression

    PubMed Central

    Lemercier, Claudie; Duncliffe, Kym; Boibessot, Isabelle; Zhang, Hui; Verdel, André; Angelov, Dimitar; Khochbin, Saadi

    2000-01-01

    The histone H10-encoding gene is expressed in vertebrates in differentiating cells during the arrest of proliferation. In the H10 promoter, a specific regulatory element, which we named the H4 box, exhibits features which implicate a role in mediating H10 gene expression in response to both differentiation and cell cycle control signals. For instance, within the linker histone gene family, the H4 box is found only in the promoters of differentiation-associated subtypes, suggesting that it is specifically involved in differentiation-dependent expression of these genes. In addition, an element nearly identical to the H4 box is conserved in the promoters of histone H4-encoding genes and is known to be involved in their cell cycle-dependent expression. The transcription factors interacting with the H10 H4 box were therefore expected to link differentiation-dependent expression of H10 to the cell cycle control machinery. The aim of this work was to identify such transcription factors and to obtain information concerning the regulatory pathway involved. Interestingly, our cloning strategy led to the isolation of a retinoblastoma protein (RB) partner known as HBP1. HBP1, a high-mobility group box transcription factor, interacted specifically with the H10 H4 box and moreover was expressed in a differentiation-dependent manner. We also showed that the HBP1-encoding gene is able to produce different forms of HBP1. Finally, we demonstrated that both HBP1 and RB were involved in the activation of H10 gene expression. We therefore propose that HBP1 mediates a link between the cell cycle control machinery and cell differentiation signals. Through modulating the expression of specific chromatin-associated proteins such as histone H10, HBP1 plays a vital role in chromatin remodeling events during the arrest of cell proliferation in differentiating cells. PMID:10958660

  13. 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.

  14. Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major

    PubMed Central

    Aoki, Juliana Ide; Coelho, Adriano Cappellazzo; Muxel, Sandra Marcia; Zampieri, Ricardo Andrade; Sanchez, Eduardo Milton Ramos; Nerland, Audun Helge; Floeter-Winter, Lucile Maria; Cotrim, Paulo Cesar

    2016-01-01

    Background Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis. Methodology/Principal findings After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway. Conclusions/Significance This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how

  15. 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

  16. Spa2p Interacts with Cell Polarity Proteins and Signaling Components Involved in Yeast Cell Morphogenesis

    PubMed Central

    Sheu, Yi-Jun; Santos, Beatriz; Fortin, Nathalie; Costigan, Christine; Snyder, Michael

    1998-01-01

    The yeast protein Spa2p localizes to growth sites and is important for polarized morphogenesis during budding, mating, and pseudohyphal growth. To better understand the role of Spa2p in polarized growth, we analyzed regions of the protein important for its function and proteins that interact with Spa2p. Spa2p interacts with Pea2p and Bud6p (Aip3p) as determined by the two-hybrid system; all of these proteins exhibit similar localization patterns, and spa2Δ, pea2Δ, and bud6Δ mutants display similar phenotypes, suggesting that these three proteins are involved in the same biological processes. Coimmunoprecipitation experiments demonstrate that Spa2p and Pea2p are tightly associated with each other in vivo. Velocity sedimentation experiments suggest that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, raising the possibility that these proteins form a large, 12S multiprotein complex. Bud6p has been shown previously to interact with actin, suggesting that the 12S complex functions to regulate the actin cytoskeleton. Deletion analysis revealed that multiple regions of Spa2p are involved in its localization to growth sites. One of the regions involved in Spa2p stability and localization interacts with Pea2p; this region contains a conserved domain, SHD-II. Although a portion of Spa2p is sufficient for localization of itself and Pea2p to growth sites, only the full-length protein is capable of complementing spa2 mutant defects, suggesting that other regions are required for Spa2p function. By using the two-hybrid system, Spa2p and Bud6p were also found to interact with components of two mitogen-activated protein kinase (MAPK) pathways important for polarized cell growth. Spa2p interacts with Ste11p (MAPK kinase [MEK] kinase) and Ste7p (MEK) of the mating signaling pathway as well as with the MEKs Mkk1p and Mkk2p of the Slt2p (Mpk1p) MAPK pathway; for both Mkk1p and Ste7p, the Spa2p-interacting region was mapped to the N-terminal putative regulatory domain

  17. Shewanella oneidensis MR-1 sensory box protein involved in aerobic and anoxic growth.

    PubMed

    Sundararajan, A; Kurowski, J; Yan, T; Klingeman, D M; Joachimiak, M P; Zhou, J; Naranjo, B; Gralnick, J A; Fields, M W

    2011-07-01

    Although little is known of potential function for conserved signaling proteins, it is hypothesized that such proteins play important roles to coordinate cellular responses to environmental stimuli. In order to elucidate the function of a putative sensory box protein (PAS domains) in Shewanella oneidensis MR-1, the physiological role of SO3389 was characterized. The predicted open reading frame (ORF) encodes a putative sensory box protein that has PAS, GGDEF, and EAL domains, and an in-frame deletion mutant was constructed (ΔSO3389) with approximately 95% of the ORF deleted. Under aerated conditions, wild-type and mutant cultures had similar growth rates, but the mutant culture had a lower growth rate under static, aerobic conditions. Oxygen consumption rates were lower for mutant cultures (1.5-fold), and wild-type cultures also maintained lower dissolved oxygen concentrations under aerated growth conditions. When transferred to anoxic conditions, the mutant did not grow with fumarate, iron(III), or dimethyl sulfoxide (DMSO) as electron acceptors. Biochemical assays demonstrated the expression of different c-type cytochromes as well as decreased fumarate reductase activity in the mutant transferred to anoxic growth conditions. Transcriptomic studies showed the inability of the mutant to up-express and down-express genes, including c-type cytochromes (e.g., SO4047/SO4048, SO3285/SO3286), reductases (e.g., SO0768, SO1427), and potential regulators (e.g., SO1329). The complemented strain was able to grow when transferred from aerobic to anoxic growth conditions with the tested electron acceptors. The modeled structure for the SO3389 PAS domains was highly similar to the crystal structures of FAD-binding PAS domains that are known O2/redox sensors. Based on physiological, genomic, and bioinformatic results, we suggest that the sensory box protein, SO3389, is an O2/redox sensor that is involved in optimization of aerobic growth and transitions to anoxia in S

  18. Mechanosensitive Molecular Networks Involved in Transducing Resistance Exercise-Signals into Muscle Protein Accretion

    PubMed Central

    Rindom, Emil; Vissing, Kristian

    2016-01-01

    Loss of skeletal muscle myofibrillar protein with disease and/or inactivity can severely deteriorate muscle strength and function. Strategies to counteract wasting of muscle myofibrillar protein are therefore desirable and invite for considerations on the potential superiority of specific modes of resistance exercise and/or the adequacy of low load resistance exercise regimens as well as underlying mechanisms. In this regard, delineation of the potentially mechanosensitive molecular mechanisms underlying muscle protein synthesis (MPS), may contribute to an understanding on how differentiated resistance exercise can transduce a mechanical signal into stimulation of muscle accretion. Recent findings suggest specific upstream exercise-induced mechano-sensitive myocellular signaling pathways to converge on mammalian target of rapamycin complex 1 (mTORC1), to influence MPS. This may e.g. implicate mechanical activation of signaling through a diacylglycerol kinase (DGKζ)-phosphatidic acid (PA) axis or implicate integrin deformation to signal through a Focal adhesion kinase (FAK)-Tuberous Sclerosis Complex 2 (TSC2)-Ras homolog enriched in brain (Rheb) axis. Moreover, since initiation of translation is reliant on mRNA, it is also relevant to consider potentially mechanosensitive signaling pathways involved in muscle myofibrillar gene transcription and whether some of these pathways converge with those affecting mTORC1 activation for MPS. In this regard, recent findings suggest how mechanical stress may implicate integrin deformation and/or actin dynamics to signal through a Ras homolog gene family member A protein (RhoA)-striated muscle activator of Rho signaling (STARS) axis or implicate deformation of Notch to affect Bone Morphogenetic Protein (BMP) signaling through a small mother of decapentaplegic (Smad) axis. PMID:27909410

  19. Mechanosensitive Molecular Networks Involved in Transducing Resistance Exercise-Signals into Muscle Protein Accretion.

    PubMed

    Rindom, Emil; Vissing, Kristian

    2016-01-01

    Loss of skeletal muscle myofibrillar protein with disease and/or inactivity can severely deteriorate muscle strength and function. Strategies to counteract wasting of muscle myofibrillar protein are therefore desirable and invite for considerations on the potential superiority of specific modes of resistance exercise and/or the adequacy of low load resistance exercise regimens as well as underlying mechanisms. In this regard, delineation of the potentially mechanosensitive molecular mechanisms underlying muscle protein synthesis (MPS), may contribute to an understanding on how differentiated resistance exercise can transduce a mechanical signal into stimulation of muscle accretion. Recent findings suggest specific upstream exercise-induced mechano-sensitive myocellular signaling pathways to converge on mammalian target of rapamycin complex 1 (mTORC1), to influence MPS. This may e.g. implicate mechanical activation of signaling through a diacylglycerol kinase (DGKζ)-phosphatidic acid (PA) axis or implicate integrin deformation to signal through a Focal adhesion kinase (FAK)-Tuberous Sclerosis Complex 2 (TSC2)-Ras homolog enriched in brain (Rheb) axis. Moreover, since initiation of translation is reliant on mRNA, it is also relevant to consider potentially mechanosensitive signaling pathways involved in muscle myofibrillar gene transcription and whether some of these pathways converge with those affecting mTORC1 activation for MPS. In this regard, recent findings suggest how mechanical stress may implicate integrin deformation and/or actin dynamics to signal through a Ras homolog gene family member A protein (RhoA)-striated muscle activator of Rho signaling (STARS) axis or implicate deformation of Notch to affect Bone Morphogenetic Protein (BMP) signaling through a small mother of decapentaplegic (Smad) axis.

  20. IGF-1 modulates gene expression of proteins involved in inflammation, cytoskeleton, and liver architecture.

    PubMed

    Lara-Diaz, V J; Castilla-Cortazar, I; Martín-Estal, I; García-Magariño, M; Aguirre, G A; Puche, J E; de la Garza, R G; Morales, L A; Muñoz, U

    2017-05-01

    Even though the liver synthesizes most of circulating IGF-1, it lacks its receptor under physiological conditions. However, according to previous studies, a damaged liver expresses the receptor. For this reason, herein, we examine hepatic histology and expression of genes encoding proteins of the cytoskeleton, extracellular matrix, and cell-cell molecules and inflammation-related proteins. A partial IGF-1 deficiency murine model was used to investigate IGF-1's effects on liver by comparing wild-type controls, heterozygous igf1(+/-), and heterozygous mice treated with IGF-1 for 10 days. Histology, microarray for mRNA gene expression, RT-qPCR, and lipid peroxidation were assessed. Microarray analyses revealed significant underexpression of igf1 in heterozygous mice compared to control mice, restoring normal liver expression after treatment, which then normalized its circulating levels. IGF-1 receptor mRNA was overexpressed in Hz mice liver, while treated mice displayed a similar expression to that of the controls. Heterozygous mice showed overexpression of several genes encoding proteins related to inflammatory and acute-phase proteins and underexpression or overexpression of genes which coded for extracellular matrix, cytoskeleton, and cell junction components. Histology revealed an altered hepatic architecture. In addition, liver oxidative damage was found increased in the heterozygous group. The mere IGF-1 partial deficiency is associated with relevant alterations of the hepatic architecture and expression of genes involved in cytoskeleton, hepatocyte polarity, cell junctions, and extracellular matrix proteins. Moreover, it induces hepatic expression of the IGF-1 receptor and elevated acute-phase and inflammation mediators, which all resulted in liver oxidative damage.

  1. Quantitative LC-MS/MS Analysis of Proteins Involved in Metastasis of Breast Cancer

    PubMed Central

    Goto, Rieko; Nakamura, Yasushi; Takami, Tomonori; Sanke, Tokio; Tozuka, Zenzaburo

    2015-01-01

    The purpose of this study was to develop quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the analysis of proteins involved in metastasis of breast cancer for diagnosis and determining disease prognosis, as well as to further our understand of metastatic mechanisms. We have previously demonstrated that the protein type XIV collagen may be specifically expressed in metastatic tissues by two dimensional LC-MS/MS. In this study, we developed quantitative LC-MS/MS methods for type XIV collagen. Type XIV collagen was quantified by analyzing 2 peptides generated by digesting type XIV collagen using stable isotope-labeled peptides. The individual concentrations were equivalent between 2 different peptides of type XIV collagen by evaluation of imprecise transitions and using the best transition for the peptide concentration. The results indicated that type XIV collagen is highly expressed in metastatic tissues of patients with massive lymph node involvement compared to non-metastatic tissues. These findings were validated by quantitative real-time RT-PCR. Further studies on type XIV collagen are desired to verify its role as a prognostic factor and diagnosis marker for metastasis. PMID:26176947

  2. CUTI-1: A Novel Tetraspan Protein Involved in C. elegans CUTicle Formation and Epithelial Integrity

    PubMed Central

    Fritz, Julie-Anne; Behm, Carolyn A.

    2009-01-01

    The nematode cuticle is a tough extracellular matrix composed primarily of cross-linked collagens and non-collagenous cuticulins. It is required for nematode motility and protection from the external environment. Little is known about how the complex process of cuticle formation has been adapted to the specialized requirements of the nematode cuticle, which is structurally and compositionally unique from other organisms. The C. elegans gene cuti-1 (CUTicle and epithelial Integrity) encodes a nematode-specific protein. We have shown that CUTI-1 is expressed in the epithelia and in seam cells. Within these tissues the expression of cuti-1 mRNA cycles throughout development in line with the molting cycle, a process that involves synthesis of a new cuticle. In addition, knockdown of cuti-1 by RNA interference (RNAi) results in worms that display post-embryonic phenotypes related to cuticle dysfunction and defects in epithelial integrity. This is one of the first reports of a nematode-specific protein involved in extracellular matrix formation. It provides further insight into how novel ways have evolved to regulate the formation of the cuticle, which is the primary protective barrier and skeletal component of nematodes. PMID:19357781

  3. Bicalutamide failure in prostate cancer treatment: involvement of Multi Drug Resistance proteins.

    PubMed

    Colabufo, Nicola Antonio; Pagliarulo, Vincenzo; Berardi, Francesco; Contino, Marialessandra; Inglese, Carmela; Niso, Mauro; Ancona, Patrizia; Albo, Giancarlo; Pagliarulo, Arcangelo; Perrone, Roberto

    2008-12-28

    Prolonged bicalutamide treatment induced pathology regression although relapses with a more aggressive form of prostate cancer have been observed. This failure could be due to androgen receptor mutation. In the present work we hypothesized an alternative mechanism responsible for bicalutamide failure involving activity of ATP-binding cassette (ABC) pumps such as P-glycoprotein, Breast Cancer Receptor Protein (BCRP), and Multi Resistant Proteins (MRPs) that extrude the androgen antagonist from the cell membrane. As experimental models androgen-dependent (LnCap) and androgen-independent (PC-3) prostate cancer cell lines have been employed. Bicalutamide has been tested in the cell lines mentioned above in the absence and in the presence of MC18, our potent P-glycoprotein/BCRP/MRP1 inhibitor. The results displayed that bicalutamide antiproliferative effect at 72 h was ameliorated in LnCap cells (EC(50) from 51.9+/-6.1 microM to 17.8+/-2.6 microM in the absence and in the presence of MC18, respectively) and restored in PC-3 cells (EC(50) from 150+/-2.4 microM to 60+/-3.5 microM in the absence and in the presence of MC18, respectively). Moreover, we established the contribution of each transporter employing stable transfected cells (MDCK) overexpressing P-glycoprotein or BCRP or MRP1 pump. The results displayed that P-glycoprotein and BCRP were involved in bicalutamide efflux while MRP1 was unable to bind the antiandrogen drug.

  4. CUTI-1: A novel tetraspan protein involved in C. elegans CUTicle formation and epithelial integrity.

    PubMed

    Fritz, Julie-Anne; Behm, Carolyn A

    2009-01-01

    The nematode cuticle is a tough extracellular matrix composed primarily of cross-linked collagens and non-collagenous cuticulins. It is required for nematode motility and protection from the external environment. Little is known about how the complex process of cuticle formation has been adapted to the specialized requirements of the nematode cuticle, which is structurally and compositionally unique from other organisms. The C. elegans gene cuti-1 (CUTicle and epithelial Integrity) encodes a nematode-specific protein. We have shown that CUTI-1 is expressed in the epithelia and in seam cells. Within these tissues the expression of cuti-1 mRNA cycles throughout development in line with the molting cycle, a process that involves synthesis of a new cuticle. In addition, knockdown of cuti-1 by RNA interference (RNAi) results in worms that display post-embryonic phenotypes related to cuticle dysfunction and defects in epithelial integrity. This is one of the first reports of a nematode-specific protein involved in extracellular matrix formation. It provides further insight into how novel ways have evolved to regulate the formation of the cuticle, which is the primary protective barrier and skeletal component of nematodes.

  5. Identification of a novel thylakoid protein gene involved in cold acclimation in cyanobacteria.

    PubMed

    Li, Weizhi; Gao, Hong; Yin, Chuntao; Xu, Xudong

    2012-09-01

    In cyanobacteria, genes involved in cold acclimation can be upregulated in response to cold stress with or without light. By inactivating 17 such genes in Synechocystis sp. PCC 6803, slr0815 (ccr2) was identified to be a novel gene required for survival at 15 °C. It was upregulated by cold stress in the light. Upon exposure to low temperature, a ccr2-null mutant showed greatly reduced photosynthetic and respiratory activities within 12 h relative to the wild-type. At 48 h, the photosystem (PS)II-mediated electron transport in the mutant was reduced to less than one-third of the wild-type level, and the duration of electron transfer from the Q(B) binding site of PSII to PSI was increased to about eight times the wild-type level, whereas the PSI-mediated electron transport remained unchanged. Using an antibody against GFP, a Ccr2-GFP fusion protein was localized to the thylakoid membrane rather than the cytoplasmic and outer membranes. Homologues to Ccr2 can be found in most cyanobacteria, algae and higher plants with sequenced genomes. Ccr2 is probably representative of a group of novel thylakoid proteins involved in acclimation to cold or other stresses.

  6. The fasciclin-like arabinogalactan protein gene, FLA3, is involved in microspore development of Arabidopsis.

    PubMed

    Li, Jun; Yu, Miao; Geng, Ling-Ling; Zhao, Jie

    2010-11-01

    Arabinogalactan proteins are widely distributed in plant tissues and cells, and may function in the growth and development of higher plants. To our knowledge, there is currently no direct evidence concerning the involvement of fasciclin-like arabinogalactan proteins (FLA) in sexual reproduction in Arabidopsis. In this study, Arabidopsis FLA3 was found to be specifically expressed in pollen grains and tubes. Subcellular localization showed that FLA3 anchors tightly to the plasma membrane, and its glycosylphosphatidylinositol anchor may affect its localization. FLA3-RNA interference transgenic plants had approximately 50% abnormal pollen grains (including shrunken and wrinkled phenotypes) which lacked viability. Cytological observations revealed that pollen abortion occurred during the transition from uninucleate microspores to bicellular pollens, with abnormal cellulose distribution seen by calcofluor white staining. Transmission electron microscopy showed that the basic structure of the exine layer in aberrant pollen was normal, but the intine layer appeared to have some abnormalities. Taken together, these results suggest that FLA3 is involved in microspore development and may affect pollen intine formation, possibly by participating in cellulose deposition. In FLA3-overexpressing transgenic plants, defective elongation of the stamen filament and reduced female fertility led to short siliques with low seed set, which suggested that ectopic expression of FLA3 in tissues may reduce or disrupt cell growth and then result in defects throughout the plant. © 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.

  7. Identification of proteins involved in the adhesionof Candida species to different medical devices.

    PubMed

    Núñez-Beltrán, Arianna; López-Romero, Everardo; Cuéllar-Cruz, Mayra

    2017-04-07

    Adhesion is the first step for Candida species to form biofilms on medical devices implanted in the human host. Both the physicochemical nature of the biomaterial and cell wall proteins (CWP) of the pathogen play a determinant role in the process. While it is true that some CWP have been identified in vitro, little is known about the CWP of pathogenic species of Candida involved in adhesion. On this background, we considered it important to investigate the potential role of CWP of C. albicans, C. glabrata, C. krusei and C. parapsilosis in adhesion to different medical devices. Our results indicate that the four species strongly adher to polyvinyl chloride (PVC) devices, followed by polyurethane and finally by silicone. It was interesting to identify fructose-bisphosphate aldolase (Fba1) and enolase 1 (Eno1) as the CWP involved in adhesion of C. albicans, C. glabrata and C. krusei to PVC devices whereas phosphoglycerate kinase (Pgk) and Eno1 allow C. parapsilosis to adher to silicone-made implants. Results presented here suggest that these CWP participate in the initial event of adhesion and are probably followed by other proteins that covalently bind to the biomaterial thus providing conditions for biofilm formation and eventually the onset of infection.

  8. The cyanobacterial Fluorescence Recovery Protein has two distinct activities: Orange Carotenoid Protein amino acids involved in FRP interaction.

    PubMed

    Thurotte, Adrien; Bourcier de Carbon, Céline; Wilson, Adjélé; Talbot, Léa; Cot, Sandrine; López-Igual, Rocio; Kirilovsky, Diana

    2017-04-01

    To deal with fluctuating light condition, cyanobacteria have developed a photoprotective mechanism which, under high light conditions, decreases the energy arriving at the photochemical centers. It relies on a photoswitch, the Orange Carotenoid Protein (OCP). Once photoactivated, OCP binds to the light harvesting antenna, the phycobilisome (PBS), and triggers the thermal dissipation of the excess energy absorbed. Deactivation of the photoprotective mechanism requires the intervention of a third partner, the Fluorescence Recovery Protein (FRP). FRP by interacting with the photoactivated OCP accelerates its conversion to the non-active form and its detachment from the phycobilisome. We have studied the interaction of FRP with free and phycobilisome-bound OCP. Several OCP variants were constructed and characterized. In this article we show that OCP amino acid F299 is essential and D220 important for OCP deactivation mediated by FRP. Mutations of these amino acids did not affect FRP activity as helper to detach OCP from phycobilisomes. In addition, while mutated R60L FRP is inactive on OCP deactivation, its activity on the detachment of the OCP from the phycobilisomes is not affected. Thus, our results demonstrate that FRP has two distinct activities: it accelerates OCP detachment from phycobilisomes and then it helps deactivation of the OCP. They also suggest that different OCP and FRP amino acids could be involved in these two activities.

  9. A novel pax-like protein involved in transcriptional activation of cyst wall protein genes in Giardia lamblia.

    PubMed

    Wang, Yi-Ting; Pan, Yu-Jiao; Cho, Chao-Cheng; Lin, Bo-Chi; Su, Li-Hsin; Huang, Yu-Chang; Sun, Chin-Hung

    2010-10-15

    Giardia lamblia differentiates into infectious cysts to survive outside of the host. It is of interest to identify factors involved in up-regulation of cyst wall proteins (CWPs) during this differentiation. Pax proteins are important regulators of development and cell differentiation in Drosophila and vertebrates. No member of this gene family has been reported to date in yeast, plants, or protozoan parasites. We have identified a pax-like gene (pax1) encoding a putative paired domain in the G. lamblia genome. Epitope-tagged Pax1 localized to nuclei during both vegetative growth and encystation. Recombinant Pax1 specifically bound to the AT-rich initiator elements of the encystation-induced cwp1 to -3 and myb2 genes. Interestingly, overexpression of Pax1 increased cwp1 to -3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of the transactivation function of Pax1. Our results indicate that the Pax family has been conserved during evolution, and Pax1 could up-regulate the key encystation-induced genes to regulate differentiation of the protozoan eukaryote, G. lamblia.

  10. 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

  11. 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.

  12. Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma

    PubMed Central

    Neira, José L.; Bintz, Jennifer; Arruebo, María; Rizzuti, Bruno; Bonacci, Thomas; Vega, Sonia; Lanas, Angel; Velázquez-Campoy, Adrián; Iovanna, Juan L.; Abián, Olga

    2017-01-01

    Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the ‘fuzzy’ interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs. PMID:28054562

  13. 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.

  14. HNE-modified proteins in Down syndrome: Involvement in development of Alzheimer disease neuropathology.

    PubMed

    Barone, Eugenio; Head, Elizabeth; Butterfield, D Allan; Perluigi, Marzia

    2016-11-10

    Down syndrome (DS), trisomy of chromosome 21, is the most common genetic form of intellectual disability. The neuropathology of DS involves multiple molecular mechanisms, similar to AD, including the deposition of beta-amyloid (Aβ) into senile plaques and tau hyperphosphorylationg in neurofibrillary tangles. Interestingly, many genes encoded by chromosome 21, in addition to being primarily linked to amyloid-beta peptide (Aβ) pathology, are responsible for increased oxidative stress (OS) conditions that also result as a consequence of reduced antioxidant system efficiency. However, redox homeostasis is disturbed by overproduction of Aβ, which accumulates into plaques across the lifespan in DS as well as in AD, thus generating a vicious cycle that amplifies OS-induced intracellular changes. The present review describes the current literature that demonstrates the accumulation of oxidative damage in DS with a focus on the lipid peroxidation by-product, 4-hydroxy-2-nonenal (HNE). HNE reacts with proteins and can irreversibly impair their functions. We suggest that among different post-translational modifications, HNE-adducts on proteins accumulate in DS brain and play a crucial role in causing the impairment of glucose metabolism, neuronal trafficking, protein quality control and antioxidant response. We hypothesize that dysfunction of these specific pathways contribute to accelerated neurodegeneration associated with AD neuropathology.

  15. Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma.

    PubMed

    Neira, José L; Bintz, Jennifer; Arruebo, María; Rizzuti, Bruno; Bonacci, Thomas; Vega, Sonia; Lanas, Angel; Velázquez-Campoy, Adrián; Iovanna, Juan L; Abián, Olga

    2017-01-05

    Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the 'fuzzy' interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs.

  16. 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.

  17. Looking for prosocial genes: ITRAQ analysis of proteins involved in MDMA-induced sociability in mice.

    PubMed

    Kuteykin-Teplyakov, Konstantin; Maldonado, Rafael

    2014-11-01

    Social behavior plays a fundamental role in life of many animal species, allowing the interaction between individuals and sharing of experiences, needs, and goals across them. In humans, some neuropsychiatric diseases, including anxiety, posttraumatic stress disorder and autism spectrum disorders, are often characterized by impaired sociability. Here we report that N-Methyl-3,4-methylenedioxyamphetamine (MDMA, "Ecstasy") at low dose (3mg/kg) has differential effects on mouse social behavior. In some animals, MDMA promotes sociability without hyperlocomotion, whereas in other mice it elevates locomotor activity without affecting sociability. Both WAY-100635, a selective antagonist of 5-HT1A receptor, and L-368899, a selective oxytocin receptor antagonist, abolish prosocial effects of MDMA. Differential quantitative analysis of brain proteome by isobaric tag for relative and absolute quantification technology (iTRAQ) revealed 21 specific proteins that were highly correlated with sociability, and allowed to distinguish between entactogenic prosocial and hyperlocomotor effects of MDMA on proteome level. Our data suggest particular relevance of neurotransmission mediated by GABA B receptor, as well as proteins involved in energy maintenance for MDMA-induced sociability. Functional association network for differentially expressed proteins in cerebral cortex, hippocampus and amygdala were identified. These results provide new information for understanding the neurobiological substrate of sociability and may help to discover new therapeutic approaches to modulate social behavior in patients suffering from social fear and low sociability.

  18. The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton

    PubMed Central

    1996-01-01

    Arp2p is an essential yeast actin-related protein. Disruption of the corresponding ARP2 gene leads to a terminal phenotype characterized by the presence of a single large bud. Thus, Arp2p may be important for a late stage of the cell cycle (Schwob, E., and R.P. Martin, 1992. Nature (Lond.). 355:179-182). We have localized Arp2p by indirect immunofluorescence. Specific peptide antibodies revealed punctate staining under the plasma membrane, which partially colocalizes with actin. Temperature-sensitive arp2 mutations were created by PCR mutagenesis and selected by an ade2/SUP11 sectoring screen. One temperature-sensitive mutant that was characterized, arp2-H330L, was osmosensitive and had an altered actin cytoskeleton at a nonpermissive temperature, suggesting a role of Arp2p in the actin cytoskeleton. Random budding patterns were observed in both haploid and diploid arp2- H330L mutant cells. Endocytosis, as judged by Lucifer yellow uptake, was severely reduced in the mutant, at all temperatures. In addition, genetic interaction was observed between temperature-sensitive alleles arp2-H330L and cdc10-1. CDC10 is a gene encoding a neck filament- associated protein that is necessary for polarized growth and cytokinesis. Overall, the immunolocalization, mutant phenotypes, and genetic interaction suggest that the Arp2 protein is an essential component of the actin cytoskeleton that is involved in membrane growth and polarity, as well as in endocytosis. PMID:8698808

  19. Nuclear pore proteins are involved in the biogenesis of functional tRNA.

    PubMed Central

    Simos, G; Tekotte, H; Grosjean, H; Segref, A; Sharma, K; Tollervey, D; Hurt, E C

    1996-01-01

    Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide-specific and intron-dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre-tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(-) pus1(-) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA. Images PMID:8641292

  20. A novel protein involved in heart development in Ambystoma mexicanum is localized in endoplasmic reticulum.

    PubMed

    Jia, P; Zhang, C; Huang, X P; Poda, M; Akbas, F; Lemanski, S L; Erginel-Unaltuna, N; Lemanski, L F

    2008-11-01

    The discovery of the naturally occurring cardiac non-function (c) animal strain in Ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. In homozygous mutant animals (c/c), rhythmic contractions of the embryonic heart are absent due to a lack of organized myofibrils. We have previously cloned a partial sequence of a peptide cDNA (N1) from an anterior-endoderm-conditioned-medium RNA library that had been shown to be able to rescue the mutant phenotype. In the current studies we have fully cloned the N1 full length cDNA sequence from the library. N1 protein has been detected in both adult heart and skeletal muscle but not in any other adult tissues. GFP-tagged expression of the N1 protein has revealed localization of the N1 protein in the endoplasmic reticulum (ER). Results from in situ hybridization experiments have confirmed the dramatic decrease of expression of N1 mRNA in mutant (c/c) embryos indicating that the N1 gene is involved in heart development.

  1. The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation.

    PubMed

    Scherfer, Christoph; Qazi, Mousumi R; Takahashi, Kuniaki; Ueda, Ryu; Dushay, Mitchell S; Theopold, Ulrich; Lemaitre, Bruno

    2006-07-01

    Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.

  2. 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.

  3. Arabidopsis Membrane Steroid Binding Protein 1 Is Involved in Inhibition of Cell ElongationW⃞

    PubMed Central

    Yang, Xiao-Hua; Xu, Zhi-Hong; Xue, Hong-Wei

    2005-01-01

    A putative Membrane Steroid Binding Protein (designated MSBP1) was identified and functionally characterized as a negative regulator of cell elongation in Arabidopsis thaliana. The MSBP1 gene encodes a 220–amino acid protein that can bind to progesterone, 5-dihydrotestosterone, 24-epi-brassinolide (24-eBL), and stigmasterol with different affinities in vitro. Transgenic plants overexpressing MSBP1 showed short hypocotyl phenotype and increased steroid binding capacity in membrane fractions, whereas antisense MSBP1 transgenic plants showed long hypocotyl phenotypes and reduced steroid binding capacity, indicating that MSBP1 negatively regulates hypocotyl elongation. The reduced cell elongation of MSBP1-overexpressing plants was correlated with altered expression of genes involved in cell elongation, such as expansins and extensins, indicating that enhanced MSBP1 affected a regulatory pathway for cell elongation. Suppression or overexpression of MSBP1 resulted in enhanced or reduced sensitivities, respectively, to exogenous progesterone and 24-eBL, suggesting a negative role of MSBP1 in steroid signaling. Expression of MSBP1 in hypocotyls is suppressed by darkness and activated by light, suggesting that MSBP1, as a negative regulator of cell elongation, plays a role in plant photomorphogenesis. This study demonstrates the functional roles of a steroid binding protein in growth regulation in higher plants. PMID:15608331

  4. Activation of a calcium-dependent protein kinase involved in the Azospirillum growth promotion in rice.

    PubMed

    Ribaudo, Claudia M; Curá, José A; Cantore, María L

    2017-02-01

    Rice seedlings (Oryza sativa) inoculated with the plant growth-promoting rhizobacteria Azospirillum brasilense FT326 showed an enhanced development of the root system 3 days after inoculation. Later on, a remarkable enlargement of shoots was also evident. An increase in the Ca(2+)-dependent histone kinase activity was also detected as a result of inoculation. The biochemical characterization and Western-blot analysis of the kinase strongly supports the hypothesis that it belongs to a member of the rice CDPK family. The fact that the amount of the protein did not change upon inoculation seems to indicate that a posttranslational activation is responsible for the change in the enzymatic activity. An in-gel kinase experiment identified a 46 kDa CDPK like protein kinase as a putative component of the signal transduction pathway triggered by Azospirillum inoculation. To our knowledge, this is the first report on the possible involvement of a Ca(2+)-dependent protein kinase in promotion of rice plants growth by A. brasilense.

  5. New shrimp IgE-binding proteins involved in mite-seafood cross-reactivity.

    PubMed

    Gámez, Cristina; Zafra, M Paz; Boquete, Manuel; Sanz, Verónica; Mazzeo, Carla; Ibáñez, M Dolores; Sánchez-García, Silvia; Sastre, Joaquín; del Pozo, Victoria

    2014-09-01

    Shrimp is a seafood consumed worldwide and the main cause of severe allergenic reactions to crustaceans. Seafood allergy has been related to mite sensitization, mainly mediated by tropomyosin, but other proteins could be involved. The aim of the study was to identify new shrimp allergens implicated in mite-seafood cross-reactivity (CR) in two different climate populations: dry and humid climates. Shrimp and mite IgE-binding profiles of patients from continental dry and humid climates were analyzed by immunoblotting, and the most frequently recognized Solenocera melantho shrimp proteins were identified by MS as α-actinin, β-actin, fructose biphosphate aldolase, arginine kinase, sarcoplasmic calcium-binding protein, and ubiquitin. Using inhibition immunoblot assays, we demonstrate that tropomyosin and ubiquitin were responsible for mite-seafood CR from both climates; but also α-actinin and arginine kinase are implicated in dry- and humid-climate populations, respectively. Reciprocal inhibition assays demonstrated that mites are the primary sensitizer in humid-climate, as shrimp is in continental dry-climate population. Several new shrimp allergens have been identified and should be considered in the diagnosis and treatment of shrimp allergy and mite-seafood CR. Differences in mite-seafood CR were founded to be based on the climate. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Adaptive expression pattern of different proteins involved in cellular calcium homeostasis in denervated rat vas deferens.

    PubMed

    Quintas, Luis Eduardo M; Cunha, Valéria M N; Scaramello, Christianne B V; da Silva, Cláudia L M; Caricati-Neto, Afonso; Lafayette, Simone S L; Jurkiewicz, Aron; Noël, François

    2005-11-21

    The activity and protein expression of plasma membrane and sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPases and ryanodine receptors were investigated in surgically denervated rat vas deferens. The function of thapsigargin-sensitive but not thapsigargin-resistant (Ca2+-Mg2+)ATPase (from sarco(endo)plasmic reticulum and plasma membrane, respectively), evidenced by enzyme activity and Ca2+ uptake experiments, was significantly depressed by 30-50% when compared to innervated vas. Western blots showed that such reduction in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase performance was accompanied by a decrement of similar magnitude in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase type 2 protein expression, without any significant change in plasma membrane (Ca2+-Mg2+)ATPase expression. Finally, [3H]ryanodine binding revealed that the density of ryanodine binding sites was reduced by 45% after denervation without modification in affinity. The present findings demonstrate that sarco(endo)plasmic reticulum proteins involved in intracellular calcium homeostasis are clearly down-regulated and brings further evidence of a modified calcium translocation in denervated rat vas deferens.

  7. 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.

  8. Kainate receptor-mediated depression of glutamatergic transmission involving protein kinase A in the lateral amygdala.

    PubMed

    Negrete-Díaz, José Vicente; Duque-Feria, Paloma; Andrade-Talavera, Yuniesky; Carrión, Miriam; Flores, Gonzalo; Rodríguez-Moreno, Antonio

    2012-04-01

    Kainate receptors (KARs) have been described as modulators of synaptic transmission at different synapses. However, this role of KARs has not been well characterized in the amygdala. We have explored the effect of kainate receptor activation at the synapse established between fibers originating at medial geniculate nucleus and the principal cells in the lateral amygdala. We have observed an inhibition of evoked excitatory postsynaptic currents (eEPSCs) amplitude after a brief application of KARs agonists KA and ATPA. Paired-pulse recordings showed a clear pair pulse facilitation that was enhanced after KA or ATPA application. When postsynaptic cells were loaded with BAPTA, the depression of eEPSC amplitude observed after the perfusion of KAR agonists was not prevented. We have also observed that the inhibition of the eEPSCs by KARs agonists was prevented by protein kinase A but not by protein kinase C inhibitors. Taken together our results indicate that KARs present at this synapse are pre-synaptic and their activation mediate the inhibition of glutamate release through a mechanism that involves the activation of protein kinase A. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

  9. 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

  10. Plasmodium falciparum proteins involved in cytoadherence of infected erythrocytes to chemokine CX3CL1

    PubMed Central

    Hermand, Patricia; Cicéron, Liliane; Pionneau, Cédric; Vaquero, Catherine; Combadière, Christophe; Deterre, Philippe

    2016-01-01

    Malaria caused by Plasmodium falciparum is associated with cytoadherence of infected red blood cells (iRBC) to endothelial cells. Numerous host molecules have been involved in cytoadherence, including the adhesive chemokine CX3CL1. Most of the identified parasite ligands are from the multigenic and hypervariable Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family which makes them poor targets for the development of a broadly protective vaccine. Using proteomics, we have identified two 25-kDa parasite proteins with adhesive properties for CX3CL1, called CBP for CX3CL1 Binding Proteins. CBPs are coded by single-copy genes with little polymorphic variation and no homology with other P. falciparum gene products. Specific antibodies raised against epitopes from the predicted extracellular domains of each CBP efficiently stain the surface of RBC infected with trophozoites or schizonts, which is a strong indication of CBP expression at the surface of iRBC. These anti-CBP antibodies partially neutralize iRBC adherence to CX3CL1. This adherence is similarly inhibited in the presence of peptides from the CBP extracellular domains, while irrelevant peptides had no such effect. CBP1 and CBP2 are new P. falciparum ligands for the human chemokine CX3CL1. The identification of this non-polymorphic P. falciparum factors provides a new avenue for innovative vaccination approaches. PMID:27653778

  11. Human axillary apocrine glands: proteins involved in the apocrine secretory mechanism.

    PubMed

    Stoeckelhuber, Mechthild; Schubert, Christoph; Kesting, Marco R; Loeffelbein, Denys J; Nieberler, Markus; Koehler, Claudia; Welsch, Ulrich

    2011-02-01

    The apocrine secretory mechanism is a mode of secretion by which the apical part of the cell cytoplasm is pinched off, which leads to the formation of an aposome. The distinct mechanism of formation and decapitation of the aposome is not well investigated. Only few proteins are known that are involved in this secretory mechanism. We studied the human axillary apocrine gland and looked at proteins associated with cytokinesis, a process that is comparable to the pinching-off mechanism of apocrine glandular cells. By immunohistochemistry, we detected actin, myosin II, cytokeratin 7 and 19, α- and β-tubulin, anillin, cofilin, syntaxin 2, vamp8/endobrevin and septin 2. In highly active glandular cells, these proteins are located at the base of the apical protrusion when the aposome is in the process of being released or are concentrated in the cap of the apical protrusion. These findings demonstrate new insights on apocrine secretory mechanisms and point to similarities to the terminal step of cytokinesis, which is regulated by a SNARE-mediated membrane fusion event.

  12. Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma

    NASA Astrophysics Data System (ADS)

    Neira, José L.; Bintz, Jennifer; Arruebo, María; Rizzuti, Bruno; Bonacci, Thomas; Vega, Sonia; Lanas, Angel; Velázquez-Campoy, Adrián; Iovanna, Juan L.; Abián, Olga

    2017-01-01

    Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the ‘fuzzy’ interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs.

  13. 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

  14. 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

    2013-12-02

    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. Copyright © 2013

  15. Involvement of FGF and BMP family proteins and VEGF in early human kidney development.

    PubMed

    Carev, Dominko; Saraga, Marijan; Saraga-Babic, Mirna

    2008-07-01

    The spatial and temporal pattern of the appearance of the fibroblast growth factor proteins (FGF-8 and FGF-10), the bone morphogenetic proteins (BMP-2/4 subfamily and BMP-7) and the vascular endothelial growth factor protein (VEGF) was investigated in the human mesonephros and metanephros of the 5-9 week-old conceptuses. In the mesonephros, both FGF's and BMP's were found in all structures and their expression slightly decreased in the early fetal period. VEGF positivity appeared in all mesonephric structures, and increased in the fetal period coincidently with formation of the mesonephric blood vessel network. In the metanephros, FGF-8 first appeared only in the metanephric mesenchyme, but from the 7th week on, its reactivity increased and spread to other metanephric structures. FGF-10 positive cells appeared in all metanephric structures already in the 5th week, and slightly intensified with progression of development. Cell survival and nephrogenesis in the permanent kidney might be associated with the appearance of both growth factors. Both BMP-2/4 and BMP-7 displayed a similar pattern of reactivity in all metanephric structures, and their reactivity intensified with advancing development. Alterations in their pattern of appearance might lead to the formation of small and dysplastic kidneys. Already in the earliest developmental stages, VEGF protein appeared in all metanephric structures. At later stages, VEGF showed more intense reaction in the collecting system than in the differentiating nephrons and interstitium. Due to VEGF involvement in vasculogenesis and angiogenesis, abnormal VEGF appearance might lead to impaired formation of the blood vessel network in the human permanent kidney.

  16. Mutations in Three Genes Encoding Proteins Involved in Hair Shaft Formation Cause Uncombable Hair Syndrome.

    PubMed

    Ü Basmanav, F Buket; Cau, Laura; Tafazzoli, Aylar; Méchin, Marie-Claire; Wolf, Sabrina; Romano, Maria Teresa; Valentin, Frederic; Wiegmann, Henning; Huchenq, Anne; Kandil, Rima; Garcia Bartels, Natalie; Kilic, Arzu; George, Susannah; Ralser, Damian J; Bergner, Stefan; Ferguson, David J P; Oprisoreanu, Ana-Maria; Wehner, Maria; Thiele, Holger; Altmüller, Janine; Nürnberg, Peter; Swan, Daniel; Houniet, Darren; Büchner, Aline; Weibel, Lisa; Wagner, Nicola; Grimalt, Ramon; Bygum, Anette; Serre, Guy; Blume-Peytavi, Ulrike; Sprecher, Eli; Schoch, Susanne; Oji, Vinzenz; Hamm, Henning; Farrant, Paul; Simon, Michel; Betz, Regina C

    2016-12-01

    Uncombable hair syndrome (UHS), also known as "spun glass hair syndrome," "pili trianguli et canaliculi," or "cheveux incoiffables" is a rare anomaly of the hair shaft that occurs in children and improves with age. UHS is characterized by dry, frizzy, spangly, and often fair hair that is resistant to being combed flat. Until now, both simplex and familial UHS-affected case subjects with autosomal-dominant as well as -recessive inheritance have been reported. However, none of these case subjects were linked to a molecular genetic cause. Here, we report the identification of UHS-causative mutations located in the three genes PADI3 (peptidylarginine deiminase 3), TGM3 (transglutaminase 3), and TCHH (trichohyalin) in a total of 11 children. All of these individuals carry homozygous or compound heterozygous mutations in one of these three genes, indicating an autosomal-recessive inheritance pattern in the majority of UHS case subjects. The two enzymes PADI3 and TGM3, responsible for posttranslational protein modifications, and their target structural protein TCHH are all involved in hair shaft formation. Elucidation of the molecular outcomes of the disease-causing mutations by cell culture experiments and tridimensional protein models demonstrated clear differences in the structural organization and activity of mutant and wild-type proteins. Scanning electron microscopy observations revealed morphological alterations in hair coat of Padi3 knockout mice. All together, these findings elucidate the molecular genetic causes of UHS and shed light on its pathophysiology and hair physiology in general. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  17. Differential neuronal expression of receptor interacting protein 3 in rat retina: involvement in ischemic stress response

    PubMed Central

    2013-01-01

    Background Receptor-interacting protein 3 (RIP3), a member of RIP family proteins, has been shown to participate in programmed necrosis or necroptosis in cell biology studies. Evidence suggests that necroptosis may be a mode of neuronal death in the retina. Results In the present study we determined the expression of RIP3 in normal rat retina and its changes following acute high intraocular pressure (aHIOP). RIP3 immunoreactivity (IR) was largely present in the inner retinal layers, localized to subsets of cells expressing neuron-specific nuclear antigen (NeuN), parvalbumin and calbindin in the ganglion cell layer (GCL) and inner nuclear layer (INL). No double labeling was detected for RIP3 with PKC-α or rhodopsin. RIP3 immunoreactivity was increased in the GCL at 6 hr and 12 hr, but reduced at 24 hr in the retina, without apparent alteration in laminar or cellular distribution pattern. Western blot analysis confirmed the above time-dependent alteration in RIP3 protein expression. RIP3 expressing cells frequently co-localized with propidium iodide (PI). A few co-localized cells were observed between RIP3 and Bax or cleaved caspase-3 in the GCL in 12 hr following aHIOP. Conclusions The results indicate that RIP3 is expressed differentially in retinal neurons in adult rats, including subsets of ganglion cells, amacrine and horizontal cells. RIP3 protein levels are elevated rapidly following aHIOP. RIP3 labeling co-localized with PI, Bax or cleaved caspase-3 among cells in the ganglion cell layer following aHIOP, which suggest its involvement of RIP3 in neuronal responses to acute ischemic insults. PMID:23374330

  18. 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

  19. Involvement of cyclic nucleotide-dependent protein kinases in cyclic AMP-mediated vasorelaxation

    PubMed Central

    Eckly-Michel, Anita; Martin, Viviane; Lugnier, Claire

    1997-01-01

    The involvement of cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase (PKG) in the effects of cyclic AMP-elevating agents on vascular smooth muscle relaxation, cyclic nucleotide dependent-protein kinase activities and ATP-induced calcium signalling ([Ca2+]i) was studied in rat aorta. Cyclic AMP-elevating agents used were a β-adrenoceptor agonist (isoprenaline), a phosphodiesterase 3 (PDE3) inhibitor (SK&F 94120) and a PDE4 inhibitor (rolipram). In rat intact aorta, the relaxant effect induced by isoprenaline (0.01–0.3 μM) was decreased by a specific inhibitor of PKA, H-89, whereas a specific inhibitor of PKG, Rp-8-Br-cyclic GMPS, was without effect. No significant difference in PKA and PKG activity ratios was detected in aortic rings when isoprenaline 10 μM was used. At the same concentration, isoprenaline did not modify ATP-induced changes in [Ca2+]i in smooth muscle cells. Neither H-89 nor Rp-8-Br-cyclic GMPS modified this response. These findings suggest that PKA is only involved in the relaxant effect induced by low concentrations of isoprenaline (0.01–0.3 μM), whereas for higher concentrations, other mechanisms independent of PKA and PKG are involved. The relaxant effects induced by SK&F 94120 and rolipram were inhibited by Rp-8-Br-cyclic GMPS with no significant effect of H-89. Neither SK&F 94120, nor rolipram at 30 μM significantly modified the activity ratios of PKA and PKG. Rolipram inhibited the ATP-induced transient increase in [Ca2+]i. This decrease was abolished by Rp-8-Br-cyclic GMPS whereas H-89 had no significant effect. These results suggest that PKG is involved in the vascular effects induced by the inhibitors of PDE3 and PDE4. Moreover, since it was previously shown that PDE3 and PDE4 inhibitors only increased cyclic AMP levels with no change in cyclic GMP level, these data also suggest a cross-activation of PKG by cyclic AMP in rat aorta. The combination of 5 μM SK&F 94120 with rolipram markedly

  20. 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.

  1. Aedes aegypti Piwi4 Is a Noncanonical PIWI Protein Involved in Antiviral Responses.

    PubMed

    Varjak, Margus; Maringer, Kevin; Watson, Mick; Sreenu, Vattipally B; Fredericks, Anthony C; Pondeville, Emilie; Donald, Claire L; Sterk, Jelle; Kean, Joy; Vazeille, Marie; Failloux, Anna-Bella; Kohl, Alain; Schnettler, Esther

    2017-01-01

    The small interfering RNA (siRNA) pathway is a major antiviral response in mosquitoes; however, another RNA interference pathway, the PIWI-interacting RNA (piRNA) pathway, has been suggested to be antiviral in mosquitoes. Piwi4 has been reported to be a key mediator of this response in mosquitoes, but it is not involved in the production of virus-specific piRNAs. Here, we show that Piwi4 associates with members of the antiviral exogenous siRNA pathway (Ago2 and Dcr2), as well as with proteins of the piRNA pathway (Ago3, Piwi5, and Piwi6) in an Aedes aegypti-derived cell line, Aag2. Analysis of small RNAs captured by Piwi4 revealed that it is predominantly associated with virus-specific siRNAs in Semliki Forest virus-infected cells and, to a lesser extent, with viral piRNAs. By using a Dcr2 knockout cell line, we showed directly that Ago2 lost its antiviral activity, as it was no longer bound to siRNAs, but Piwi4 retained its antiviral activity in the absence of the siRNA pathway. These results demonstrate a complex interaction between the siRNA and piRNA pathways in A. aegypti and identify Piwi4 as a noncanonical PIWI protein that interacts with members of the siRNA and piRNA pathways, and its antiviral activities may be independent of either pathway. IMPORTANCE Mosquitoes transmit several pathogenic viruses, for example, the chikungunya and Zika viruses. In mosquito cells, virus replication intermediates in the form of double-stranded RNA are cleaved by Dcr2 into 21-nucleotide-long siRNAs, which in turn are used by Ago2 to target the virus genome. A different class of virus-derived small RNAs, PIWI-interacting RNAs (piRNAs), have also been found in infected insect cells. These piRNAs are longer and are produced in a Dcr2-independent manner. The only known antiviral protein in the PIWI family is Piwi4, which is not involved in piRNA production. It is associated with key proteins of the siRNA and piRNA pathways, although its antiviral function is independent of

  2. Probing the Sites of Interactions of Rotaviral Proteins Involved in Replication

    PubMed Central

    Viskovska, Maria; Anish, Ramakrishnan; Hu, Liya; Chow, Dar-Chone; Hurwitz, Amy M.; Brown, Nicholas G.; Palzkill, Timothy; Estes, Mary K.

    2014-01-01

    ABSTRACT Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms, which form during virus infection. These processes are orchestrated by yet-to-be-understood complex networks of interactions involving nonstructural proteins (NSPs) 2, 5, and 6 and structural proteins (VPs) 1, 2, 3, and 6. The multifunctional enzyme NSP2, an octamer with RNA binding activity, is critical for viroplasm formation with its binding partner, NSP5, and for genome replication/packaging through its interactions with replicating RNA, the viral polymerase VP1, and the inner core protein VP2. Using isothermal calorimetry, biolayer interferometry, and peptide array screening, we examined the interactions between NSP2, VP1, VP2, NSP5, and NSP6. These studies provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from reciprocal peptide arrays were found to be in close proximity to the RNA template entry and double-stranded RNA (dsRNA) exit tunnels of VP1 and near the catalytic cleft and RNA-binding grooves of NSP2; these sites are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1. Peptide screening of VP2 identified NSP2-binding sites in the regions close to the intersubunit junctions, suggesting that NSP2 binding could be a regulatory mechanism for preventing the premature self-assembly of VP2. The binding sites on NSP2 for NSP6 were found to overlap that of VP1, and the NSP5-binding sites overlap those of VP2 and VP1, suggesting that interaction of these proteins with NSP2 is likely spatially and/or temporally regulated. IMPORTANCE Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms that form during virus infection and are orchestrated by complex networks of interactions involving nonstructural proteins (NSPs) and structural proteins (VPs). A multifunctional RNA

  3. Membrane proteins involved in transport, vesicle traffic and Ca(2+) signaling increase in beetroots grown in saline soils.

    PubMed

    Lino, Bárbara; Chagolla, Alicia; E González de la Vara, Luis

    2016-07-01

    By separating plasma membrane proteins according to their hydropathy from beetroots grown in saline soils, several proteins probably involved in salt tolerance were identified by mass spectrometry. Beetroots, as a salt-tolerant crop, have developed mechanisms to cope with stresses associated with saline soils. To observe which plasma membrane (PM) proteins were more abundant in beet roots grown in saline soils, beet root plants were irrigated with water or 0.2 M NaCl. PM-enriched membrane preparations were obtained from these plants, and their proteins were separated according to their hydropathy by serial phase partitioning with Triton X-114. Some proteins whose abundance increased visibly in membranes from salt-grown beetroots were identified by mass spectrometry. Among them, there was a V-type H(+)-ATPase (probably from contaminating vacuolar membranes), which increased with salt at all stages of beetroots' development. Proteins involved in solute transport (an H(+)-transporting PPase and annexins), vesicle traffic (clathrin and synaptotagmins), signal perception and transduction (protein kinases and phospholipases, mostly involved in calcium signaling) and metabolism, appeared to increase in salt-grown beetroot PM-enriched membranes. These results suggest that PM and vacuolar proteins involved in transport, metabolism and signal transduction increase in beet roots adapted to saline soils. In addition, these results show that serial phase partitioning with Triton X-114 is a useful method to separate membrane proteins for their identification by mass spectrometry.

  4. 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.

  5. The Chlamydomonas reinhardtii Nar1 Gene Encodes a Chloroplast Membrane Protein Involved in Nitrite Transport

    PubMed Central

    Rexach, Jesus; Fernández, Emilio; Galván, Aurora

    2000-01-01

    A key step for nitrate assimilation in photosynthetic eukaryotes occurs within chloroplasts, where nitrite is reduced to ammonium, which is incorporated into carbon skeletons. The Nar1 gene from Chlamydomonas reinhardtii is clustered with five other genes for nitrate assimilation, all of them regulated by nitrate. Sequence analysis of genomic DNA and cDNA of Nar1 and comparative studies of strains having or lacking Nar1 have been performed. The deduced amino acid sequence indicates that Nar1 encodes a chloroplast membrane protein with substantial identity to putative formate and nitrite transporters in bacteria. Use of antibodies against NAR1 has corroborated its location in the plastidic membrane. Characterization of strains having or lacking this gene suggests that NAR1 is involved in nitrite transport in plastids, which is critical for cell survival under limiting nitrate conditions, and controls the amount of nitrate incorporated by the cells under limiting CO2 conditions. PMID:10948261

  6. Involvement of retinoblastoma-associated protein 48 during photodynamic therapy of cervical cancer cells.

    PubMed

    Wu, Shuxia; Wang, Lijun; Ren, Xingye; Pan, Yulu; Peng, Yan; Zou, Xiaoyan; Shi, Cuige; Zhang, Youzhong

    2017-03-01

    5-Aminolevulinic acid-mediated photodynamic therapy (ALA‑PDT) is an effective treatment option for cervical intraepithelial neoplasia, the precancerous lesion of cervical cancer, and early cervical cancer, particularly for young or nulliparous women who want to remain fertile. A previous report described the involvement of histone deacetylases (HDAC) during ALA‑PDT mediated apoptosis in the cerebral cortex of a mouse model. Retinoblastoma‑associated protein 48 (RbAp48), a highly abundant component of HDACs, is a critical mediator that controls the transforming activity of human papillomavirus 16 in cervical cancer cells. The aim of the present study was to investigate the involvement of RbAp48 in ALA‑PDT‑induced cell death in cervical cancer cells. RbAp48 was significantly upregulated in cervical cancer cell lines treated with ALA‑PDT, including SiHa and HeLa cells. To establish the relevance of RbAp48 and the efficacy of ALA‑PDT in cervical cancer cells, the effect of ALA‑PDT was investigated in SiHa or HeLa cells following the depletion of RbAp48 by small interfering RNA (siRNA). Reduction of RbAp48 led to the reduced suppression of proliferation and apoptosis induced by ALA‑PDT in cervical cancer cells, which was associated with a reduction in tumor suppressor protein 53 (p53), retinoblastoma (Rb), apoptosis‑related enzyme caspase‑3, and increased levels of the oncogenic genes, human papillomavirus E6 and E7. These results provide evidence that RbAp48 is an important contributor to the efficacy of ALA‑PDT in cervical cancer cells. RbAp48 may be a therapeutic target that may help to improve the treatment of cervical cancer.

  7. Matrix Gla protein is involved in elastic fiber calcification in the dermis of pseudoxanthoma elasticum patients.

    PubMed

    Gheduzzi, Dealba; Boraldi, Federica; Annovi, Giulia; DeVincenzi, Chiara Paolinelli; Schurgers, Leon J; Vermeer, Cees; Quaglino, Daniela; Ronchetti, Ivonne Pasquali

    2007-10-01

    Mature MGP (Matrix gamma-carboxyglutamic acid protein) is known to inhibit soft connective tissues calcification. We investigated its possible involvement in pseudoxanthoma elasticum (PXE), a genetic disorder whose clinical manifestations are due to mineralization of elastic fibers. PXE patients have lower serum concentration of total MGP compared to controls (P<0.001). Antibodies specific for the noncarboxylated (Glu-MGP) and for the gamma-carboxylated (Gla-MGP) forms of MGP were assayed on ultrathin sections of dermis from controls and PXE patients. Normal elastic fibers in controls and patients were slightly positive for both forms of MGP, whereas Gla-MGP was more abundant within control's than within patient's elastic fibers (P<0.001). In patients' calcified elastic fibers, Glu-MGP intensively colocalized with mineral precipitates, whereas Gla-MGP precisely localized at the mineralization front. Data suggest that MGP is present within elastic fibers and is associated with calcification of dermal elastic fibers in PXE. To investigate whether local cells produce MGP, dermal fibroblasts were cultured in vitro and MGP was assayed at mRNA and protein levels. In spite of very similar MGP mRNA expression, cells from PXE patients produced 30% less of Gla-MGP compared to controls. Data were confirmed by immunocytochemistry on ultrathin sections. Normal fibroblasts in vitro were positive for both forms of MGP. PXE fibroblasts were positive for Glu-MGP and only barely positive for Gla-MGP (P<0.001). In conclusion, MGP is involved in elastic fiber calcification in PXE. The lower ratio of Gla-MGP over Glu-MGP in pathological fibroblasts compared to controls suggests these cells may play an important role in the ectopic calcification in PXE.

  8. Involvement of eicosanoids and surfactant protein D in extrinsic allergic alveolitis.

    PubMed

    Higashi, A; Higashi, N; Tsuburai, T; Takeuchi, Y; Taniguchi, M; Mita, H; Saito, A; Takatori, K; Arimura, K; Akiyama, K

    2005-12-01

    The pathophysiology of extrinsic allergic alveolitis (EAA) involves oxidative lung damage as well as interstitial and alveolar inflammation. Macrophages and mast cells are inflammatory components of EAA that produce both leukotrienes (LTs) and prostaglandin D2 (PGD2). In addition, PGD2 is also produced by the free-radical-catalysed peroxidation of arachidonic acid during oxidative stress. Urinary 8-iso prostaglandin F2alpha (8-isoPGF2alpha) and serum surfactant protein D (SP-D) are considered appropriate biomarkers of oxidative stress and interstitial lung disease activity, respectively. The present study aimed to assess the association of these biomarkers with the pathophysiology of EAA. Two cases of acute EAA caused by the inhalation of fungi spores were reported. Eight asthmatic patients and six healthy control subjects were also enrolled in the current study. The serum SP-D and urinary eicosanoid (LTE4, PGD2 metabolite (9alpha,11betaPGF2), 8-isoPGF2alpha) concentrations markedly increased during the acute exacerbation phase. These concentrations decreased following corticosteroid therapy in the EAA patients. There was a significant correlation between serum SP-D and urinary 9alpha,11betaPGF2 concentrations in the EAA patients. In conclusion, although the present study proposes that serum surfactant protein-D and urinary eicosanoids are new biomarkers involved in the various immunological responses in extrinsic allergic alveolitis, further large-scale studies are needed to investigate the role of these compounds, not just as biomarkers, but also as biological potentiators of extrinsic allergic alveolitis.

  9. Identify in Breast Cancer Stem Cell-Like Cells the Proteins Involved in Non-Homologous End Joining DNA Repair

    DTIC Science & Technology

    2007-09-01

    Cells the Proteins Involved in Non - Homologous End Joining DNA Repair PRINCIPAL INVESTIGATOR: Hong Yin, M.D., PH.D...5a. CONTRACT NUMBER Identify in Breast Cancer Stem Cell-Like Cells the Proteins Involved in Non - Homologous End Joining DNA Repair 5b. GRANT NUMBER...subpopulations. 15. SUBJECT TERMS CD44/CD24, Cancer stem-like cells, radiation sensitivity, non - homologous end joining DNA repair, response to DNA

  10. Adenylate cyclase-associated protein 1 overexpressed in pancreatic cancers is involved in cancer cell motility.

    PubMed

    Yamazaki, Ken; Takamura, Masaaki; Masugi, Yohei; Mori, Taisuke; Du, Wenlin; Hibi, Taizo; Hiraoka, Nobuyoshi; Ohta, Tsutomu; Ohki, Misao; Hirohashi, Setsuo; Sakamoto, Michiie

    2009-04-01

    Pancreatic cancer has the worst prognosis among cancers due to the difficulty of early diagnosis and its aggressive behavior. To characterize the aggressiveness of pancreatic cancers on gene expression, pancreatic cancer xenografts transplanted into severe combined immunodeficient mice served as a panel for gene-expression profiling. As a result of profiling, the adenylate cyclase-associated protein 1 (CAP1) gene was shown to be overexpressed in all of the xenografts. The expression of CAP1 protein in all 73 cases of pancreatic cancer was recognized by immunohistochemical analyses. The ratio of CAP1-positive tumor cells in clinical specimens was correlated with the presence of lymph node metastasis and neural invasion, and also with the poor prognosis of patients. Immunocytochemical analyses in pancreatic cancer cells demonstrated that CAP1 colocalized to the leading edge of lamellipodia with actin. Knockdown of CAP1 by RNA interference resulted in the reduction of lamellipodium formation, motility, and invasion of pancreatic cancer cells. This is the first report demonstrating the overexpression of CAP1 in pancreatic cancers and suggesting the involvement of CAP1 in the aggressive behavior of pancreatic cancer cells.

  11. HTLV-1 Tax-mediated TAK1 activation involves TAB2 adapter protein

    SciTech Connect

    Yu Qingsheng; Minoda, Yasumasa; Yoshida, Ryoko; Yoshida, Hideyuki; Iha, Hidekatsu; Kobayashi, Takashi; Yoshimura, Akihiko; Takaesu, Giichi

    2008-01-04

    Human T cell leukemia virus type 1 (HTLV-1) Tax is an oncoprotein that plays a crucial role in the proliferation and transformation of HTLV-1-infected T lymphocytes. It has recently been reported that Tax activates a MAPKKK family, TAK1. However, the molecular mechanism of Tax-mediated TAK1 activation is not well understood. In this report, we investigated the role of TAK1-binding protein 2 (TAB2) in Tax-mediated TAK1 activation. We found that TAB2 physically interacts with Tax and augments Tax-induced NF-{kappa}B activity. Tax and TAB2 cooperatively activate TAK1 when they are coexpressed. Furthermore, TAK1 activation by Tax requires TAB2 binding as well as ubiquitination of Tax. We also found that the overexpression of TRAF2, 5, or 6 strongly induces Tax ubiquitination. These results suggest that TAB2 may be critically involved in Tax-mediated activation of TAK1 and that NF-{kappa}B-activating TRAF family proteins are potential cellular E3 ubiquitin ligases toward Tax.

  12. Sendai virus trailer RNA binds TIAR, a cellular protein involved in virus-induced apoptosis.

    PubMed

    Iseni, Frédéric; Garcin, Dominique; Nishio, Machiko; Kedersha, Nancy; Anderson, Paul; Kolakofsky, Daniel

    2002-10-01

    Sendai virus (SeV) leader (le) and trailer (tr) RNAs are short transcripts generated during abortive antigenome and genome synthesis, respectively. Recom binant SeV (rSeV) that express tr-like RNAs from the leader region are non-cytopathic and, moreover, prevent wild-type SeV from inducing apoptosis in mixed infections. These rSeV thus appear to have gained a function. Here we report that tr RNA binds to a cellular protein with many links to apoptosis (TIAR) via the AU-rich sequence 5' UUUUAAAUUUU. Duplication of this AU-rich sequence alone within the le RNA confers TIAR binding on this le* RNA and a non-cytopathic phenotype to these rSeV in cell culture. Transgenic overexpression of TIAR during SeV infection promotes apoptosis and reverses the anti-apoptotic effects of le* RNA expression. More over, TIAR overexpression and SeV infection act synergistically to induce apoptosis. These short viral RNAs may act by sequestering TIAR, a multivalent RNA recognition motif (RRM) family RNA-binding protein involved in SeV-induced apoptosis. In this view, tr RNA is not simply a by-product of abortive genome synthesis, but is also an antigenome transcript that modulates the cellular antiviral response.

  13. The RNA binding protein TIAR is involved in the regulation of human iNOS expression.

    PubMed

    Fechir, M; Linker, K; Pautz, A; Hubrich, T; Kleinert, H

    2005-09-05

    Human inducible NO synthase (iNOS) expression is regulated by post-transcriptional mechanisms. The 3'-untranslated region (3'-UTR) of the human iNOS mRNA contains AU-rich elements (ARE), which are known to be important for the regulation of mRNA stability. The 3'-UTR of the human iNOS mRNA has been shown to regulate human iNOS mRNA expression post-transcriptionally. One RNA-binding protein known to interact with AREs and to regulate mRNA stability is the T cell intracellular antigen-1-related protein (TIAR). In RNA binding studies TIAR displayed high affinity binding to the human iNOS 3'-UTR sequence. In RNase protection experiments, the cytokine incubation needed for iNOS expression did not change TIAR expression in DLD-1 cells. However, overexpression of TIAR in human DLD-1 colon carcinoma cells resulted in enhanced cytokine-induced iNOS expression. In conclusion, TIAR seems to be involved in the post-transcriptional regulation of human iNOS expression.

  14. ZAS: C2H2 zinc finger proteins involved in growth and development.

    PubMed

    Wu, Lai-Chu

    2002-01-01

    A ZAS gene encodes a large protein with two separate C2H2 zinc finger pairs that independently bind to specific DNA sequences, including the kappaB motif. Three paralogous mammalian genes, ZAS1, ZAS2, and ZAS3, and a related Drosophila gene, Schnurri, have been cloned and characterized. The ZAS genes encode transcriptional proteins that activate or repress the transcription of a variety of genes involved in growth, development, and metastasis. In addition, ZAS3 associates with a TNF receptor-associated factor to inhibit NF-kappaB- and JNK/ SAPK-mediated signaling of TNF-alpha. Genetic experiments show that ZAS3 deficiency leads to proliferation of cells and tumor formation in mice. The data suggest that ZAS3 is important in controlling cell growth, apoptosis, and inflammation. The potent vasoactive hormone endothelin and transcription factor AP2 gene families also each consist of three members. The ZAS, endothelin, and transcription factor AP2 genes form several linkage groups. Knowledge of the chromosomal locations of these genes provides valuable clues to the evolution of the vertebrate genome.

  15. Involvement of the cellular prion protein in the migration of brain microvascular endothelial cells.

    PubMed

    Watanabe, Takuya; Yasutaka, Yuki; Nishioku, Tsuyoshi; Kusakabe, Sae; Futagami, Koujiro; Yamauchi, Atsushi; Kataoka, Yasufumi

    2011-06-01

    The conversion of cellular prion protein (PrP(C)) to its protease-resistant isoform is involved in the pathogenesis of prion disease. Although PrP(C) is a ubiquitous glycoprotein that is present in various cell types, the physiological role of PrP(C) remains obscure. The present study aimed to determine whether PrP(C) mediates migration of brain microvascular endothelial cells. Small interfering RNAs (siRNAs) targeting PrP(C) were transfected into a mouse brain microvascular endothelial cell line (bEND.3 cells). siPrP1, selected among three siRNAs, reduced mRNA and protein levels of PrP(C) in bEND.3 cells. Cellular migration was evaluated with a scratch-wound assay. siPrP1 suppressed migration without significantly affecting cellular proliferation. This study provides the first evidence that PrP(C) may be necessary for brain microvascular endothelial cells to migrate into damaged regions in the brain. This function of PrP(C) in the brain endothelium may be a mechanism by which the neurovascular unit recovers from an injury such as an ischemic insult.

  16. Equatorial segment protein (ESP) is a human alloantigen involved in sperm-egg binding and fusion.

    PubMed

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

    2008-01-01

    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.

  17. Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis

    PubMed Central

    Guo, Caixia; Fischhaber, Paula L.; Luk-Paszyc, Margaret J.; Masuda, Yuji; Zhou, Jing; Kamiya, Kenji; Kisker, Caroline; Friedberg, Errol C.

    2003-01-01

    Polκ and Rev1 are members of the Y family of DNA polymerases involved in tolerance to DNA damage by replicative bypass [translesion DNA synthesis (TLS)]. We demonstrate that mouse Rev1 protein physically associates with Polκ. We show too that Rev1 interacts independently with Rev7 (a subunit of a TLS polymerase, Polζ) and with two other Y-family polymerases, Polι and Polη. Mouse Polκ, Rev7, Polι and Polη each bind to the same ∼100 amino acid C-terminal region of Rev1. Furthermore, Rev7 competes directly with Polκ for binding to the Rev1 C-terminus. Notwith standing the physical interaction between Rev1 and Polκ, the DNA polymerase activity of each measured by primer extension in vitro is unaffected by the complex, either when extending normal primer-termini, when bypassing a single thymine glycol lesion, or when extending certain mismatched primer termini. Our observations suggest that Rev1 plays a role(s) in mediating protein–protein interactions among DNA polymerases required for TLS. The precise function(s) of these interactions during TLS remains to be determined. PMID:14657033

  18. Novel outer membrane protein involved in cellulose and cellooligosaccharide degradation by Cytophaga hutchinsonii.

    PubMed

    Ji, Xiaofei; Wang, Ying; Zhang, Cong; Bai, Xinfeng; Zhang, Weican; Lu, Xuemei

    2014-08-01

    Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium which was reported to use a novel contact-dependent strategy to degrade cellulose. It was speculated that cellooligosaccharides were transported into the periplasm for further digestion. In this study, we reported that most of the endoglucanase and -glucosidase activity was distributed on the cell surface of C. hutchinsonii.Cellobiose and part of the cellulose could be hydrolyzed to glucose on the cell surface. However, the cell surface cellulolytic enzymes were not sufficient for cellulose degradation by C. hutchinsonii. An outer membrane protein, CHU_1277, was disrupted by insertional mutation. Although the mutant maintained the same endoglucanase activity and most of the -glucosidase activity,it failed to digest cellulose, and its cellooligosaccharide utilization ability was significantly reduced, suggesting that CHU_1277 was essential for cellulose degradation and played an important role in cellooligosaccharide utilization. Further study of cellobiose hydrolytic ability of the mutant on the enzymatic level showed that the -glucosidase activity in the outer membrane of the mutant was not changed. It revealed that CHU_1277 played an important role in assisting cell surface -glucosidase to exhibit its activity sufficiently. Studies on the outer membrane proteins involved in cellulose and cellooligosaccharide utilization could shed light on the mechanism of cellulose degradation by C. hutchinsonii.

  19. Activation of Holliday junction recognizing protein involved in the chromosomal stability and immortality of cancer cells.

    PubMed

    Kato, Tatsuya; Sato, Nagato; Hayama, Satoshi; Yamabuki, Takumi; Ito, Tomoo; Miyamoto, Masaki; Kondo, Satoshi; Nakamura, Yusuke; Daigo, Yataro

    2007-09-15

    We identified a novel gene HJURP (Holliday junction-recognizing protein) whose activation seemed to play a pivotal role in the immortality of cancer cells. HJURP was considered a possible downstream target for ataxia telangiectasia mutated signaling, and its expression was increased by DNA double-strand breaks (DSB). HJURP was involved in the homologous recombination pathway in the DSB repair process through interaction with hMSH5 and NBS1, which is a part of the MRN protein complex. HJURP formed nuclear foci in cells at S phase and those subjected to DNA damage. In vitro assays implied that HJURP bound directly to the Holliday junction and rDNA arrays. Treatment of cancer cells with small interfering RNA (siRNA) against HJURP caused abnormal chromosomal fusions and led to genomic instability and senescence. In addition, HJURP overexpression was observed in a majority of lung cancers and was associated with poor prognosis as well. We suggest that HJURP is an indispensable factor for chromosomal stability in immortalized cancer cells and is a potential novel therapeutic target for the development of anticancer drugs.

  20. 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

  1. 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

  2. Proteins from the Wnt pathway are involved in the pathogenesis and progression of mammary phyllodes tumours.

    PubMed

    Karim, R Z; Gerega, S K; Yang, Y H; Horvath, L; Spillane, A; Carmalt, H; Scolyer, R A; Lee, C S

    2009-11-01

    The Wnt pathway is important in cell signalling transduction and is involved in the pathogenesis of multiple tumour types. A comprehensive analysis of the expression of Wnt signalling pathway proteins in mammary phyllodes tumours (PTs) has not been previously performed. To evaluate the immunohistochemical expression of Wnt pathway proteins in a cohort of PTs, to determine their role in tumour pathogenesis and to identify any associations with patient outcome. 65 PTs (34 benign, 23 borderline and 8 malignant) diagnosed at a single institution between 1990 and 2006 were analysed. Immunohistochemical stains were performed on tissue microarrays for beta-catenin, Wnt1, Wnt5a, SFRP4 and E-cadherin. Stroma and epithelium were scored separately. Stromal cytoplasmic Wnt5a and SFRP4 expression showed significant progressive increases in expression with increasing grade (p = 0.002 and p = 0.02 respectively). Epithelial membranous and stromal nuclear beta-catenin, epithelial cytoplasmic Wnt1 and epithelial E-cadherin all also showed increasing expression with increasing tumour grade, however, the differences were not significant. Disease-free survival was significantly decreased (p = 0.0017) with positive epithelial E-cadherin staining. Results suggest that alterations in the Wnt pathway are important in the progression and in the epithelial and stromal interactions in PTs. They have important implications for understanding the pathogenesis of these uncommon but clinically important tumours.

  3. A Vicilin-Like Seed Storage Protein, PAP85, Is Involved in Tobacco Mosaic Virus Replication

    PubMed Central

    Chen, Cheng-En; Yeh, Kuo-Chen; Wu, Shu-Hsing; Wang, Hsiang-Iu

    2013-01-01

    One striking feature of viruses with RNA genomes is the modification of the host membrane structure during early infection. This process requires both virus- and host-encoded proteins; however, the host factors involved and their role in this process remain largely unknown. On infection with Tobacco mosaic virus (TMV), a positive-strand RNA virus, the filamentous and tubular endoplasmic reticulum (ER) converts to aggregations at the early stage and returns to filamentous at the late infectious stage, termed the ER transition. Also, membrane- or vesicle-packaged viral replication complexes (VRCs) are induced early during infection. We used microarray assays to screen the Arabidopsis thaliana gene(s) responding to infection with TMV in the initial infection stage and identified an Arabidopsis gene, PAP85 (annotated as a vicilin-like seed storage protein), with upregulated expression during 0.5 to 6 h of TMV infection. TMV accumulation was reduced in pap85-RNA interference (RNAi) Arabidopsis and restored to wild-type levels when PAP85 was overexpressed in pap85-RNAi Arabidopsis. We did not observe the ER transition in TMV-infected PAP85-knockdown Arabidopsis protoplasts. In addition, TMV accumulation was reduced in PAP85-knockdown protoplasts. VRC accumulation was reduced, but not significantly (P = 0.06), in PAP85-knockdown protoplasts. Coexpression of PAP85 and the TMV main replicase (P126), but not their expression alone in Arabidopsis protoplasts, could induce ER aggregations. PMID:23576511

  4. Molecular heterogeneity in major urinary proteins of Mus musculus subspecies: potential candidates involved in speciation

    PubMed Central

    Hurst, Jane L.; Beynon, Robert J.; Armstrong, Stuart D.; Davidson, Amanda J.; Roberts, Sarah A.; Gómez-Baena, Guadalupe; Smadja, Carole M.; Ganem, Guila

    2017-01-01

    When hybridisation carries a cost, natural selection is predicted to favour evolution of traits that allow assortative mating (reinforcement). Incipient speciation between the two European house mouse subspecies, Mus musculus domesticus and M.m.musculus, sharing a hybrid zone, provides an opportunity to understand evolution of assortative mating at a molecular level. Mouse urine odours allow subspecific mate discrimination, with assortative preferences evident in the hybrid zone but not in allopatry. Here we assess the potential of MUPs (major urinary proteins) as candidates for signal divergence by comparing MUP expression in urine samples from the Danish hybrid zone border (contact) and from allopatric populations. Mass spectrometric characterisation identified novel MUPs in both subspecies involving mostly new combinations of amino acid changes previously observed in M.m.domesticus. The subspecies expressed distinct MUP signatures, with most MUPs expressed by only one subspecies. Expression of at least eight MUPs showed significant subspecies divergence both in allopatry and contact zone. Another seven MUPs showed divergence in expression between the subspecies only in the contact zone, consistent with divergence by reinforcement. These proteins are candidates for the semiochemical barrier to hybridisation, providing an opportunity to characterise the nature and evolution of a putative species recognition signal. PMID:28337988

  5. Novel Outer Membrane Protein Involved in Cellulose and Cellooligosaccharide Degradation by Cytophaga hutchinsonii

    PubMed Central

    Ji, Xiaofei; Wang, Ying; Zhang, Cong; Bai, Xinfeng; Zhang, Weican

    2014-01-01

    Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium which was reported to use a novel contact-dependent strategy to degrade cellulose. It was speculated that cellooligosaccharides were transported into the periplasm for further digestion. In this study, we reported that most of the endoglucanase and β-glucosidase activity was distributed on the cell surface of C. hutchinsonii. Cellobiose and part of the cellulose could be hydrolyzed to glucose on the cell surface. However, the cell surface cellulolytic enzymes were not sufficient for cellulose degradation by C. hutchinsonii. An outer membrane protein, CHU_1277, was disrupted by insertional mutation. Although the mutant maintained the same endoglucanase activity and most of the β-glucosidase activity, it failed to digest cellulose, and its cellooligosaccharide utilization ability was significantly reduced, suggesting that CHU_1277 was essential for cellulose degradation and played an important role in cellooligosaccharide utilization. Further study of cellobiose hydrolytic ability of the mutant on the enzymatic level showed that the β-glucosidase activity in the outer membrane of the mutant was not changed. It revealed that CHU_1277 played an important role in assisting cell surface β-glucosidase to exhibit its activity sufficiently. Studies on the outer membrane proteins involved in cellulose and cellooligosaccharide utilization could shed light on the mechanism of cellulose degradation by C. hutchinsonii. PMID:24837387

  6. Solute carriers involved in energy transfer of mitochondria form a homologous protein family.

    PubMed

    Aquila, H; Link, T A; Klingenberg, M

    1987-02-09

    The sequences of three mitochondrial carriers involved in energy transfer, the ADP/ATP carrier, phosphate carrier and uncoupling carrier, are analyzed. Similarly to what has been previously reported for the ADP/ATP carrier and the uncoupling protein, now also the phosphate carrier is found to have a tripartite structure comprising three similar repeats of approx. 100 residues each. The three sequences show a fair overall homology with each other. More significant homologies are found by comparing the repeats within and between the carriers in a scheme where the sequences are spliced into repeats, which are arranged for maximum homology by allowing possible insertions or deletions. A striking conservation of critical residues, glycine, proline, of charged and of aromatic residues is found throughout all nine repeats. This is indicative of a similar structural principle in the repeats. Hydropathy profiles of the three proteins and a search for amphipathic alpha-spans reveal six membrane-spanning segments for each carrier, providing further support for the basic structural identity of the repeats. The proposed folding pattern of the carriers in the membrane is exemplified with the phosphate carrier. A possible tertiary arrangement of the repeats and the membrane-spanning helices is shown. The emergence of a mitochondrial carrier family by triplication and by divergent evolution from a common gene of about 100 residues is discussed.

  7. 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.

  8. Hepatitis C virus nonstructural protein 5B is involved in virus morphogenesis.

    PubMed

    Gouklani, Hamed; Bull, Rowena A; Beyer, Claudia; Coulibaly, Fasséli; Gowans, Eric J; Drummer, Heidi E; Netter, Hans J; White, Peter A; Haqshenas, Gholamreza

    2012-05-01

    The p7 protein of hepatitis C virus (HCV) is a viroporin that is dispensable for viral genome replication but plays a critical role in virus morphogenesis. In this study, we generated a JFH1-based intergenotypic chimeric genome that encoded a heterologous genotype 1b (GT1b) p7. The parental intergenotypic chimeric genome was nonviable in human hepatoma cells, and infectious chimeric virions were produced only when cells transfected with the chimeric genomes were passaged several times. Sequence analysis of the entire polyprotein-coding region of the recovered chimeric virus revealed one predominant amino acid substitution in nonstructural protein 2 (NS2), T23N, and one in NS5B, K151R. Forward genetic analysis demonstrated that each of these mutations per se restored the infectivity of the parental chimeric genome, suggesting that interactions between p7, NS2, and NS5B were required for virion assembly/maturation. p7 and NS5B colocalized in cellular compartments, and the NS5B mutation did not affect the colocalization pattern. The NS5B K151R mutation neither increased viral RNA replication in human hepatoma cells nor altered the polymerase activity of NS5B in an in vitro assay. In conclusion, this study suggests that HCV NS5B is involved in virus morphogenesis.

  9. Hepatitis C Virus Nonstructural Protein 5B Is Involved in Virus Morphogenesis

    PubMed Central

    Gouklani, Hamed; Bull, Rowena A.; Beyer, Claudia; Coulibaly, Fasséli; Gowans, Eric J.; Drummer, Heidi E.; Netter, Hans J.; White, Peter A.

    2012-01-01

    The p7 protein of hepatitis C virus (HCV) is a viroporin that is dispensable for viral genome replication but plays a critical role in virus morphogenesis. In this study, we generated a JFH1-based intergenotypic chimeric genome that encoded a heterologous genotype 1b (GT1b) p7. The parental intergenotypic chimeric genome was nonviable in human hepatoma cells, and infectious chimeric virions were produced only when cells transfected with the chimeric genomes were passaged several times. Sequence analysis of the entire polyprotein-coding region of the recovered chimeric virus revealed one predominant amino acid substitution in nonstructural protein 2 (NS2), T23N, and one in NS5B, K151R. Forward genetic analysis demonstrated that each of these mutations per se restored the infectivity of the parental chimeric genome, suggesting that interactions between p7, NS2, and NS5B were required for virion assembly/maturation. p7 and NS5B colocalized in cellular compartments, and the NS5B mutation did not affect the colocalization pattern. The NS5B K151R mutation neither increased viral RNA replication in human hepatoma cells nor altered the polymerase activity of NS5B in an in vitro assay. In conclusion, this study suggests that HCV NS5B is involved in virus morphogenesis. PMID:22345449

  10. The novel heart-specific RING finger protein 207 is involved in energy metabolism in cardiomyocytes.

    PubMed

    Mizushima, Wataru; Takahashi, Hidehisa; Watanabe, Masashi; Kinugawa, Shintaro; Matsushima, Shouji; Takada, Shingo; Yokota, Takashi; Furihata, Takaaki; Matsumoto, Junichi; Tsuda, Masaya; Chiba, Ikuru; Nagashima, Shun; Yanagi, Shigeru; Matsumoto, Masaki; Nakayama, Keiichi I; Tsutsui, Hiroyuki; Hatakeyama, Shigetsugu

    2016-11-01

    A failing heart shows severe energy insufficiency, and it is presumed that this energy shortage plays a critical role in the development of cardiac dysfunction. However, little is known about the mechanisms that cause energy metabolic alterations in the failing heart. Here, we show that the novel RING-finger protein 207 (RNF207), which is specifically expressed in the heart, plays a role in cardiac energy metabolism. Depletion of RNF207 in neonatal rat cardiomyocytes (NRCs) leads to a reduced cellular concentration of adenosine triphosphate (ATP) and mitochondrial dysfunction. Consistent with this result, we observed here that the expression of RNF207 was significantly reduced in mice with common cardiac diseases including heart failure. Intriguingly, proteomic approaches revealed that RNF207 interacts with the voltage-dependent anion channel (VDAC), which is considered to be a key regulator of mitochondria function, as an RNF207-interacting protein. Our findings indicate that RNF207 is involved in ATP production by cardiomyocytes, suggesting that RNF207 plays an important role in the development of heart failure. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. A review of the electrophilic reaction chemistry involved in covalent protein binding relevant to toxicity.

    PubMed

    Enoch, S J; Ellison, C M; Schultz, T W; Cronin, M T D

    2011-10-01

    Several pieces of legislation have led to an increased interest in the use of in silico methods, specifically the formation of chemical categories for the assessment of toxicological endpoints. For a number of endpoints, this requires a detailed knowledge of the electrophilic reaction chemistry that governs the ability of an exogenous chemical to form a covalent adduct. Historically, this chemistry has been defined as compilations of structural alerts without documenting the associated electrophilic chemistry mechanisms. To address this, this article has reviewed the literature defining the structural alerts associated with covalent protein binding and detailed the associated electrophilic reaction chemistry. This information is useful to both toxicologists and regulators when using the chemical category approach to fill data gaps for endpoints involving covalent protein binding. The structural alerts and associated electrophilic reaction chemistry outlined in this review have been incorporated into the OECD (Q)SAR Toolbox, a freely available software tool designed to fill data gaps in a regulatory environment without the need for further animal testing.

  12. RNA interference identifies a calcium-dependent protein kinase involved in Medicago truncatula root development.

    PubMed

    Ivashuta, Sergey; Liu, Jinyuan; Liu, Junqi; Lohar, Dasharath P; Haridas, Sajeet; Bucciarelli, Bruna; VandenBosch, Kathryn A; Vance, Carroll P; Harrison, Maria J; Gantt, J Stephen

    2005-11-01

    Changes in cellular or subcellular Ca2+ concentrations play essential roles in plant development and in the responses of plants to their environment. However, the mechanisms through which Ca2+ acts, the downstream signaling components, as well as the relationships among the various Ca2+-dependent processes remain largely unknown. Using an RNA interference-based screen for gene function in Medicago truncatula, we identified a gene that is involved in root development. Silencing Ca2+-dependent protein kinase1 (CDPK1), which is predicted to encode a Ca2+-dependent protein kinase, resulted in significantly reduced root hair and root cell lengths. Inactivation of CDPK1 is also associated with significant diminution of both rhizobial and mycorrhizal symbiotic colonization. Additionally, microarray analysis revealed that silencing CDPK1 alters cell wall and defense-related gene expression. We propose that M. truncatula CDPK1 is a key component of one or more signaling pathways that directly or indirectly modulates cell expansion or cell wall synthesis, possibly altering defense gene expression and symbiotic interactions.

  13. Involvement of protein kinase C in the response of Neurospora crassa to blue light.

    PubMed

    Arpaia, G; Cerri, F; Baima, S; Macino, G

    1999-09-01

    As a first step towards understanding the process of blue light perception, and the signal transduction mechanisms involved, in Neurospora crassa we have used a pharmacological approach to screen a wide range of second messengers and chemical compounds known to interfere with the activity of well-known signal transducing molecules in vivo. We tested the influence of these compounds on the induction of the al-3 gene, a key step in light-induced carotenoid biosynthesis. This approach has implicated protein kinase C (PKC) as a component of the light transduction machinery. The conclusion is based on the effects of specific inhibitors (calphostin C and chelerythrine chloride) and activators of PKC (1,2-dihexanoyl-sn-glycerol). During vegetative growth PKC may be responsible for desensitization to light because inhibitors of the enzyme cause an increase in the total amount of mRNA transcribed after illumination. PKC is therefore proposed here to be an important regulator of transduction of the blue light signal, and may act through modification of the protein White Collar-1, which we show to be a substrate for PKC in N. crassa.

  14. 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.

  15. Human DCXR - another 'moonlighting protein' involved in sugar metabolism, carbonyl detoxification, cell adhesion and male fertility?

    PubMed

    Ebert, Bettina; Kisiela, Michael; Maser, Edmund

    2015-02-01

    Dicarbonyl/L-xylulose reductase (DCXR; SDR20C1), a member of the short-chain dehydrogenase/reductase (SDR) superfamily catalyzes the reduction of α-dicarbonyl compounds and monosaccharides. Its role in the metabolism of L-xylulose has been known since 1970, when essential pentosuria was found to be associated with DCXR deficiency. Despite its early discovery, our knowledge about the role of human DCXR in normal physiology and pathophysiology is still incomplete. Sporadic studies have demonstrated aberrant expression in several cancers, but their physiological significance is unknown. In reproductive medicine, where DCXR is commonly referred to as 'sperm surface protein P34H', it serves as marker for epididymal sperm maturation and is essential for gamete interaction and successful fertilization. DCXR exhibits a multifunctional nature, both acting as a carbonyl reductase and also performing non-catalytic functions, possibly resulting from interactions with other proteins. Recent observations associate DCXR with a role in cell adhesion, pointing to a novel function involving tumour progression and possibly metastasis. This review summarizes the current knowledge about human DCXR and its orthologs from mouse and Caenorhabditis elegans (DHS-21) with an emphasis on its multifunctional characteristics. Due to its close structural relationship with DCXR, carbonyl reductase 2 (Cbr2), a tetrameric enzyme found in several non-primate species is also discussed. Similar to human DCXR, Cbr2 from golden hamster (P26h) and cow (P25b) is essential for sperm-zona pellucida interaction and fertilization. Because of the apparent similarity of these two proteins and the inconsistent use of alternative names previously, we provide an overview of the systematic classification of DCXR and Cbr2 and a phylogenetic analysis to illustrate their ancestry.

  16. PARP12, an interferon-stimulated gene involved in the control of protein translation and inflammation.

    PubMed

    Welsby, Iain; Hutin, David; Gueydan, Cyril; Kruys, Veronique; Rongvaux, Anthony; Leo, Oberdan

    2014-09-19

    Transcriptome analyses have recently identified PARP12, a member of a large family of ADP-ribosyl transferases, as an interferon-induced gene (ISG), whose function remains incompletely characterized. We demonstrate herein that PARP12 is a genuine ISG, whose expressed protein displays at least two distinct subcellular locations and related functions. Upon ectopic expression or exposure to oxidative stress, PARP12 is recruited to stress-granules (SGs), known sites of mRNA translational arrest. Accordingly, PARP12 was found to block mRNA translation, possibly upon association to the translational machinery. Both the N-terminal domain (containing an RNA-binding domain characterized by the presence of five CCCH-type Zn-fingers) and integrity of the catalytic domain are required for this suppressive function. In contrast, stimulation with LPS leads to the localization of PARP12 to p62/SQSTM1 (an adaptor protein involved in innate signaling and autophagy) containing structures, unrelated to SGs. Deletion of the N-terminal domain promotes the association of the protein to p62/SQSTM1, suggesting that the RNA-binding domain is responsible for the subcellular localization of PARP12. Association to p62/SQSTM1 was found to correlate with increased NF-κB signaling, suggesting a role for PARP12 in inflammation. Collectively, these observations suggest that PARP12 can alternate between two distinct subcellular compartments associated to two distinct cellular functions. The present work therefore identifies PARP12 as an ISG with a potential role in cellular defenses against viral infections.

  17. 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.

  18. 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

  19. An Ehrlichia chaffeensis tandem repeat protein interacts with multiple host targets involved in cell signaling, transcriptional regulation, and vesicle trafficking.

    PubMed

    Wakeel, Abdul; Kuriakose, Jeeba A; McBride, Jere W

    2009-05-01

    Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes forming cytoplasmic membrane-bound microcolonies called morulae. To survive and replicate within phagocytes, E. chaffeensis exploits the host cell by modulating a number of host cell processes, but the ehrlichial effector proteins involved are unknown. In this study, we determined that p47, a secreted, differentially expressed, tandem repeat (TR) protein, interacts with multiple host proteins associated with cell signaling, transcriptional regulation, and vesicle trafficking. Yeast two-hybrid analysis revealed that p47 interacts with polycomb group ring finger 5 (PCGF5) protein, Src protein tyrosine kinase FYN (FYN), protein tyrosine phosphatase non-receptor type 2 (PTPN2), and adenylate cyclase-associated protein 1 (CAP1). p47 interaction with these proteins was further confirmed by coimmunoprecipitation assays and colocalization in HeLa cells transfected with p47-green fluorescent fusion protein (AcGFP1-p47). Moreover, confocal microscopy demonstrated p47-expressing dense-cored (DC) ehrlichiae colocalized with PCGF5, FYN, PTPN2, and CAP1. An amino-terminally truncated form of p47 containing TRs interacted only with PCGF5 and not with FYN, PTPN2, and CAP1, indicating differences in p47 domains that are involved in these interactions. These results demonstrate that p47 is involved in a complex network of interactions involving numerous host cell proteins. Furthermore, this study provides a new insight into the molecular and functional distinction of DC ehrlichiae, as well as the effector proteins involved in facilitating ehrlichial survival in mononuclear phagocytes.

  20. 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

  1. Arabidopsis microtubule destabilizing protein40 is involved in brassinosteroid regulation of hypocotyl elongation.

    PubMed

    Wang, Xianling; Zhang, Jin; Yuan, Ming; Ehrhardt, David W; Wang, Zhiyong; Mao, Tonglin

    2012-10-01

    The brassinosteroid (BR) phytohormones play crucial roles in regulating plant cell growth and morphogenesis, particularly in hypocotyl cell elongation. The microtubule cytoskeleton is also known to participate in the regulation of hypocotyl elongation. However, it is unclear if BR regulation of hypocotyl elongation involves the microtubule cytoskeleton. In this study, we demonstrate that BRs mediate hypocotyl cell elongation by influencing the orientation and stability of cortical microtubules. Further analysis identified the previously undiscovered Arabidopsis thaliana microtubule destabilizing protein40 (MDP40) as a positive regulator of hypocotyl cell elongation. Brassinazole-resistant1, a key transcription factor in the BR signaling pathway, directly targets and upregulates MDP40. Overexpression of MDP40 partially rescued the shorter hypocotyl phenotype in BR-deficient mutant de-etiolated-2 seedlings. Reorientation of the cortical microtubules in the cells of MDP40 RNA interference transgenic lines was less sensitive to BR. These findings demonstrate that MDP40 is a key regulator in BR regulation of cortical microtubule reorientation and mediates hypocotyl growth. This study reveals a mechanism involving BR regulation of microtubules through MDP40 to mediate hypocotyl cell elongation.

  2. 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.

  3. BcSUN1, a B. cinerea SUN-Family Protein, Is Involved in Virulence

    PubMed Central

    Pérez-Hernández, Alicia; González, Mario; González, Celedonio; van Kan, Jan A. L.; Brito, Nélida

    2017-01-01

    BcSUN1 is a glycoprotein secreted by Botrytis cinerea, an important plant pathogen that causes severe losses in agriculture worldwide. In this work, the role of BcSUN1 in different aspects of the B. cinerea biology was studied by phenotypic analysis of Bcsun1 knockout strains. We identified BcSUN1 as the only member of the Group-I SUN family of proteins encoded in the B. cinerea genome, which is expressed both in axenic culture and during infection. BcSUN1 is also weakly attached to the cellular surface and is involved in maintaining the structure of the cell wall and/or the extracellular matrix. Disruption of the Bcsun1 gene produces different cell surface alterations affecting the production of reproductive structures and adhesion to plant surface, therefore reducing B. cinerea virulence. BcSUN1 is the first member of the SUN family reported to be involved in the pathogenesis of a filamentous fungus. PMID:28163701

  4. The MagA protein of Magnetospirilla is not involved in bacterial magnetite biomineralization.

    PubMed

    Uebe, René; Henn, Verena; Schüler, Dirk

    2012-03-01

    Magnetotactic bacteria have the ability to orient along geomagnetic field lines based on the formation of magnetosomes, which are intracellular nanometer-sized, membrane-enclosed magnetic iron minerals. The formation of these unique bacterial organelles involves several processes, such as cytoplasmic membrane invagination and magnetosome vesicle formation, the accumulation of iron in the vesicles, and the crystallization of magnetite. Previous studies suggested that the magA gene encodes a magnetosome-directed ferrous iron transporter with a supposedly essential function for magnetosome formation in Magnetospirillum magneticum AMB-1 that may cause magnetite biomineralization if expressed in mammalian cells. However, more recent studies failed to detect the MagA protein among polypeptides associated with the magnetosome membrane and did not identify magA within the magnetosome island, a conserved genomic region that is essential for magnetosome formation in magnetotactic bacteria. This raised increasing doubts about the presumptive role of magA in bacterial magnetosome formation, which prompted us to reassess MagA function by targeted deletion in Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. Contrary to previous reports, magA mutants of both strains still were able to form wild-type-like magnetosomes and had no obvious growth defects. This unambiguously shows that magA is not involved in magnetosome formation in magnetotactic bacteria.

  5. The MagA Protein of Magnetospirilla Is Not Involved in Bacterial Magnetite Biomineralization

    PubMed Central

    Uebe, René; Henn, Verena

    2012-01-01

    Magnetotactic bacteria have the ability to orient along geomagnetic field lines based on the formation of magnetosomes, which are intracellular nanometer-sized, membrane-enclosed magnetic iron minerals. The formation of these unique bacterial organelles involves several processes, such as cytoplasmic membrane invagination and magnetosome vesicle formation, the accumulation of iron in the vesicles, and the crystallization of magnetite. Previous studies suggested that the magA gene encodes a magnetosome-directed ferrous iron transporter with a supposedly essential function for magnetosome formation in Magnetospirillum magneticum AMB-1 that may cause magnetite biomineralization if expressed in mammalian cells. However, more recent studies failed to detect the MagA protein among polypeptides associated with the magnetosome membrane and did not identify magA within the magnetosome island, a conserved genomic region that is essential for magnetosome formation in magnetotactic bacteria. This raised increasing doubts about the presumptive role of magA in bacterial magnetosome formation, which prompted us to reassess MagA function by targeted deletion in Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. Contrary to previous reports, magA mutants of both strains still were able to form wild-type-like magnetosomes and had no obvious growth defects. This unambiguously shows that magA is not involved in magnetosome formation in magnetotactic bacteria. PMID:22194451

  6. Involvement of hypothalamic AMP-activated protein kinase in leptin-induced sympathetic nerve activation.

    PubMed

    Tanida, Mamoru; Yamamoto, Naoki; Shibamoto, Toshishige; Rahmouni, Kamal

    2013-01-01

    In mammals, leptin released from the white adipose tissue acts on the central nervous system to control feeding behavior, cardiovascular function, and energy metabolism. Central leptin activates sympathetic nerves that innervate the kidney, adipose tissue, and some abdominal organs in rats. AMP-activated protein kinase (AMPK) is essential in the intracellular signaling pathway involving the activation of leptin receptors (ObRb). We investigated the potential of AMPKα2 in the sympathetic effects of leptin using in vivo siRNA injection to knockdown AMPKα2 in rats, to produce reduced hypothalamic AMPKα2 expression. Leptin effects on body weight, food intake, and blood FFA levels were eliminated in AMPKα2 siRNA-treated rats. Leptin-evoked enhancements of the sympathetic nerve outflows to the kidney, brown and white adipose tissues were attenuated in AMPKα2 siRNA-treated rats. To check whether AMPKα2 was specific to sympathetic changes induced by leptin, we examined the effects of injecting MT-II, a melanocortin-3 and -4 receptor agonist, on the sympathetic nerve outflows to the kidney and adipose tissue. MT-II-induced sympatho-excitation in the kidney was unchanged in AMPKα2 siRNA-treated rats. However, responses of neural activities involving adipose tissue to MT-II were attenuated in AMPKα2 siRNA-treated rats. These results suggest that hypothalamic AMPKα2 is involved not only in appetite and body weight regulation but also in the regulation of sympathetic nerve discharges to the kidney and adipose tissue. Thus, AMPK might function not only as an energy sensor, but as a key molecule in the cardiovascular, thermogenic, and lipolytic effects of leptin through the sympathetic nervous system.

  7. Toxicant-induced acceleration of epididymal sperm transit: androgen-dependent proteins may be involved.

    PubMed

    Klinefelter, G R; Suarez, J D

    1997-01-01

    protein profile in homogenates of the caput/corpus epididymidis revealed treatment-related diminutions in two proteins CC9 (M(r) = 42 kDa, pI = 4.2) and CC34 (M(r) = 35 kDa, pI = 5.5), and the level of each of these proteins in the caput/corpus was significantly correlated with the decrease in caput/corpus sperm number. Thus, both CEMS and HFLUT accelerate sperm transit through the proximal segment of the epididymis; and, while this effect is not dependent on the testis, it may involve a lesion in androgen-dependent epididymal function.

  8. Amyloid precursor protein in Drosophila glia regulates sleep and genes involved in glutamate recycling.

    PubMed

    Farca Luna, Abud Jose; Perier, Magali; Seugnet, Laurent

    2017-03-17

    The Amyloid Precursor Protein (App) plays a crucial role in Alzheimer disease (AD) via the production and deposition of toxic β-amyloid peptides. App is heavily expressed in neurons where the vast majority of studies investigating its function have been carried out, while almost nothing is known about its function in glia, where it is also expressed, and can potentially participate in the regulation of neuronal physiology. In this report, we investigated whether Appl, the Drosophila homolog of App, could influence sleep-wake regulation when its function is manipulated in glial cells. Appl inhibition in astrocyte-like and cortex glia resulted in higher sleep amounts and longer sleep bout duration during the night, while overexpression had the opposite effect. These sleep phenotypes were not the result of developmental defects, and were correlated with changes in expression in Glutamine Synthetase (GS) in astrocyte-like glia, and in changes in the gap-junction component innexin2 in cortex glia. Downregulating both GS and innexin2, but not either one individually, resulted in higher sleep amounts, similarly to Appl inhibition. Consistent with these results the expression of GS and innexin2 are increased following sleep deprivation indicating that these two genes are dynamically linked to vigilance states. Interestingly, the reduction of GS expression and the sleep phenotype observed upon Appl inhibition could be rescued by increasing the expression of the glutamate transporter dEaat1. In contrast, reducing dEaat1 expression severely disrupted sleep. These results associate glutamate recycling, sleep and a glial function for the App family proteins.StatementThe Amyloid Precursor Protein (App) has been intensively studied for its implication in Alzheimer Disease (AD). The attributed functions of App are linked to the physiology and cellular biology of neurons where the protein is predominantly expressed. Consequences on glia in AD are generally thought to be secondary

  9. 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

  10. Polydom Is an Extracellular Matrix Protein Involved in Lymphatic Vessel Remodeling.

    PubMed

    Morooka, Nanami; Futaki, Sugiko; Sato-Nishiuchi, Ryoko; Nishino, Masafumi; Totani, Yuta; Shimono, Chisei; Nakano, Itsuko; Nakajima, Hiroyuki; Mochizuki, Naoki; Sekiguchi, Kiyotoshi

    2017-04-14

    Lymphatic vasculature constitutes a second vascular system essential for immune surveillance and tissue fluid homeostasis. Maturation of the hierarchical vascular structure, with a highly branched network of capillaries and ducts, is crucial for its function. Environmental cues mediate the remodeling process, but the mechanism that underlies this process is largely unknown. Polydom (also called Svep1) is an extracellular matrix protein identified as a high-affinity ligand for integrin α9β1. However, its physiological function is unclear. Here, we investigated the role of Polydom in lymphatic development. We generated Polydom-deficient mice. Polydom(-/-) mice showed severe edema and died immediately after birth because of respiratory failure. We found that although a primitive lymphatic plexus was formed, it failed to undergo remodeling in Polydom(-/-) embryos, including sprouting of new capillaries and formation of collecting lymphatic vessels. Impaired lymphatic development was also observed after knockdown/knockout of polydom in zebrafish. Polydom was deposited around lymphatic vessels, but secreted from surrounding mesenchymal cells. Expression of Foxc2 (forkhead box protein c2), a transcription factor involved in lymphatic remodeling, was decreased in Polydom(-/-) mice. Polydom bound to the lymphangiogenic factor Ang-2 (angiopoietin-2), which was found to upregulate Foxc2 expression in cultured lymphatic endothelial cells. Expressions of Tie1/Tie2 receptors for angiopoietins were also decreased in Polydom(-/-) mice. Polydom affects remodeling of lymphatic vessels in both mouse and zebrafish. Polydom deposited around lymphatic vessels seems to ensure Foxc2 upregulation in lymphatic endothelial cells, possibly via the Ang-2 and Tie1/Tie2 receptor system. © 2017 American Heart Association, Inc.

  11. 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.

  12. Functional characterization of rice CW-domain containing zinc finger proteins involved in histone recognition.

    PubMed

    Zhang, Zhe; Zhang, Feng; Cheng, Zhi-Jun; Liu, Ling-Long; Lin, Qi-Bing; Wu, Fu-Qing; Zhang, Huan; Wang, Jiu-Lin; Wang, Jie; Guo, Xiu-Ping; Zhang, Xin; Lei, Cai-Lin; Zhao, Zhi-Chao; Zhu, Shan-Shan; Wan, Jian-Min

    2017-10-01

    Histone recognition is important for understanding the mechanisms of histone modification, which play a pivotal role in transcriptional regulation during plant development. Here, we identified three cysteine-tryptophan (CW)-domain containing zinc finger (ZF) proteins involved in histone recognition, namely OsCW-ZF3, OsCW-ZF5 and OsCW-ZF7. Protein sequence analysis showed that they have two unknown motifs in addition to the CW domain. All three OsCW-ZFs were expressed in aerial tissues, with relatively high levels in developing panicles. Subcellular localization revealed that the OsCW-ZFs target the cell nucleus and CW domains are not necessary for their nuclear localization. In contrast to OsCW-ZF3 and OsCW-ZF5 where the CW domains bind histone H3 lysine 4 with different methylated forms (H3K4me), the CW domain from OsCW-ZF7 recognizes only trimethylated histone H3 lysine 4 (H3K4me3). Analysis of mutant suggested that three conserved tryptophan residues in the CW domain are essential for binding to H3K4me. Further study found that OsCW-ZF7 interacts with TAFII20, a transcription initiation factor TFIID 20kDa subunit. Knockout of OsCW-ZF7 caused defective development of awns. This study provides new insights into our understanding of the CW domain and lays a foundation for further investigation of its roles in rice. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. 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.

  14. Mitochondrial fission and fusion in Dictyostelium discoideum: a search for proteins involved in membrane dynamics

    PubMed Central

    2012-01-01

    Background Mitochondrial morphology is maintained by two distinct membrane events -fission and fusion. Altering these conserved processes can disrupt mitochondrial morphology and distribution, thereby disrupting the organelle’s functionality and impeding cellular function. In higher eukaryotes, these processes are mediated by a family of dynamin-related proteins (DRP’s). In the lower eukaryotes, for instance Dictyostelium discoideum, mitochondrial fission and fusion have been implicated but not yet established. To understand the overall mechanism of these dynamics across organisms, we developed an assay to identify fission and fusion events in Dictyostelium and to assess the involvement of the mitochondrial proteins, MidA, CluA, and two DRP’s, DymA and DymB. Findings Using laser scanning confocal microscopy we show, for the first time, that lower eukaryotes mediate mitochondrial fission and fusion. In Dictyostelium, these processes are balanced, occurring approximately 1 event/minute. Quantification of the rates in midA-, cluA-, dymA-, or dymB- strains established that MidA appears to play an indirect role in the regulation of fission and fusion, while the DRP’s are not essential for these processes. Rates of fission and fusion were significantly reduced in cluA-cells, indicating that CluA is necessary for maintaining both fission and fusion. Conclusions We have successfully demonstrated that Dictyostelium mitochondria undergo the dynamic processes of fission and fusion. The classical mediators of membrane dynamics - the DRP’s – are not necessary for these dynamics, whereas CluA is necessary for both processes. This work contributes to our overall understanding of mitochondrial dynamics and ultimately will provide additional insight into mitochondrial disease. PMID:22980139

  15. Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement

    PubMed Central

    Alrashdan, Yazan A.; Alkhouri, Hatem; Chen, Emily; Lalor, Daniel J.; Poniris, Maree; Henness, Sheridan; Brightling, Christopher E.; Burgess, Janette K.; Armour, Carol L.; Ammit, Alaina J.

    2012-01-01

    CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma. PMID:22387292

  16. Involvement of Synaptonemal Complex Proteins in Sex Chromosome Segregation during Marsupial Male Meiosis

    PubMed Central

    Page, Jesús; Viera, Alberto; Parra, María Teresa; de la Fuente, Roberto; Suja, José Ángel; Prieto, Ignacio; Barbero, José Luis; Rufas, Julio S; Berríos, Soledad; Fernández-Donoso, Raúl

    2006-01-01

    Marsupial sex chromosomes break the rule that recombination during first meiotic prophase is necessary to ensure reductional segregation during first meiotic division. It is widely accepted that in marsupials X and Y chromosomes do not share homologous regions, and during male first meiotic prophase the synaptonemal complex is absent between them. Although these sex chromosomes do not recombine, they segregate reductionally in anaphase I. We have investigated the nature of sex chromosome association in spermatocytes of the marsupial Thylamys elegans, in order to discern the mechanisms involved in ensuring their proper segregation. We focused on the localization of the axial/lateral element protein SCP3 and the cohesin subunit STAG3. Our results show that X and Y chromosomes never appear as univalents in metaphase I, but they remain associated until they orientate and segregate to opposite poles. However, they must not be tied by a chiasma since their separation precedes the release of the sister chromatid cohesion. Instead, we show they are associated by the dense plate, a SCP3-rich structure that is organized during the first meiotic prophase and that is still present at metaphase I. Surprisingly, the dense plate incorporates SCP1, the main protein of the central element of the synaptonemal complex, from diplotene until telophase I. Once sex chromosomes are under spindle tension, they move to opposite poles losing contact with the dense plate and undergoing early segregation. Thus, the segregation of the achiasmatic T. elegans sex chromosomes seems to be ensured by the presence in metaphase I of a synaptonemal complex-derived structure. This feature, unique among vertebrates, indicates that synaptonemal complex elements may play a role in chromosome segregation. PMID:16934004

  17. Translation of CGA codon repeats in yeast involves quality control components and ribosomal protein L1.

    PubMed

    Letzring, Daniel P; Wolf, Andrew S; Brule, Christina E; Grayhack, Elizabeth J

    2013-09-01

    Translation of CGA codon repeats in the yeast Saccharomyces cerevisiae is inefficient, resulting in dose-dependent reduction in expression and in production of an mRNA cleavage product, indicative of a stalled ribosome. Here, we use genetics and translation inhibitors to understand how ribosomes respond to CGA repeats. We find that CGA codon repeats result in a truncated polypeptide that is targeted for degradation by Ltn1, an E3 ubiquitin ligase involved in nonstop decay, although deletion of LTN1 does not improve expression downstream from CGA repeats. Expression downstream from CGA codons at residue 318, but not at residue 4, is improved by deletion of either ASC1 or HEL2, previously implicated in inhibition of translation by polybasic sequences. Thus, translation of CGA repeats likely causes ribosomes to stall and exploits known quality control systems. Expression downstream from CGA repeats at amino acid 4 is improved by paromomycin, an aminoglycoside that relaxes decoding specificity. Paromomycin has no effect if native tRNA(Arg(ICG)) is highly expressed, consistent with the idea that failure to efficiently decode CGA codons might occur in part due to rejection of the cognate tRNA(Arg(ICG)). Furthermore, expression downstream from CGA repeats is improved by inactivation of RPL1B, one of two genes encoding the universally conserved ribosomal protein L1. The effects of rpl1b-Δ and of either paromomycin or tRNA(Arg(ICG)) on CGA decoding are additive, suggesting that the rpl1b-Δ mutant suppresses CGA inhibition by means other than increased acceptance of tRNA(Arg(ICG)). Thus, inefficient decoding of CGA likely involves at least two independent defects in translation.

  18. Calmodulin and Ca2+/calmodulin-binding proteins are involved in Tetrahymena thermophila phagocytosis.

    PubMed

    Gonda, K; Komatsu, M; Numata, O

    2000-08-01

    The ciliated protist, Tetrahymena thermophila, possesses one oral apparatus for phagocytosis, one of the most important cell functions, in the anterior cell cortex. The apparatus comprises four membrane structures which consist of ciliated and unciliated basal bodies, a cytostome where food is collected by oral ciliary motility, and a cytopharynx where food vacuoles are formed. The food vacuole is thought to be transported into the cytoplasm by a deep fiber which connects with the oral apparatus. Although a large number of studies have been done on the structure of the oral apparatus, the molecular mechanisms of phagocytosis in Tetrahymena thermophila are not well understood. In this study, using indirect immunofluorescence, we demonstrated that the deep fiber consisted of actin, CaM, and Ca2+/CaM-binding proteins, p85 and EF-1alpha, which are closely involved in cytokinesis. Moreover, we showed that CaM, p85, and EF-1alpha are colocalized in the cytostome and the cytopharynx of the oral apparatus. Next, we examined whether Ca2+/CaM signal regulates Tetrahymena thermophila phagocytosis, using Ca2+/CaM inhibitors chlorpromazine, trifluoperazine, N-(6-aminohexyl)-1-naphthalenesulfonamide, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCI. In Tetrahymena, it is known that Ca2+/CaM signal is closely involved in ciliary motility and cytokinesis. The results showed that one of the inhibitors, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl, inhibited the food vacuole formation rather than the ciliary motility, while the other three inhibitors effectively prevented the ciliary motility. Considering the colocalization of CaM, p85, and EF-1alpha to the cytopharynx, these results suggest that the Ca2+/CaM signal plays a pivotal role in Tetrahymena thermophila food vacuole formation.

  19. Identification and initial characterisation of a protein involved in Campylobacter jejuni cell shape.

    PubMed

    Esson, Diane; Gupta, Srishti; Bailey, David; Wigley, Paul; Wedley, Amy; Mather, Alison E; Méric, Guillaume; Mastroeni, Pietro; Sheppard, Samuel K; Thomson, Nicholas R; Parkhill, Julian; Maskell, Duncan J; Christie, Graham; Grant, Andrew J

    2017-03-01

    Campylobacter jejuni is the leading cause of bacterial food borne illness. While helical cell shape is considered important for C. jejuni pathogenesis, this bacterium is capable of adopting other morphologies. To better understand how helical-shaped C. jejuni maintain their shape and thus any associated colonisation, pathogenicity or other advantage, it is first important to identify the genes and proteins involved. So far, two peptidoglycan modifying enzymes Pgp1 and Pgp2 have been shown to be required for C. jejuni helical cell shape. We performed a visual screen of ∼2000 transposon mutants of C. jejuni for cell shape mutants. Whole genome sequence data of the mutants with altered cell shape, directed mutants, wild type stocks and isolated helical and rod-shaped 'wild type' C. jejuni, identified a number of different mutations in pgp1 and pgp2, which result in a change in helical to rod bacterial cell shape. We also identified an isolate with a loss of curvature. In this study, we have identified the genomic change in this isolate, and found that targeted deletion of the gene with the change resulted in bacteria with loss of curvature. Helical cell shape was restored by supplying the gene in trans. We examined the effect of loss of the gene on bacterial motility, adhesion and invasion of tissue culture cells and chicken colonisation, as well as the effect on the muropeptide profile of the peptidoglycan sacculus. Our work identifies another factor involved in helical cell shape. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. The Werner Syndrome Protein Is Involved in RNA Polymerase II Transcription

    PubMed Central

    Balajee, Adayabalam S.; Machwe, Amrita; May, Alfred; Gray, Matthew D.; Oshima, Junko; Martin, George M.; Nehlin, Jan O.; Brosh, Robert; Orren, David K.; Bohr, Vilhelm A.

    1999-01-01

    Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)–dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40–60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II–dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid–protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype

  1. 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.

  2. Collagen-induced platelet activation mainly involves the protein kinase C pathway.

    PubMed Central

    Karniguian, A; Grelac, F; Levy-Toledano, S; Legrand, Y J; Rendu, F

    1990-01-01

    This study analyses early biochemical events in collagen-induced platelet activation. An early metabolic event occurring during the lag phase was the activation of PtdIns(4,5)P2-specific phospholipase C. Phosphatidic acid (PtdOH) formation, phosphorylation of P43 and P20, thromboxane B2 (TXB2) synthesis and platelet secretion began after the lag phase, and were similarly time-dependent, except for TXB2 synthesis, which was delayed. Collagen induced extensive P43 phosphorylation, whereas P20 phosphorylation was weak and always lower than with thrombin. The dose-response curves of P43 phosphorylation and granule secretion were similar, and both reached a peak at 7.5 micrograms of collagen/ml, a dose which induced half-maximal PtdOH and TXB2 formation. Sphingosine, assumed to inhibit protein kinase C, inhibited P43 phosphorylation and secretion in parallel. However, sphingosine was not specific for protein kinase C, since a 15 microM concentration, which did not inhibit P43 phosphorylation, blocked TXB2 synthesis by 50%. Sphingosine did not affect PtdOH formation at all, even at 100 microM, suggesting that collagen itself induced this PtdOH formation, independently of TXB2 generation. The absence of external Ca2+ allowed the cleavage of polyphosphoinositides and the accumulation of InsP3 to occur, but impaired P43 phosphorylation, PtdOH and TXB2 formation, and secretion; these were only restored by adding 0.11 microM-Ca2+. In conclusion, stimulation of platelet membrane receptors for collagen initiates a PtdInsP2-specific phospholipase C activation, which is independent of external Ca2+, and might be the immediate receptor-linked response. A Ca2+ influx is indispensable to the triggering of subsequent platelet responses. This stimulation predominantly involves the protein kinase C pathway associated with secretion, and appears not to be mediated by TXB2, at least during its initial stage. Images Fig. 6. PMID:2163606

  3. 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.

  4. Sensing of Fatty Acids for Octanoylation of Ghrelin Involves a Gustatory G-Protein

    PubMed Central

    Janssen, Sara; Laermans, Jorien; Iwakura, Hiroshi; Tack, Jan; Depoortere, Inge

    2012-01-01

    Background Ghrelin is an important regulator of energy – and glucose homeostasis. The octanoylation at Ser3 is essential for ghrelin’s biological effects but the mechanisms involved in the octanoylation are unknown. We investigated whether the gustatory G-protein, α-gustducin, and the free fatty acid receptors GPR40 and GPR120 are involved in the fatty acid sensing mechanisms of the ghrelin cell. Methods Wild-type (WT) and α-gustducin knockout (gust−/−) mice were fed a glyceryl trioctanoate-enriched diet (OD) during 2 weeks. Ghrelin levels and gastric emptying were determined. Co-localization between GPR40, GPR120 and ghrelin or α-gustducin/α-transducin was investigated by immunofluorescence staining. The role of GPR120 in the effect of medium and long chain fatty acids on the release of ghrelin was studied in the ghrelinoma cell line, MGN3-1. The effect of the GPR40 agonist, MEDICA16, and the GPR120 agonist, grifolic acid, on ghrelin release was studied both in vitro and in vivo. Results Feeding an OD specifically increased octanoyl ghrelin levels in the stomach of WT mice but not of gust−/− mice. Gastric emptying was accelerated in WT but not in gust−/− mice. GPR40 was colocalized with desoctanoyl but not with octanoyl ghrelin, α-gustducin or α-transducin positive cells in the stomach. GPR120 only colocalized with ghrelin in the duodenum. Addition of octanoic acid or α-linolenic acid to MGN3-1 cells increased and decreased octanoyl ghrelin levels, respectively. Both effects could not be blocked by GPR120 siRNA. MEDICA16 and grifolic acid did not affect ghrelin secretion in vitro but oral administration of grifolic acid increased plasma ghrelin levels. Conclusion This study provides the first evidence that α-gustducin is involved in the octanoylation of ghrelin and shows that the ghrelin cell can sense long- and medium-chain fatty acids directly. GPR120 but not GPR40 may play a role in the lipid sensing cascade of the ghrelin cell. PMID

  5. Biochemical and physiological characterization of a BLUF protein-EAL protein complex involved in blue light-dependent degradation of cyclic diguanylate in the purple bacterium Rhodopseudomonas palustris.

    PubMed

    Kanazawa, Takuya; Ren, Shukun; Maekawa, Mikika; Hasegawa, Koji; Arisaka, Fumio; Hyodo, Mamoru; Hayakawa, Yoshihiro; Ohta, Hiroyuki; Masuda, Shinji

    2010-12-21

    Organisms adapt their physiologies in response to the quality and quantity of environmental light. Members of a recently identified photoreceptor protein family, BLUF domain proteins, use a flavin chromophore to sense blue light. Herein, we report that PapB, which contains a BLUF domain, controls the biofilm formation of the purple photosynthetic bacterium Rhodopseudomonas palustris. Purified PapB undergoes a typical BLUF-type photocycle, and light-excited PapB enhances the phosphodiesterase activity of the EAL domain protein, PapA, which degrades the second messenger, cyclic dimeric GMP (c-di-GMP). PapB directly interacts with PapA in vitro in a light-independent manner and induces a conformational change in the preformed PapA-PapB complex. A PapA-PapB docking simulation, as well as a site-directed mutagenesis study, identified amino acids partially responsible for the interaction between the PapA EAL domain and the two C-terminal α-helices of the PapB BLUF domain. Thus, the conformational change, which involves the C-terminal α-helices, transfers the flavin-sensed blue light signal to PapA. Deletion of papB in R. palustris enhances biofilm formation under high-intensity blue light conditions, indicating that PapB functions as a blue light sensor, which negatively regulates biofilm formation. These results demonstrate that R. palustris can control biofilm formation via a blue light-dependent modulation of its c-di-GMP level by the BLUF domain protein, PapB.

  6. The involvement of multidrug and toxin extrusion protein 1 in the distribution and excretion of berberine.

    PubMed

    Xiao, Ling; Xue, Yaru; Zhang, Cuifeng; Wang, Le; Lin, Yunfei; Pan, Guoyu

    2017-03-16

    1. Berberine (BBR), an isoquinoline alkaloid, has demonstrated multiple clinical pharmacological actions. As a substrate of multiple transporters in the liver, BBR is rarely excreted into the bile but can be found in the urine. The purpose of the present study was to investigate the role of multidrug and toxin extrusion protein 1 (MATE1) in the transport of BBR in the liver and kidney. 2. Using human MATE1 (hMATE1)-transfected HEK293 cells, BBR was shown to be a substrate of hMATE1 (Km = 4.28 ± 2.18 μM). In primary rat hepatocytes, pH-dependent uptake and efflux studies suggested that the transport of BBR was driven by the exchange of H(+) and involved Mate1. In rats, we found that pyrimethamine (PYR), an inhibitor of Mate1, increased hepatic and renal distribution of BBR and decreased systematic excretion of BBR. 3. These findings indicated that BBR is a substrate of MATE1 and that hepatic and renal Mate1 promote excretion of BBR into bile and urine, respectively. In conclusion, Mate1 plays a key role in the distribution and excretion of BBR, and we speculate that drug-drug interactions (DDIs) caused by MATE1 may occur between BBR and other co-administered drugs.

  7. Aminophospholipid translocation in erythrocytes: Evidence for the involvement of a specific transporter and an endofacial protein

    SciTech Connect

    Connor, J.; Schroit, A.J. )

    1990-01-09

    The transport of exogenously supplied fluorescent analogues of aminophospholipids from the outer to inner leaflet in red blood cells (RBC) is dependent upon the oxidative status of membrane sulfhydryls. Oxidation of a sulfhydryl on a 32-kDa membrane protein by pyridyldithioethylamine (PDA) has been previously shown to inhibit the transport of NBD-labeled phosphatidylserine (NBD-PS). In the present study, other sulfhydryl oxidants were examined to determine whether additional sites are involved in the transport process. The results show that diamide inhibits the transport of NBD-PS via a mechanism that is independent of the 32-kDa site. This is shown by the inability of diamide to block labeling of the 32-kDa sulfhydryl with {sup 125}I-labeled PDA and to protect against PDA-mediated inhibition of NBD-PS transport. Diamide-mediated inhibition, but not PDA-mediated inhibition, could be reversed by reduction with cysteamine or endogenous glutathione. Once established, the asymmetric distribution of NBD-PS could not be altered by oxidation of either site. These data indicate that a second site critical to the transport of aminophospholipids residues on the endofacial surface and suggest that the transport of aminophospholipids across the bilayer membrane of RBC depends on a coordinated and complementary process between a cytoskeletal component and the 32-kDa membrane polypeptide; both must be operative for transport to proceed.

  8. Antimicrobial resistance to ceftazidime involving loss of penicillin-binding protein 3 in Burkholderia pseudomallei

    PubMed Central

    Chantratita, Narisara; Rholl, Drew A.; Sim, Bernice; Wuthiekanun, Vanaporn; Limmathurotsakul, Direk; Amornchai, Premjit; Thanwisai, Aunchalee; Chua, Hui Hoon; Ooi, Wen Fong; Holden, Matthew T. G.; Day, Nicholas P.; Tan, Patrick; Schweizer, Herbert P.; Peacock, Sharon J.

    2011-01-01

    Known mechanisms of resistance to β-lactam antibiotics include β-lactamase expression, altered drug target, decreased bacterial permeability, and increased drug efflux. Here, we describe a unique mechanism of β-lactam resistance in the biothreat organism Burkholderia pseudomallei (the cause of melioidosis), associated with treatment failure during prolonged ceftazidime therapy of natural infection. Detailed comparisons of the initial ceftazidime-susceptible infecting isolate and subsequent ceftazidime-resistant variants from six patients led us to identify a common, large-scale genomic loss involving a minimum of 49 genes in all six resistant strains. Mutational analysis of wild-type B. pseudomallei demonstrated that ceftazidime resistance was due to deletion of a gene encoding a penicillin-binding protein 3 (BPSS1219) present within the region of genomic loss. The clinical ceftazidime-resistant variants failed to grow using commonly used laboratory culture media, including commercial blood cultures, rendering the variants almost undetectable in the diagnostic laboratory. Melioidosis is notoriously difficult to cure and clinical treatment failure is common in patients treated with ceftazidime, the drug of first choice across most of Southeast Asia where the majority of cases are reported. The mechanism described here represents an explanation for ceftazidime treatment failure, and may be a frequent but undetected resistance event. PMID:21969582

  9. The Lowe syndrome protein OCRL1 is involved in primary cilia assembly.

    PubMed

    Coon, Brian G; Hernandez, Victor; Madhivanan, Kayalvizhi; Mukherjee, Debarati; Hanna, Claudia B; Barinaga-Rementeria Ramirez, Irene; Lowe, Martin; Beales, Philip L; Aguilar, R Claudio

    2012-04-15

    Lowe syndrome (LS) is a devastating, X-linked genetic disease characterized by the presence of congenital cataracts, profound learning disabilities and renal dysfunction. Unfortunately, children affected with LS often die early of health complications including renal failure. Although this syndrome was first described in the early 1950s and the affected gene, OCRL1, was identified more than 17 years ago, the mechanism by which Ocrl1 defects lead to LS's symptoms remains unknown. Here we show that LS display characteristics of a ciliopathy. Specifically, we found that patients' cells have defects in the assembly of primary cilia and this phenotype was reproduced in cell lines by knock-down of Ocrl1. Importantly, this defect could be rescued by re-introduction of WT Ocrl1 in both patient and Ocrl1 knock-down cells. In addition, a zebrafish animal model of LS exhibited cilia defects and multiple morphological and anatomical abnormalities typically seen in ciliopathies. Mechanistically, we show that Ocrl1 is involved in protein trafficking to the primary cilia in an Rab8-and IPIP27/Ses-dependent manner. Taking into consideration the relevance of the signaling pathways hosted by the primary cilium, our results suggest hitherto unrecognized mechanisms by which Ocrl1 deficiency may contribute to the phenotypic characteristics of LS. This conceptual change in our understanding of the disease etiology may provide an alternative avenue for the development of therapies.

  10. 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

  11. G Protein-Coupled Receptors Involved in GnRH Regulation: Molecular Insights from Human Disease

    PubMed Central

    Noel, Sekoni D.; Kaiser, Ursula B.

    2011-01-01

    In the past two decades, an increasing body of evidence has demonstrated that several G protein-coupled receptor (GPCR)-ligand pairs are critical for normal human reproductive development and function. Patients harboring genetic insults in either the receptors or their cognate ligands have presented with reproductive disorders characterized by varying degrees of GnRH deficiency. These disorders include idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann Syndrome (KS). Conversely, mutations in some of these ligand-receptor pairs have been associated with accelerated reproductive maturation, manifested as central precocious puberty (CPP). To date, a series of elegant studies have characterized four GPCRs that play important roles in the neuroendocrine control of human reproductive development and function: GnRHR, KISS1R, PROKR2 and NK3R. Furthermore, these studies provide insights into the mechanisms by which mutations in these receptors give rise to reproductive disease phenotypes. This report will review mutations identified in GPCRs involved in the neuroendocrine control of the human reproductive axis with the aims of elucidating structure-function relationships of these GPCRs and identifying correlations between these structure-function relationships and the genotypic-phenotypic characterization of the patients. PMID:21736917

  12. Nicotine-induced plasticity in the retinocollicular pathway: Evidence for involvement of amyloid precursor protein.

    PubMed

    Gonçalves, R G J; Vasques, J F; Trindade, P; Serfaty, C A; Campello-Costa, P; Faria-Melibeu, A C

    2016-01-28

    During early postnatal development retinocollicular projections undergo activity-dependent synaptic refinement that results in the formation of precise topographical maps in the visual layers of the superior colliculus (SC). Amyloid Precursor Protein (APP) is a widely expressed transmembrane glycoprotein involved in the regulation of several aspects of neural development, such as neurite outgrowth, synapse formation and plasticity. Stimulation of cholinergic system has been found to alter the expression and processing of APP in different cell lines. Herein, we investigated the effect of nicotine on the development of retinocollicular pathway and on APP metabolism in the SC of pigmented rats. Animals were submitted to intracranial Elvax implants loaded with nicotine or phosphate-buffered saline (vehicle) at postnatal day (PND) 7. The ipsilateral retinocollicular pathway of control and experimental groups was anterogradely labeled either 1 or 3 weeks after surgery (PND 14 or PND 28). Local nicotine exposure produces a transitory sprouting of uncrossed retinal axons outside their main terminal zones. Nicotine also increases APP content and its soluble neurotrophic fragment sAPPα. Furthermore, nicotine treatment upregulates nicotinic acetylcholine receptor α7 and β2 subunits. Taken together, these data indicate that nicotine disrupts the ordering and topographic mapping of axons in the retinocollicular pathway and facilitates APP processing through the nonamyloidogenic pathway, suggesting that sAPPα may act as a trophic agent that mediates nicotine-induced morphological plasticity.

  13. Direct involvement of retinoblastoma family proteins in DNA repair by non-homologous end-joining.

    PubMed

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

    2015-03-31

    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. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Myocardial protection evoked by hyperoxic exposure involves signaling through nitric oxide and mitogen activated protein kinases.

    PubMed

    Ruusalepp, Arno; Czibik, Gabor; Flatebø, Torun; Vaage, Jarle; Valen, Guro

    2007-07-01

    Hyperoxic exposure in vivo (> 95% oxygen) attenuates ischemia-reperfusion injury, but the signaling mechanisms of this cardioprotection are not fully determined. We studied a possible role of nitric oxide (NO) and mitogen activated protein kinases (MAPK) in hyperoxic protection. Mice (n = 7-9 in each group) were kept in normoxic or hyperoxic environments for 15 min prior to harvesting the heart and Langendorff perfusion with global ischemia (45 min) and reperfusion (60 min). Endpoints were cardiac function and infarct size. Additional hearts were collected to evaluate MAPK phosphorylation (immunoblot). The nitric oxide synthase inhibitor L-NAME, the ERK1/2 inhibitor PD98059 and the p38 MAPK inhibitor FR167653 were injected intraperitoneally before hyperoxia or normoxia. Hyperoxia improved postischemic functional recovery and reduced infarct size (p < 0.05). Hyperoxic exposure caused cardiac phosphorylation of the MAPK family members p38 and ERK1/2, but not JNK. L-NAME, PD98059 and FR167653 all reduced the protection afforded by hyperoxic exposure, but did not influence performance or infarction in hearts of normoxic mice. The hyperoxia-induced phosphorylation of ERK1/2 and p38 was reduced by L-NAME and both MAPK inhibitors. Nitric oxide triggers hyperoxic protection, and ERK1/2 and p38 MAPK are involved in signaling of protection against ischemia-reperfusion injury.

  15. The Mechanism of L-Arginine Modulates Signal Proteins Involved in Glucose and Lipid Metabolic Imbalance.

    PubMed

    Hu, Shengdi; Han, Meng; Rezaei, Arash; Li, Defa; Guoyao, W; Ma, Xi

    2016-06-26

    Type 2 diabetes has become a global public health problem affecting approximately 380 million people throughout the world. It can cause many complications and lead to greater mortality. At present, there is no available medicine for effectively preventing diabetes. L-arginine, a functional amino acid, the precursor of nitric oxide, plays a crucial role in maintenance, reproduction, growth, anti-aging and immunity in animals. Growing clinical evidence indicates that dietary L-arginine supplementation can reduce obesity, decrease arterial blood pressure, resist oxidation and normalize endothelial dysfunction to cause remission of type 2 diabetes. The potential molecular mechanism may play a role in modulating glucose homeostasis, promoting lipolysis, maintaining hormone level, ameliorating insulin resistance, and fetal programing in early stage. The possible signaling pathway of the beneficial effects of L-arginine likely involves L-arginine-nitric oxide pathway through which cell signal protein can be activated. Accumulating studies have indicated that L-arginine may have potential to prevent and/or relieve type 2 diabetes via restoring insulin sensitivity in vivo.

  16. Protein SUMOylation is Involved in Cell-cycle Progression and Cell Morphology in Giardia lamblia.

    PubMed

    Di Genova, Bruno M; da Silva, Richard C; da Cunha, Júlia P C; Gargantini, Pablo R; Mortara, Renato A; Tonelli, Renata R

    2017-07-01

    The unicellular protozoa Giardia lamblia is a food- and waterborne parasite that causes giardiasis. This illness is manifested as acute and self-limited diarrhea and can evolve to long-term complications. Successful establishment of infection by Giardia trophozoites requires adhesion to host cells and colonization of the small intestine, where parasites multiply by mitotic division. The tight binding of trophozoites to host cells occurs by means of the ventral adhesive disc, a spiral array of microtubules and associated proteins such as giardins. In this work we show that knock down of the Small Ubiquitin-like MOdifier (SUMO) results in less adhesive trophzoites, decreased cell proliferation and deep morphological alterations, including at the ventral disc. Consistent with the reduced proliferation, SUMO knocked-down trophozoites were arrested in G1 and in S phases of the cell cycle. Mass spectrometry analysis of anti-SUMO immunoprecipitates was performed to identify SUMO substrates possibly involved in these events. Among the identified SUMOylation targets, α-tubulin was further validated by Western blot and confirmed to be a SUMO target in Giardia trophozoites. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

  17. 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.

  18. 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.

  19. Using co-expression analysis and stress-based screens to uncover Arabidopsis peroxisomal proteins involved in drought response

    DOE PAGES

    Li, Jiying; Hu, Jianping; Bassham, Diane

    2015-09-14

    Peroxisomes are essential organelles that house a wide array of metabolic reactions important for plant growth and development. However, our knowledge regarding the role of peroxisomal proteins in various biological processes, including plant stress response, is still incomplete. Recent proteomic studies of plant peroxisomes significantly increased the number of known peroxisomal proteins and greatly facilitated the study of peroxisomes at the systems level. The objectives of this study were to determine whether genes that encode peroxisomal proteins with related functions are co-expressed in Arabidopsis and identify peroxisomal proteins involved in stress response using in silico analysis and mutant screens. Usingmore » microarray data from online databases, we performed hierarchical clustering analysis to generate a comprehensive view of transcript level changes for Arabidopsis peroxisomal genes during development and under abiotic and biotic stress conditions. Many genes involved in the same metabolic pathways exhibited co-expression, some genes known to be involved in stress response are regulated by the corresponding stress conditions, and function of some peroxisomal proteins could be predicted based on their coexpression pattern. Since drought caused expression changes to the highest number of genes that encode peroxisomal proteins, we subjected a subset of Arabidopsis peroxisomal mutants to a drought stress assay. Mutants of the LON2 protease and the photorespiratory enzyme hydroxypyruvate reductase 1 (HPR1) showed enhanced susceptibility to drought, suggesting the involvement of peroxisomal quality control and photorespiration in drought resistance. Lastly, our study provided a global view of how genes that encode peroxisomal proteins respond to developmental and environmental cues and began to reveal additional peroxisomal proteins involved in stress response, thus opening up new avenues to investigate the role of peroxisomes in plant adaptation to

  20. Using co-expression analysis and stress-based screens to uncover Arabidopsis peroxisomal proteins involved in drought response

    SciTech Connect

    Li, Jiying; Hu, Jianping; Bassham, Diane

    2015-09-14

    Peroxisomes are essential organelles that house a wide array of metabolic reactions important for plant growth and development. However, our knowledge regarding the role of peroxisomal proteins in various biological processes, including plant stress response, is still incomplete. Recent proteomic studies of plant peroxisomes significantly increased the number of known peroxisomal proteins and greatly facilitated the study of peroxisomes at the systems level. The objectives of this study were to determine whether genes that encode peroxisomal proteins with related functions are co-expressed in Arabidopsis and identify peroxisomal proteins involved in stress response using in silico analysis and mutant screens. Using microarray data from online databases, we performed hierarchical clustering analysis to generate a comprehensive view of transcript level changes for Arabidopsis peroxisomal genes during development and under abiotic and biotic stress conditions. Many genes involved in the same metabolic pathways exhibited co-expression, some genes known to be involved in stress response are regulated by the corresponding stress conditions, and function of some peroxisomal proteins could be predicted based on their coexpression pattern. Since drought caused expression changes to the highest number of genes that encode peroxisomal proteins, we subjected a subset of Arabidopsis peroxisomal mutants to a drought stress assay. Mutants of the LON2 protease and the photorespiratory enzyme hydroxypyruvate reductase 1 (HPR1) showed enhanced susceptibility to drought, suggesting the involvement of peroxisomal quality control and photorespiration in drought resistance. Lastly, our study provided a global view of how genes that encode peroxisomal proteins respond to developmental and environmental cues and began to reveal additional peroxisomal proteins involved in stress response, thus opening up new avenues to investigate the role of peroxisomes in plant adaptation to

  1. The Maize Floury1 Gene Encodes a Novel Endoplasmic Reticulum Protein Involved in Zein Protein Body Formation[W

    PubMed Central

    Holding, David R.; Otegui, Marisa S.; Li, Bailin; Meeley, Robert B.; Dam, Thao; Hunter, Brenda G.; Jung, Rudolf; Larkins, Brian A.

    2007-01-01

    The maize (Zea mays) floury1 (fl1) mutant was first reported almost 100 years ago, but its molecular identity has remained unknown. We report the cloning of Fl1, which encodes a novel zein protein body membrane protein with three predicted transmembrane domains and a C-terminal plant-specific domain of unknown function (DUF593). In wild-type endosperm, the FL1 protein accumulates at a high level during the period of zein synthesis and protein body development and declines to a low level at kernel maturity. Immunogold labeling showed that FL1 resides in the endoplasmic reticulum surrounding the protein body. Zein protein bodies in fl1 mutants are of normal size, shape, and abundance. However, mutant protein bodies ectopically accumulate 22-kD α-zeins in the γ-zein–rich periphery and center of the core, rather than their normal discrete location in a ring at outer edge of the core. The 19-kD α-zein is uniformly distributed throughout the core in wild-type protein bodies, and this distribution is unaffected in fl1 mutants. Pairwise yeast two-hybrid experiments showed that FL1 DUF593 interacts with the 22-kD α-zein. Results of these studies suggest that FL1 participates in protein body formation by facilitating the localization of 22-kD α-zein and that this is essential for the formation of vitreous endosperm. PMID:17693529

  2. Overproduction, purification, and ATPase activity of the Escherichia coli RuvB protein involved in DNA repair.

    PubMed Central

    Iwasaki, H; Shiba, T; Makino, K; Nakata, A; Shinagawa, H

    1989-01-01

    The ruvA and ruvB genes of Escherichia coli constitute an operon which belongs to the SOS regulon. Genetic evidence suggests that the products of the ruv operon are involved in DNA repair and recombination. To begin biochemical characterization of these proteins, we developed a plasmid system that overproduced RuvB protein to 20% of total cell protein. Starting from the overproducing system, we purified RuvB protein. The purified RuvB protein behaved like a monomer in gel filtration chromatography and had an apparent relative molecular mass of 38 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the value predicted from the DNA sequence. The amino acid sequence of the amino-terminal region of the purified protein was analyzed, and the sequence agreed with the one deduced from the DNA sequence. Since the deduced sequence of RuvB protein contained the consensus sequence for ATP-binding proteins, we examined the ATP-binding and ATPase activities of the purified RuvB protein. RuvB protein had a stronger affinity to ADP than to ATP and weak ATPase activity. The results suggest that the weak ATPase activity of RuvB protein is at least partly due to end product inhibition by ADP. Images PMID:2529252

  3. IP3 production in the hypersensitive response of lemon seedlings against Alternaria alternata involves active protein tyrosine kinases but not a G-protein.

    PubMed

    Ortega, Ximena; Velásquez, Juan Carlos; Pérez, Luz M

    2005-01-01

    IP3 increase and de novo synthesis of scoparone are produced in the hypersensitive response (HR) of lemon seedlings against the fungus Alternaria alternata. To elucidate whether a G-protein and/or a protein tyrosine kinase (PTK) are involved in signal transduction leading to the production of such a defensive response, we studied the HR in this plant system after treatment with G-protein activators alone and PTK inhibitors in the presence of fungal conidia. No changes in the level of IP3 were detected in response to the treatment with the G-protein activators cholera toxin or mastoparan, although the HR was observed in response to these compounds as determined by the scoparone synthesis. On the contrary, the PTK inhibitors lavendustin A and 2,5-dihidroxy methyl cinnamate (DHMC) not only prevented the IP3 changes observed in response to the fungal inoculation of lemon seedlings but also blocked the development of the HR. These results suggest that the IP3 changes observed in response to A. alternata require a PTK activity and are the result of a G-protein independent Phospholipase C activity, even though the activation of a G-protein can also lead to the development of a HR. Therefore, it appears that more than one signaling pathway may be activated for the development of HR in lemon seedlings: one involving a G-protein and the other involving a PTK-dependent PLC.

  4. 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. © 2016 Wiley Periodicals, Inc.

  5. Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains.

    PubMed Central

    Stenberg, R M; Fortney, J; Barlow, S W; Magrane, B P; Nelson, J A; Ghazal, P

    1990-01-01

    trans activation of promoters by viral regulatory proteins provides a useful tool to study coordinate control of gene expression. Immediate-early (IE) regions 1 and 2 of human cytomegalovirus (CMV) code for a series of proteins that originate from differentially spliced mRNAs. These IE proteins are proposed to regulate the temporal expression of the viral genome. To examine the structure and function of the IE proteins, we used linker insertion mutagenesis of the IE gene region as well as cDNA expression vector cloning of the abundant IE mRNAs. We showed that IE1 and IE2 proteins of CMV exhibit promoter-specific differences in their modes of action by either trans activating early and IE promoters or repressing the major IE promoter (MIEP). Transient cotransfection experiments with permissive human cells revealed a synergistic interaction between the 72- and the 86-kilodalton (kDa) IE proteins in trans activating an early promoter. In addition, transfection studies revealed that the 72-kDa protein was capable of trans activating the MIEP. In contrast, the 86-kDa protein specifically repressed the MIEP and this repression was suppressed by the 72-kDa protein. Furthermore, observations based on the primary sequence structure revealed a modular arrangement of putative regulatory motifs that could either potentiate or repress gene expression. These modular domains are either shared or unique among the IE proteins. From these data, we propose a model for IE protein function in the coordinate control of CMV gene expression. Images PMID:2157043

  6. 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

  7. PIAS proteins are involved in the SUMO-1 modification, intracellular translocation and transcriptional repressive activity of RET finger protein

    SciTech Connect

    Matsuura, Tetsuo; Shimono, Yohei; Kawai, Kumi; Murakami, Hideki; Urano, Takeshi; Niwa, Yasumasa; Goto, Hidemi; Takahashi, Masahide . E-mail: mtakaha@med.nagoya-u.ac.jp

    2005-08-01

    Ret finger protein (RFP) is a nuclear protein that is highly expressed in testis and in various tumor cell lines. RFP functions as a transcriptional repressor and associates with Enhancer of Polycomb 1 (EPC1), a member of the Polycomb group proteins, and Mi-2{beta}, a main component of the nucleosome remodeling and deacetylase (NuRD) complex. We show that RFP binds with PIAS (protein inhibitor of activated STAT) proteins, PIAS1, PIAS3, PIASx{alpha} and PIASy at their carboxyl-terminal region and is covalently modified by SUMO-1 (sumoylation). PIAS proteins enhance the sumoylation of RFP in a dose-dependent manner and induce the translocation of RFP into nuclear bodies reminiscent of the PML bodies. In addition, co-expression of PIAS proteins or SUMO-1 strengthened the transcriptional repressive activity of RFP. Finally, our immunohistochemical results show that RFP, SUMO-1 and PIASy localize in a characteristic nuclear structure juxtaposed with the inner nuclear membrane (XY body) of primary spermatocytes in mouse testis. These results demonstrate that the intracellular location and the transcriptional activity of RFP are modified by PIAS proteins which possess SUMO E3 ligase activities and suggest that they may play a co-operative role in spermatogenesis.

  8. The microtubule motor protein KIF13A is involved in intracellular trafficking of the Lassa virus matrix protein Z.

    PubMed

    Fehling, Sarah Katharina; Noda, Takeshi; Maisner, Andrea; Lamp, Boris; Conzelmann, Karl-Klaus; Kawaoka, Yoshihiro; Klenk, Hans-Dieter; Garten, Wolfgang; Strecker, Thomas

    2013-02-01

    The small matrix protein Z of arenaviruses has been identified as the main driving force to promote viral particle production at the plasma membrane. Although multiple functions of Z in the arenaviral life cycle have been uncovered, the mechanism of intracellular transport of Z to the site of virus budding is poorly understood and cellular motor proteins that mediate Z trafficking remain to be identified. In the present study, we report that the Z protein of the Old World arenavirus Lassa virus (LASV) interacts with the kinesin family member 13A (KIF13A), a plus-end-directed microtubule-dependent motor protein. Plasmid-driven overexpression of KIF13A results in relocalization of Z to the cell periphery, while functional blockage of endogenous KIF13A by overexpression of a dominant-negative mutant or KIF13A-specific siRNA causes a perinuclearaccumulation and decreased production of both Z-induced virus-like particles and infectious LASV. The interaction of KIF13A with Z proteins from both Old and New World arenaviruses suggests a conserved intracellular transport mechanism. In contrast, the intracellular distribution of the matrix proteins of prototypic members of the paramyxo- and rhabdovirus family is independent of KIF13A. In summary, our studies identify for the first time a molecular motor protein as a critical mediator for intracellular microtubule-dependent transport of arenavirus matrix proteins.

  9. The mechanism of binding staphylococcal protein A to immunoglobin G does not involve helix unwinding.

    PubMed

    Jendeberg, L; Tashiro, M; Tejero, R; Lyons, B A; Uhlén, M; Montelione, G T; Nilsson, B

    1996-01-09

    Structural changes in staphylococcal protein A (SpA) upon its binding to the constant region (Fc) of immunoglobulin G (IgG) have been studied by nuclear magnetic resonance and circular dichroism (CD) spectroscopy. The NMR solution structure of the engineered IgG-binding domain of SpA, the Z domain (an analogue of the B domain of SpA), has been determined by simulated annealing with molecular dynamics, using 599 distance and dihedral angle constraints. Domain Z contains three alpha-helices in the polypeptide segments Lys7 to His18 (helix 1), Glu25 to Asp36 (helix 2), and Ser41 to Ala54 (helix 3). The overall chain fold is an antiparallel three-helical bundle. This is in contrast to the previously determined X-ray structure of the similar SpA domain B in complex with Fc, where helix 3 is not observed in the electron density map [Deisenhofer, J. (1981) Biochemistry 20, 2361-2370], but similar to the solution NMR structure of domain B, which is also a three-helical bundle structure [Gouda, H., et al. (1992) Biochemistry 31, 9665-9672]. In order to characterize possible secondary structural changes associated with IgG binding, far-UV CD spectra were collected for the Z domain, an engineered repeat of this molecule (ZZ), recombinant Fc from IgG subclass 1 (Fc1), recombinant Fc from IgG subclass 3 (Fc3), and mixtures of Z/Fc1, Z/Fc3, ZZ/Fc1, and ZZ/Fc3. Fc3 was included as a control for possible changes of the CD spectrum in the mixture of noncomplexed molecules, since SpA is known not to bind Fc3. From these CD spectra, it was concluded that the third alpha-helix in Z is not disrupted in its complexes with Fc1. Similar results were obtained for the ZZ molecule. However, in both Z and ZZ there are some perturbations in CD spectra at high energy wavelengths (i.e., lambda < 215 nm) accompanying complex formation. On the basis of the combined CD and NMR results, as well as previously described binding studies of Z mutant proteins to Fc1, we conclude that the Z domain

  10. 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

  11. Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

    PubMed

    Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

    2014-01-01

    G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Sieve element occlusion (SEO) genes encode structural phloem proteins involved in wound sealing of the phloem.

    PubMed

    Ernst, Antonia M; Jekat, Stephan B; Zielonka, Sascia; Müller, Boje; Neumann, Ulla; Rüping, Boris; Twyman, Richard M; Krzyzanek, Vladislav; Prüfer, Dirk; Noll, Gundula A

    2012-07-10

    The sieve element occlusion (SEO) gene family originally was delimited to genes encoding structural components of forisomes, which are specialized crystalloid phloem proteins found solely in the Fabaceae. More recently, SEO genes discovered in various non-Fabaceae plants were proposed to encode the common phloem proteins (P-proteins) that plug sieve plates after wounding. We carried out a comprehensive characterization of two tobacco (Nicotiana tabacum) SEO genes (NtSEO). Reporter genes controlled by the NtSEO promoters were expressed specifically in immature sieve elements, and GFP-SEO fusion proteins formed parietal agglomerates in intact sieve elements as well as sieve plate plugs after wounding. NtSEO proteins with and without fluorescent protein tags formed agglomerates similar in structure to native P-protein bodies when transiently coexpressed in Nicotiana benthamiana, and the analysis of these protein complexes by electron microscopy revealed ultrastructural features resembling those of native P-proteins. NtSEO-RNA interference lines were essentially devoid of P-protein structures and lost photoassimilates more rapidly after injury than control plants, thus confirming the role of P-proteins in sieve tube sealing. We therefore provide direct evidence that SEO genes in tobacco encode P-protein subunits that affect translocation. We also found that peptides recently identified in fascicular phloem P-protein plugs from squash (Cucurbita maxima) represent cucurbit members of the SEO family. Our results therefore suggest a common evolutionary origin for P-proteins found in the sieve elements of all dicotyledonous plants and demonstrate the exceptional status of extrafascicular P-proteins in cucurbits.

  13. Functional definition of outer membrane proteins involved in preprotein import into mitochondria.

    PubMed

    Lister, Ryan; Carrie, Chris; Duncan, Owen; Ho, Lois H M; Howell, Katharine A; Murcha, Monika W; Whelan, James

    2007-11-01

    The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of mitochondrial precursor proteins and likely plays a role in the assembly of beta-barrel proteins into the outer membrane. An outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy. Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of mitochondrial protein import.

  14. Overexpression of G protein-coupled receptors in cancer cells: involvement in tumor progression.

    PubMed

    Li, Shuyu; Huang, Shuguang; Peng, Sheng-Bin

    2005-11-01

    G protein-coupled receptors (GPCRs) play important roles in a variety of biological and pathological processes. They are considered among the most desirable targets for drug development. Recent studies have demonstrated that many GPCRs, such as endothelin receptors, chemokine receptors and lysophosphatidic acid receptors have been implicated in the tumorigenesis and metastasis of multiple human cancers. In this study, we conducted an in silico analysis of GPCR gene expression in primary human tumors by analyzing some publicly available gene expression profiling data. Statistical analysis was performed on eight microarray data sets of non-small cell lung cancer, breast cancer, prostate cancer, melanoma, gastric cancer and diffused large B cell lymphoma to identify GPCRs that are up-regulated in primary or metastatic cancer cells. Our analysis has demonstrated overexpression of several GPCRs in primary tumor cells, including chemokine receptors and protease-activated receptors that were shown to be important for tumorigenesis by previous studies. In addition, we have uncovered several GPCRs, such as neuropeptide receptors, adenosine A2B receptor, P2Y purinoceptor, calcium-sensing receptor and metabotropic glutamate receptors, that are expressed at a significantly higher level in some cancer tissue and may play a role in cancer progression. Analysis of cancer samples in different disease stages also suggests that some GPCRs, such as endothelin receptor A, may be involved in early tumor progression and others, such as CXCR4, may play a critical role in tumor invasion and metastasis. The present study demonstrates the value of publicly available microarray data as a resource to gain more understanding of cancer biology, to validate previous findings from in vitro experiments, and to identify potential novel anticancer targets and biomarkers.

  15. Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization.

    PubMed

    Tena-Campos, Mercè; Ramon, Eva; Lupala, Cecylia S; Pérez, Juan J; Koch, Karl-W; Garriga, Pere

    2016-04-01

    5-Hydroxytryptamine 1A receptor and galanin receptor 1 belong to the G protein-coupled receptors superfamily, and they have been described to heterodimerize triggering an anomalous physiological state that would underlie depression. Zinc supplementation has been widely reported to improve treatment against major depressive disorder. Our work has focused on the study and characterization of these receptors and its relationships with zinc both under purified conditions and in cell culture. To this aim, we have designed a strategy to purify the receptors in a conformationally active state. We have used receptors tagged with the monoclonal Rho-1D4 antibody and employed ligand-assisted purification in order to successfully purify both receptors in a properly folded and active state. The interaction between both purified receptors has been analyzed by surface plasmon resonance in order to determine the kinetics of dimerization. Zinc effect on heteromer has also been tested using the same methodology but exposing the 5-hydroxytryptamine 1A receptor to zinc before the binding experiment. These results, combined with Förster resonance energy transfer (FRET) measurements, in the absence and presence of zinc, suggest that this ion is capable of disrupting this interaction. Moreover, molecular modeling suggests that there is a coincidence between zinc-binding sites and heterodimerization interfaces for the serotonin receptor. Our results establish a rational explanation for the role of zinc in the molecular processes associated with receptor-receptor interactions and its relationship with depression, in agreement with previously reported evidence for the positive effects of zinc in depression treatment, and the involvement of our target dimer in the same disease.

  16. Targeting of a polytopic membrane protein to the inner envelope membrane of chloroplasts in vivo involves multiple transmembrane segments

    PubMed Central

    Okawa, Kumiko; Inoue, Hitoshi; Adachi, Fumi; Nakayama, Katsuhiro; Ito-Inaba, Yasuko; Schnell, Danny J.; Uehara, Susumu; Inaba, Takehito

    2014-01-01

    The inner envelope membrane (IEM) of the chloroplast plays crucial roles in forming an osmotic barrier and controlling metabolite exchange between the organelle and the cytosol. The IEM therefore harbours a number of membrane proteins and requires the import and integration of these nuclear-encoded proteins for its biogenesis. Recent studies have demonstrated that the transmembrane segment of single-spanning IEM proteins plays key roles in determining their IEM localization. However, few studies have focused on the molecular mechanisms by which polytopic membrane proteins are targeted to the IEM. In this study, we investigated the targeting mechanism of polytopic IEM proteins using the protein Cor413im1 as a model substrate. Cor413im1 does not utilize a soluble intermediate for its targeting to the IEM. Furthermore, we show that the putative fifth transmembrane segment of Cor413im1 is necessary for its targeting to the IEM. The C-terminal portion containing this transmembrane segment is also able to deliver Cor413im1 protein to the IEM. However, the fifth transmembrane segment of Cor413im1 itself is insufficient to target a fusion protein to the IEM. These data suggest that the targeting of polytopic membrane proteins to the chloroplast IEM in vivo involves multiple transmembrane segments and that chloroplasts have evolved a unique mechanism for the integration of polytopic proteins to the IEM. PMID:25013120

  17. Proteomic profile of carbonylated proteins in rat liver: exercise attenuated oxidative stress may be involved in fatty liver improvement.

    PubMed

    Hu, Xiaofei; Duan, Zhigui; Hu, Hui; Li, Guolin; Yan, Siyu; Wu, Jinfeng; Wang, Jun; Yin, Dazhong; Xie, Qingji

    2013-05-01

    To screen target proteins of oxidative stress which mediate the effects of exercise on preventing nonalcoholic fatty liver disease (NAFLD), the methods for selecting carbonylated proteins were modified, and carbonylated proteins were profiled. The results showed that treadmill training reduced oxidative stress and the levels of intrahepatic triglyceride (IHTG). The changes in IHTG showed a significant positive correlation with oxidative stress as indicated by malondialdehyde level. Further results from proteomics illustrated that 17 functional proteins were susceptible to oxidative modification, and exercise protected three proteins from carbonylation. The latter three proteins may serve as both direct target proteins of oxidative stress and mediators contributing to the beneficial effects of exercise. In particular, a long-chain specific acyl-CoA dehydrogenase (ACADL) which was a key enzyme in lipid metabolism was not carbonylated and with higher activities in exercise group. These findings indicate that this modified technique is practical and powerful in selecting carbonylated proteins. Long-term treadmill training is effective in ameliorating oxidative stress and preventing the accumulation of IHTG. Among the 17 target proteins of oxidative modification, three proteins contribute to the beneficial effects of exercise. Preventing ACADL from carbonylation may be involved in the physiological mechanism of exercise-induced NAFLD improvement.

  18. A brief history of the search for the protein(s) involved in the acute regulation of steroidogenesis.

    PubMed

    Stocco, Douglas M; Zhao, Amy H; Tu, Lan N; Morohaku, Kanako; Selvaraj, Vimal

    2017-02-05

    The synthesis of steroid hormones occurs in specific cells and tissues in the body in response to trophic hormones and other signals. In order to synthesize steroids de novo, cholesterol, the precursor of all steroid hormones, must be mobilized from cellular stores to the inner mitochondrial membrane (IMM) to be converted into the first steroid formed, pregnenolone. This delivery of cholesterol to the IMM is the rate-limiting step in this process, and has long been known to require the rapid synthesis of a new protein(s) in response to stimulation. Although several possibilities for this protein have arisen over the past few decades, most of the recent attention to fill this role has centered on the candidacies of the proteins the Translocator Protein (TSPO) and the Steroidogenic Acute Regulatory Protein (StAR). In this review, the process of regulating steroidogenesis is briefly described, the characteristics of the candidate proteins and the data supporting their candidacies summarized, and some recent findings that propose a serious challenge for the role of TSPO in this process are discussed. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  19. Characterization of a periplasmic protein involved in iron utilization of Actinobacillus actinomycetemcomitans.

    PubMed

    Willemsen, P T; Vulto, I; Boxem, M; de Graaff, J

    1997-08-01

    The periodontopathic bacterium Actinobacillus actinomycetemcomitans possesses a 35-kDa periplasmic iron-repressible protein. Its regulation is mediated by the Fur protein, as was inferred from the Fur-binding consensus sequence at the -35 position of the gene for the 35-kDa protein and from the relaxed expression of the gene in a mutant with an altered Fur-binding sequence. The 35-kDa protein, designated AfuA, has strong homology to HitA and FbpA of Haemophilus influenzae and Neisseria meningitidis, respectively, which serve as periplasmic iron transport proteins.

  20. Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl–like molecules binding

    PubMed Central

    2013-01-01

    Background Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. Method We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Results Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. Conclusions The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins. PMID:23768251

  1. Export of virulence proteins by malaria-infected erythrocytes involves remodeling of host actin cytoskeleton.

    PubMed

    Rug, Melanie; Cyrklaff, Marek; Mikkonen, Antti; Lemgruber, Leandro; Kuelzer, Simone; Sanchez, Cecilia P; Thompson, Jennifer; Hanssen, Eric; O'Neill, Matthew; Langer, Christine; Lanzer, Michael; Frischknecht, Friedrich; Maier, Alexander G; Cowman, Alan F

    2014-11-27

    Following invasion of human red blood cells (RBCs) by the malaria parasite, Plasmodium falciparum, a remarkable process of remodeling occurs in the host cell mediated by trafficking of several hundred effector proteins to the RBC compartment. The exported virulence protein, P falciparum erythrocyte membrane protein 1 (PfEMP1), is responsible for cytoadherence of infected cells to host endothelial receptors. Maurer clefts are organelles essential for protein trafficking, sorting, and assembly of protein complexes. Here we demonstrate that disruption of PfEMP1 trafficking protein 1 (PfPTP1) function leads to severe alterations in the architecture of Maurer's clefts. Furthermore, 2 major surface antigen families, PfEMP1 and STEVOR, are no longer displayed on the host cell surface leading to ablation of cytoadherence to host receptors. PfPTP1 functions in a large complex of proteins and is required for linking of Maurer's clefts to the host actin cytoskeleton.

  2. NMR Identification of the Binding Surfaces Involved in the Salmonella and Shigella Type III Secretion Tip-Translocon Protein-Protein Interactions

    PubMed Central

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

    2017-01-01

    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. PMID:27093649

  3. Tau deficiency leads to the upregulation of BAF-57, a protein involved in neuron-specific gene repression.

    PubMed

    de Barreda, Elena Gómez; Dawson, Hana N; Vitek, Michel P; Avila, Jesús

    2010-06-03

    Although tau is mainly located in the cell cytoplasm, mostly bound to tubulin, it may also be found in the nucleus of neurons. Hence, we tested whether tau might play a role in regulating the expression of certain genes by comparing gene expression in mice containing or lacking the tau protein. Our results identified a significant difference in the expression of the smarce1 gene, which codes for the BAF-57 protein, a protein involved in the repression of neuron specific genes. These data suggest a role for tau in neuron maturation. Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  4. 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. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Architecture and regulation of HtrA-family proteins involved in protein quality control and stress response.

    PubMed

    Hansen, Guido; Hilgenfeld, Rolf

    2013-03-01

    Protein quality control is vital for all living cells and sophisticated molecular mechanisms have evolved to prevent the excessive accumulation of unfolded proteins. High-temperature requirement A (HtrA) proteases have been identified as important ATP-independent quality-control factors in most species. HtrA proteins harbor a serine-protease domain and at least one peptide-binding PDZ domain to ensure efficient removal of misfolded or damaged proteins. One distinctive property of HtrAs is their ability to assemble into complex oligomers. Whereas all examined HtrAs are capable of forming pyramidal 3-mers, higher-order complexes consisting of up to 24 molecules have been reported. Tight control of chaperone and protease function is of pivotal importance in preventing deleterious HtrA-protease activity. In recent years, structural biology provided detailed insights into the molecular basis of the regulatory mechanisms, which include unique intramolecular allosteric signaling cascades and the dynamic switching of oligomeric states of HtrA proteins. Based on these results, functional models for many family members have been developed. The HtrA protein family represents a remarkable example of how structural and functional diversity is attained from the assembly of simple molecular building blocks.

  6. 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.

  7. 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.

  8. 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

  9. Involvement of histone phosphorylation in thymocyte apoptosis by protein phosphatase inhibitors.

    PubMed

    Lee, E; Nakatsuma, A; Hiraoka, R; Ishikawa, E; Enomoto, R; Yamauchi, A

    1999-07-01

    Incubation of rat thymocytes with the inhibitors of protein phosphatase such as calyculin A and okadaic acid resulted in an increase in DNA fragmentation. These effects were dependent on the concentration of the inhibitors and the incubation time. Analyses of the fragmented DNA revealed the production of approximately 50 kbp of DNA and a 180 bp DNA ladder. In addition, a laser scanning-microscopic analysis showed that these compounds caused nuclear condensation. Thus, these results demonstrated that protein phosphatase inhibitors induced thymocyte apoptosis. The inhibitors of protein phosphatase increased the phosphorylation of proteins of approximately 15 kDa. The phosphorylation of proteins preceded the DNA fragmentation induced by these inhibitors. Judging from acetic acid-urea-Triton X-100 gel electrophoresis, the phosphorylated proteins were histone H1 and H2A/H3. Therefore, these results suggest that phosphorylation of histones triggers the DNA fragmentation of thymocytes undergoing apoptosis.

  10. Ubiquitin-Like Protein Involved in the Proteasome Pathway of Mycobacterium tuberculosis

    PubMed Central

    Pearce, Michael J.; Mintseris, Julian; Ferreyra, Jessica; Gygi, Steven P.; Darwin, K. Heran

    2009-01-01

    The protein modifier ubiquitin is a signal for proteasome-mediated degradation in eukaryotes. Proteasome-bearing prokaryotes have been thought to degrade proteins via a ubiquitin-independent pathway. We have identified a prokaryotic ubiquitin-like protein, Pup (Rv2111c), which was specifically conjugated to proteasome substrates in the pathogen Mycobacterium tuberculosis. Pupylation occurred on lysines and required proteasome accessory factor A (PafA). In a pafA mutant, pupylated proteins were absent and substrates accumulated, thereby connecting pupylation with degradation. Although analogous to ubiquitylation, pupylation appears to proceed by a different chemistry. Thus, like eukaryotes, bacteria may use a small-protein modifier to control protein stability. PMID:18832610

  11. Conservation of proteins involved in oocyst wall formation in Eimeria maxima, Eimeria tenella and Eimeria acervulina.

    PubMed

    Belli, Sabina I; Ferguson, David J P; Katrib, Marilyn; Slapetova, Iveta; Mai, Kelly; Slapeta, Jan; Flowers, Sarah A; Miska, Kate B; Tomley, Fiona M; Shirley, Martin W; Wallach, Michael G; Smith, Nicholas C

    2009-08-01

    Vaccination with proteins from gametocytes of Eimeria maxima protects chickens, via transfer of maternal antibodies, against infection with several species of Eimeria. Antibodies to E. maxima gametocyte proteins recognise proteins in the wall forming bodies of macrogametocytes and oocyst walls of E. maxima, Eimeria tenella and Eimeria acervulina. Homologous genes for two major gametocyte proteins - GAM56 and GAM82 - were found in E. maxima, E. tenella and E. acervulina. Alignment of the predicted protein sequences of these genes reveals that, as well as sharing regions of tyrosine richness, strong homology exists in their amino-terminal regions, where protective antibodies bind. This study confirms the conservation of the roles of GAM56 and GAM82 in oocyst wall formation and shows that antibodies to gametocyte antigens of E. maxima cross-react with homologous proteins in other species, helping to explain cross-species maternal immunity.

  12. A proteomic approach to apoplastic proteins involved in cell wall regeneration in protoplasts of Arabidopsis suspension-cultured cells.

    PubMed

    Kwon, Hye-Kyoung; Yokoyama, Ryusuke; Nishitani, Kazuhiko

    2005-06-01

    To clarify the mechanisms of cell wall construction, we used a proteomic approach to investigate the proteins secreted into cell wall spaces during cell wall regeneration from the protoplasts of Arabidopsis suspension-cultured cells. We focused on cell wall proteins loosely bound to the cell wall architecture and extractable with 1 M KCl solutions from: (i) native suspension cultured cells; (ii) protoplasts that had been allowed to regenerate their cell walls for 1 h; and (iii) protoplasts allowed to regenerate their cell walls for 3 h. We adopted a non-destructive extraction procedure without disrupting cellular integrity, thereby avoiding contamination from cytoplasmic proteins. Using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), we separated, mapped and identified 71 proteins derived from the native cell wall, and 175 and 212 proteins derived from the 1 and 3 h regenerated protoplasts, respectively. Quite different sets of proteins with differing status of their post-translational modifications, including phosphorylation and glycosylation, were identified in the three protein fractions. This indicated dynamic in muro changes in the cell wall proteins during cell wall regeneration in the protoplasts. The analysis revealed a set of enzymes specifically involved in cell wall expansion and construction in suspension-cultured cells. This approach has also determined a set of cell wall proteins that had not been predicted to be localized in cell wall spaces.

  13. Subcellular localization based comparative study on radioresistant bacteria: A novel approach to mine proteins involve in radioresistance.

    PubMed

    Vishambra, Divya; Srivastava, Malay; Dev, Kamal; Jaiswal, Varun

    2017-08-01

    Radioresistant bacteria (RRB) are among the most radioresistant organisms and has a unique role in evolution. Along with the evolutionary role, radioresistant organisms play important role in paper industries, bioremediation, vaccine development and possibility in anti-aging and anti-cancer treatment. The study of radiation resistance in RRB was mainly focused on cytosolic mechanisms such as DNA repair mechanism, cell cleansing activity and high antioxidant activity. Although it was known that protein localized on outer areas of cell play role in resistance towards extreme condition but the mechanisms/proteins localized on the outer area of cells are not studied for radioresistance. Considering the fact that outer part of cell is more exposed to radiations and proteins present in outer area of the cell may have role in radioresistance. Localization based comparative study of proteome from RRB and non-radio resistant bacteria was carried out. In RRB 20 unique proteins have been identified. Further domain, structural, and pathway analysis of selected proteins were carried out. Out of 20 proteins, 8 proteins were direct involvement in radioresistance and literature study strengthens this, however, 1 proteins had assumed relation in radioresistance. Selected radioresistant proteins may be helpful for optimal use of RRB in industry and health care. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. 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.

  15. Protein mobilization in germinating mung bean seeds involves vacuolar sorting receptors and multivesicular bodies.

    PubMed

    Wang, Junqi; Li, Yubing; Lo, Sze Wan; Hillmer, Stefan; Sun, Samuel S M; Robinson, David G; Jiang, Liwen

    2007-04-01

    Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating seeds. We demonstrate that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean (Vigna radiata) seed germination. Immunogold electron microscopy with VSR antibodies demonstrate that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in day 1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at day 3 germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating seeds. Further confocal immunofluorescence and immunogold electron microscopy studies demonstrate that VSR and aleurain colocalize to MVBs as well as PSVs in germinating seeds. Thus, MVBs in germinating seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination.

  16. 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

  17. 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

  18. Structure and function of prostatic- and seminal vesicle-secreted proteins involved in the gelation and liquefaction of human semen.

    PubMed

    Lilja, H

    1988-01-01

    At ejaculation, the epididymal spermatozoa mix with the secretions produced by the seminal vesicles and the prostate. The glandular secretions immediately turns into a gel-like structure which entraps the spermatozoa. The ejaculated spermatozoa become progressively motile as the gel dissolves. The discovery, structure, and interactions of proteins involved in these post-ejaculatory phase reactions of semen are here reported.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  20. Proteins involved in wine aroma compounds metabolism by a Saccharomyces cerevisiae flor-velum yeast strain grown in two conditions.

    PubMed

    Moreno-García, Jaime; García-Martínez, Teresa; Millán, M Carmen; Mauricio, Juan Carlos; Moreno, Juan

    2015-10-01

    A proteomic and exometabolomic study was conducted on Saccharomyces cerevisiae flor yeast strain growing under biofilm formation condition (BFC) with ethanol and glycerol as carbon sources and results were compared with those obtained under no biofilm formation condition (NBFC) containing glucose as carbon source. By using modern techniques, OFFGEL fractionator and LTQ-Orbitrap for proteome and SBSE-TD-GC-MS for metabolite analysis, we quantified 84 proteins including 33 directly involved in the metabolism of glycerol, ethanol and 17 aroma compounds. Contents in acetaldehyde, acetic acid, decanoic acid, 1,1-diethoxyethane, benzaldehyde and 2-phenethyl acetate, changed above their odor thresholds under BFC, and those of decanoic acid, ethyl octanoate, ethyl decanoate and isoamyl acetate under NBFC. Of the twenty proteins involved in the metabolism of ethanol, acetaldehyde, acetoin, 2,3-butanediol, 1,1-diethoxyethane, benzaldehyde, organic acids and ethyl esters, only Adh2p, Ald4p, Cys4p, Fas3p, Met2p and Plb1p were detected under BFC and as many Acs2p, Ald3p, Cem1p, Ilv2p, Ilv6p and Pox1p, only under NBFC. Of the eight proteins involved in glycerol metabolism, Gut2p was detected only under BFC while Pgs1p and Rhr2p were under NBFC. Finally, of the five proteins involved in the metabolism of higher alcohols, Thi3p was present under BFC, and Aro8p and Bat2p were under NBFC.

  1. Dynamic localization of two tobamovirus ORF6 proteins involves distinct organellar compartments.

    PubMed

    Gushchin, Vladimir A; Lukhovitskaya, Nina I; Andreev, Dmitri E; Wright, Kathryn M; Taliansky, Michael E; Solovyev, Andrey G; Morozov, Sergey Y; MacFarlane, Stuart A

    2013-01-01

    ORF6 is a small gene that overlaps the movement and coat protein genes of subgroup 1a tobamoviruses. The ORF6 protein of tomato mosaic virus (ToMV) strain L (L-ORF6), interacts in vitro with eukaryotic elongation factor 1α, and mutation of the ORF6 gene of tobacco mosaic virus (TMV) strain U1 (U1-ORF6) reduces the pathogenicity in vivo of TMV, whereas expression of this gene from two other viruses, tobacco rattle virus (TRV) and potato virus X (PVX), increases their pathogenicity. In this work, the in vivo properties of the L-ORF6 and U1-ORF6 proteins were compared to identify sequences that direct the proteins to different subcellular locations and also influence virus pathogenicity. Site-specific mutations in the ORF6 protein were made, hybrid ORF6 proteins were created in which the N-terminal and C-terminal parts were derived from the two proteins, and different subregions of the protein were examined, using expression either from a recombinant TRV vector or as a yellow fluorescent protein fusion from a binary plasmid in Agrobacterium tumefaciens. L-ORF6 caused mild necrotic symptoms in Nicotiana benthamiana when expressed from TRV, whereas U1-ORF6 caused severe symptoms including death of the plant apex. The difference in symptoms was associated with the C-terminal region of L-ORF6, which directed the protein to the endoplasmic reticulum (ER), whereas U1-ORF6 was directed initially to the nucleolus and later to the mitochondria. Positively charged residues at the N terminus allowed nucleolar entry of both U1-ORF6 and L-ORF6, but hydrophobic residues at the C terminus of L-ORF6 directed this protein to the ER.

  2. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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 (...

  5. Involvement of C-Terminal Histidines in Soybean PM1 Protein Oligomerization and Cu2+ Binding.

    PubMed

    Liu, Guobao; Liu, Ke; Gao, Yang; Zheng, Yizhi

    2017-04-06

    Late embryogenesis abundant (LEA) proteins are widely distributed among plant species, where they contribute to abiotic stress tolerance. LEA proteins can be classified into seven groups according to conserved sequence motifs. The PM1 protein from soybean, which belongs to the Pfam LEA_1 group, has been shown previously to be at least partially natively unfolded, to bind metal ions and potentially to stabilize proteins and membranes. Here, we investigated the role of the PM1 C-terminal domain and in particular the multiple histidine residues in this half of the protein. We constructed recombinant plasmids expressing full-length PM1 and two truncated forms, PM1-N and PM1-C, which represent the N- and C-terminal halves of the protein, respectively. Immunoblotting and cross-linking experiments showed that full-length PM1 forms oligomers and high molecular weight (HMW) complexes in vitro and in vivo, while PM1-C, but not PM1-N, also formed oligomers and HMW complexes in vitro. When the histidine residues in PM1 and PM1-C were chemically modified, oligomerization was abolished, suggesting that histidines play a key role in this process. Furthermore, we demonstrated that high Cu2+ concentrations promote oligomerization and induce PM1 and PM1-C to form HMW complexes. Therefore, we speculate that PM1 proteins not only maintain ion homeostasis in the cytoplasm, but also potentially stabilize and protect other proteins during abiotic stress by forming a large, oligomeric molecular shield around biological targets.

  6. Interactome of the amyloid precursor protein APP in brain reveals a protein network involved in synaptic vesicle turnover and a close association with Synaptotagmin-1.

    PubMed

    Kohli, Bernhard M; Pflieger, Delphine; Mueller, Lukas N; Carbonetti, Giovanni; Aebersold, Ruedi; Nitsch, Roger M; Konietzko, Uwe

    2012-08-03

    Knowledge of the protein networks interacting with the amyloid precursor protein (APP) in vivo can shed light on the physiological function of APP. To date, most proteins interacting with the APP intracellular domain (AICD) have been identified by Yeast Two Hybrid screens which only detect direct interaction partners. We used a proteomics-based approach by biochemically isolating tagged APP from the brains of transgenic mice and subjecting the affinity-purified complex to mass spectrometric (MS) analysis. Using two different quantitative MS approaches, we compared the protein composition of affinity-purified samples isolated from wild-type mice versus transgenic mice expressing tagged APP. This enabled us to assess truly enriched proteins in the transgenic sample and yielded an overlapping set of proteins containing the major proteins involved in synaptic vesicle endo- and exocytosis. Confocal microscopy analyses of cotransfected primary neurons showed colocalization of APP with synaptic vesicle proteins in vesicular structures throughout the neurites. We analyzed the interaction of APP with these proteins using pulldown experiments from transgenic mice or cotransfected cells followed by Western blotting. Synaptotagmin-1 (Stg1), a resident synaptic vesicle protein, was found to directly bind to APP. We fused Citrine and Cerulean to APP and the candidate proteins and measured fluorescence resonance energy transfer (FRET) in differentiated SH-SY5Y cells. Differentially tagged APPs showed clear sensitized FRET emission, in line with the described dimerization of APP. Among the candidate APP-interacting proteins, again only Stg1 was in close proximity to APP. Our results strongly argue for a function of APP in synaptic vesicle turnover in vivo. Thus, in addition to the APP cleavage product Aβ, which influences synaptic transmission at the postsynapse, APP interacts with the calcium sensor of synaptic vesicles and might thus play a role in the regulation of synaptic

  7. Different glycosyltransferases are involved in lipid glycosylation and protein N-glycosylation in the halophilic archaeon Haloferax volcanii.

    PubMed

    Naparstek, Shai; Vinagradov, Evguenii; Eichler, Jerry

    2010-07-01

    Both the lipid and the protein components of biological membranes can be modified by the covalent addition of polysaccharides. Whereas eukaryal and bacterial pathways of lipid and protein glycosylation are relatively well defined, considerably less is known of the parallel processes in Archaea. Recent efforts have identified glycosyltransferases involved in N-glycosylation of the surface-layer glycoprotein of the halophilic archaeon Haloferax volcanii. In the present study, the involvement of these same glycosyltransferases in the biosynthesis of Hfx. volcanii glycolipids was considered by performing nuclear magnetic resonance analysis of the glycolipid fraction of Hfx. volcanii cells deleted of genes encoding those glycosyltransferases, as well as the oligosaccharyltransferase, AglB. The results reveal that different glycosyltransferases are involved in the biosynthesis of N-linked glycoproteins and glycolipids in Archaea.

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

    PubMed Central

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

    2016-01-01

    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

  9. IBT-based quantitative proteomics identifies potential regulatory proteins involved in pigmentation of purple sea cucumber, Apostichopus japonicus.

    PubMed

    Xing, Lili; Sun, Lina; Liu, Shilin; Li, Xiaoni; Zhang, Libin; Yang, Hongsheng

    2017-09-01

    Sea cucumbers are an important economic species and exhibit high yield value among aquaculture animals. Purple sea cucumbers are very rare and beautiful and have stable hereditary patterns. In this study, isobaric tags (IBT) were first used to reveal the molecular mechanism of pigmentation in the body wall of the purple sea cucumber. We analyzed the proteomes of purple sea cucumber in early pigmentation stage (Pa), mid pigmentation stage (Pb) and late pigmentation stage (Pc), resulting in the identification of 5580 proteins, including 1099 differentially expressed proteins in Pb: Pa and 339 differentially expressed proteins in Pc: Pb. GO and KEGG analyses revealed possible differentially expressed proteins, including"melanogenesis", "melanosome", "melanoma", "pigment-biosynthetic process", "Epidermis development", "Ras-signaling pathway", "Wnt-signaling pathway", "response to UV light", and "tyrosine metabolism", involved in pigment synthesis and regulation in purple sea cucumbers. The large number of differentially expressed proteins identified here should be highly useful in further elucidating the mechanisms underlying pigmentation in sea cucumbers. Furthermore, these results may also provide the base for further identification of proteins involved in resistance mechanisms against melanoma, albinism, UV damage, and other diseases in sea cucumbers. Copyright © 2017. Published by Elsevier Inc.

  10. 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

  11. 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

    2015-10-07

    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.

  12. The involvement of FANCM, FANCI, and checkpoint proteins in the interstrand DNA crosslink repair pathway is conserved in C. elegans.

    PubMed

    Lee, Kyong Yun; Chung, Kee Yang; Koo, Hyeon-Sook

    2010-04-04

    Fanconi anemia (FA) patients are specifically defective in the repair of interstrand DNA crosslinks (ICLs), a complex process involving at least 13 FA proteins and other repair/checkpoint proteins. Of the 13 FA proteins, FANCD1/BRCA2, FANCD2, and FANCJ were previously found to be functionally conserved in C. elegans. We have also identified C. elegans homologs of FANCM and FANCI, and determined their epistatic relationships with homologs of FANCD2, checkpoint proteins, and RAD51 upon DNA crosslinking. The counterparts of FANCM, FANCI, and three checkpoint proteins (RPA, ATR and CHK1) are required for focus formation and ubiquitination associated with FANCD2 in C. elegans. However, C. elegans FANCM affects neither RPA focus formation nor CHK1 phosphorylation induced by ICLs, unlike the reported role of human FANCM, which influences ATR-CHK1 signaling at stalled replication forks. Although focus formation by both FANCD2 and RAD51 requires ATR-CHK1 signaling, FANCD2 and RAD51 acted independently in the formation of their respective foci. Thus, the FANCD2 activation pathway involving FANCM, FANCI, and the checkpoint proteins is conserved in C. elegans but with distinct differences.

  13. Functional dissection of protein domains involved in the immunomodulatory properties of PE_PGRS33 of Mycobacterium tuberculosis.

    PubMed

    Zumbo, Antonella; Palucci, Ivana; Cascioferro, Alessandro; Sali, Michela; Ventura, Marcello; D'Alfonso, Pamela; Iantomasi, Raffaella; Di Sante, Gabriele; Ria, Francesco; Sanguinetti, Maurizio; Fadda, Giovanni; Manganelli, Riccardo; Delogu, Giovanni

    2013-12-01

    PE_PGRSs are a large family of proteins identified in Mycobacterium tuberculosis complex and in few other pathogenic mycobacteria. The PE domain of PE_PGRS33 mediates localization of the protein on the mycobacterial cell surface, where the PGRS domain is available to interact with host components. In this study, PE_PGRS33 and its functional deletion mutants were expressed in M. smegmatis, and in vitro and in vivo assays were used to dissect the protein domains involved in the immunomodulatory properties of the protein. We demonstrate that PE_PGRS33-mediated secretion of TNF-α by macrophages occurs by extracellular interaction with TLR2. Our results also show that while the PGRS domain of the protein is required for triggering TNF-α secretion, mutation in the PE domain affects the pro-inflammatory properties of the protein. These results indicate that PE_PGRS33 is a protein with immunomodulatory activity and that protein stability and localization on the mycobacterial surface can affect these properties.

  14. KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes*

    PubMed Central

    Sanchez, Marco A.; Tran, Khoa D.; Valli, Jessica; Hobbs, Sam; Johnson, Errin; Gluenz, Eva; Landfear, Scott M.

    2016-01-01

    African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability. PMID:27489106

  15. A Universal Stress Protein Involved in Oxidative Stress Is a Phosphorylation Target for Protein Kinase CIPK6.

    PubMed

    Gutiérrez-Beltrán, Emilio; Personat, José María; de la Torre, Fernando; Del Pozo, Olga

    2017-01-01

    Calcineurin B-like interacting protein kinases (CIPKs) decode calcium signals upon interaction with the calcium sensors calcineurin B like proteins into phosphorylation events that result into adaptation to environmental stresses. Few phosphorylation targets of CIPKs are known and therefore the molecular mechanisms underlying their downstream output responses are not fully understood. Tomato (Solanum lycopersicum) Cipk6 regulates immune and susceptible Programmed cell death in immunity transforming Ca(2+) signals into reactive oxygen species (ROS) signaling. To investigate SlCipk6-induced molecular mechanisms and identify putative substrates, a yeast two-hybrid approach was carried on and a protein was identified that contained a Universal stress protein (Usp) domain present in bacteria, protozoa and plants, which we named "SlRd2". SlRd2 was an ATP-binding protein that formed homodimers in planta. SlCipk6 and SlRd2 interacted using coimmunoprecipitation and bimolecular fluorescence complementation (BiFC) assays in Nicotiana benthamiana leaves and the complex localized in the cytosol. SlCipk6 phosphorylated SlRd2 in vitro, thus defining, to our knowledge, a novel target for CIPKs. Heterologous SlRd2 overexpression in yeast conferred resistance to highly toxic LiCl, whereas SlRd2 expression in Escherichia coli UspA mutant restored bacterial viability in response to H2O2 treatment. Finally, transient expression of SlCipk6 in transgenic N benthamiana SlRd2 overexpressors resulted in reduced ROS accumulation as compared to wild-type plants. Taken together, our results establish that SlRd2, a tomato UspA, is, to our knowledge, a novel interactor and phosphorylation target of a member of the CIPK family, SlCipk6, and functionally regulates SlCipk6-mediated ROS generation.

  16. The Arabidopsis Tellurite resistance C protein together with ALB3 is involved in photosystem II protein synthesis.

    PubMed

    Schneider, Anja; Steinberger, Iris; Strissel, Henning; Kunz, Hans-Henning; Manavski, Nikolay; Meurer, Jörg; Burkhard, Gabi; Jarzombski, Sabine; Schünemann, Danja; Geimer, Stefan; Flügge, Ulf-Ingo; Leister, Dario

    2014-04-01

    Assembly of photosystem II (PSII) occurs sequentially and requires several auxiliary proteins, such as ALB3 (ALBINO3). Here, we describe the role of the Arabidopsis thaliana thylakoid membrane protein Tellurite resistance C (AtTerC) in this process. Knockout of AtTerC was previously shown to be seedling-lethal. This phenotype was rescued by expressing TerC fused C-terminally to GFP in the terc-1 background, and the resulting terc-1TerC- GFP line and an artificial miRNA-based knockdown allele (amiR-TerC) were used to analyze the TerC function. The alterations in chlorophyll fluorescence and thylakoid ultrastructure observed in amiR-TerC plants and terc-1TerC- GFP were attributed to defects in PSII. We show that this phenotype resulted from a reduction in the rate of de novo synthesis of PSII core proteins, but later steps in PSII biogenesis appeared to be less affected. Yeast two-hybrid assays showed that TerC interacts with PSII proteins. In particular, its interaction with the PSII assembly factor ALB3 has been demonstrated by co-immunoprecipitation. ALB3 is thought to assist in incorporation of CP43 into PSII via interaction with Low PSII Accumulation2 (LPA2) Low PSII Accumulation3 (LPA3). Homozygous lpa2 mutants expressing amiR-TerC displayed markedly exacerbated phenotypes, leading to seedling lethality, indicating an additive effect. We propose a model in which TerC, together with ALB3, facilitates de novo synthesis of thylakoid membrane proteins, for instance CP43, at the membrane insertion step.

  17. Involvement of the catalytically important Asp54 residue of Mycobacterium smegmatis DevR in protein-protein interactions between DevR and DevS.

    PubMed

    Lee, Ha-Na; Lee, Na-On; Ko, In-Jeong; Kim, Si Wouk; Kang, Beom Sik; Oh, Jeong-Il

    2013-06-01

    The DevSR two-component system in Mycobacterium smegmatis consists of the DevS histidine kinase and the DevR response regulator. It is a regulatory system that is involved in the adaptation of mycobacteria to hypoxic and NO stresses. Using the yeast two-hybrid assay and pull-down assay, it was demonstrated that the phosphoaccepting Asp (Asp54) of DevR is important for protein-protein interactions between DevR and DevS. The negative charge of Asp54 of DevR was shown to play an important role in protein-protein interactions between DevR and DevS. When the Lys104 residue, which is involved in transmission of conformational changes induced by phosphorylation of the response regulator, was replaced with Ala, the mutant form of DevR was not phosphorylated by DevS and functionally inactive in vivo. However, the K104A mutation in DevR only slightly affected protein-protein interactions between DevR and DevS. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  18. 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

  19. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.

    PubMed

    Urban, Constantin F; Ermert, David; Schmid, Monika; Abu-Abed, Ulrike; Goosmann, Christian; Nacken, Wolfgang; Brinkmann, Volker; Jungblut, Peter R; Zychlinsky, Arturo

    2009-10-01

    Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.

  20. Structures and functions of protein disulfide isomerase family members involved in proteostasis in the endoplasmic reticulum.

    PubMed

    Okumura, Masaki; Kadokura, Hiroshi; Inaba, Kenji

    2015-06-01

    The endoplasmic reticulum (ER) is an essential cellular compartment in which an enormous number of secretory and cell surface membrane proteins are synthesized and subjected to cotranslational or posttranslational modifications, such as glycosylation and disulfide bond formation. Proper maintenance of ER protein homeostasis (sometimes termed proteostasis) is essential to avoid cellular stresses and diseases caused by abnormal proteins. Accumulating knowledge of cysteine-based redox reactions catalyzed by members of the protein disulfide isomerase (PDI) family has revealed that these enzymes play pivotal roles in productive protein folding accompanied by disulfide formation, as well as efficient ER-associated degradation accompanied by disulfide reduction. Each of PDI family members forms a protein-protein interaction with a preferential partner to fulfill a distinct function. Multiple redox pathways that utilize PDIs appear to function synergistically to attain the highest quality and productivity of the ER, even under various stress conditions. This review describes the structures, physiological functions, and cooperative actions of several essential PDIs, and provides important insights into the elaborate proteostatic mechanisms that have evolved in the extremely active and stress-sensitive ER. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Multiple PPR protein interactions are involved in the RNA editing system in Arabidopsis mitochondria and plastids.

    PubMed

    Andrés-Colás, Nuria; Zhu, Qiang; Takenaka, Mizuki; De Rybel, Bert; Weijers, Dolf; Van Der Straeten, Dominique

    2017-08-15

    Recent identification of several different types of RNA editing factors in plant organelles suggests complex RNA editosomes within which each factor has a different task. However, the precise protein interactions between the different editing factors are still poorly understood. In this paper, we show that the E(+)-type pentatricopeptide repeat (PPR) protein SLO2, which lacks a C-terminal cytidine deaminase-like DYW domain, interacts in vivo with the DYW-type PPR protein DYW2 and the P-type PPR protein NUWA in mitochondria, and that the latter enhances the interaction of the former ones. These results may reflect a protein scaffold or complex stabilization role of NUWA between E(+)-type PPR and DYW2 proteins. Interestingly, DYW2 and NUWA also interact in chloroplasts, and DYW2-GFP overexpressing lines show broad editing defects in both organelles, with predominant specificity for sites edited by E(+)-type PPR proteins. The latter suggests a coordinated regulation of organellar multiple site editing through DYW2, which probably provides the deaminase activity to E(+) editosomes.

  2. The essential WD40 protein Cia1 is involved in a late step of cytosolic and nuclear iron-sulfur protein assembly.

    PubMed

    Balk, Janneke; Aguilar Netz, Daili J; Tepper, Katharina; Pierik, Antonio J; Lill, Roland

    2005-12-01

    The assembly of cytosolic and nuclear iron-sulfur (Fe/S) proteins in yeast is dependent on the iron-sulfur cluster assembly and export machineries in mitochondria and three recently identified extramitochondrial proteins, the P-loop NTPases Cfd1 and Nbp35 and the hydrogenase-like Nar1. However, the molecular mechanism of Fe/S protein assembly in the cytosol is far from being understood, and more components are anticipated to take part in this process. Here, we have identified and functionally characterized a novel WD40 repeat protein, designated Cia1, as an essential component required for Fe/S cluster assembly in vivo on cytosolic and nuclear, but not mitochondrial, Fe/S proteins. Surprisingly, Nbp35 and Nar1, themselves Fe/S proteins, could assemble their Fe/S clusters in the absence of Cia1, demonstrating that these components act before Cia1. Consequently, Cia1 is involved in a late step of Fe/S cluster incorporation into target proteins. Coimmunoprecipitation assays demonstrated a specific interaction between Cia1 and Nar1. In contrast to the mostly cytosolic Nar1, Cia1 is preferentially localized to the nucleus, suggesting an additional function of Cia1. Taken together, our results indicate that Cia1 is a new member of the cytosolic Fe/S protein assembly (CIA) machinery participating in a step after Nbp35 and Nar1.

  3. The Essential WD40 Protein Cia1 Is Involved in a Late Step of Cytosolic and Nuclear Iron-Sulfur Protein Assembly

    PubMed Central

    Balk, Janneke; Aguilar Netz, Daili J.; Tepper, Katharina; Pierik, Antonio J.; Lill, Roland

    2005-01-01

    The assembly of cytosolic and nuclear iron-sulfur (Fe/S) proteins in yeast is dependent on the iron-sulfur cluster assembly and export machineries in mitochondria and three recently identified extramitochondrial proteins, the P-loop NTPases Cfd1 and Nbp35 and the hydrogenase-like Nar1. However, the molecular mechanism of Fe/S protein assembly in the cytosol is far from being understood, and more components are anticipated to take part in this process. Here, we have identified and functionally characterized a novel WD40 repeat protein, designated Cia1, as an essential component required for Fe/S cluster assembly in vivo on cytosolic and nuclear, but not mitochondrial, Fe/S proteins. Surprisingly, Nbp35 and Nar1, themselves Fe/S proteins, could assemble their Fe/S clusters in the absence of Cia1, demonstrating that these components act before Cia1. Consequently, Cia1 is involved in a late step of Fe/S cluster incorporation into target proteins. Coimmunoprecipitation assays demonstrated a specific interaction between Cia1 and Nar1. In contrast to the mostly cytosolic Nar1, Cia1 is preferentially localized to the nucleus, suggesting an additional function of Cia1. Taken together, our results indicate that Cia1 is a new member of the cytosolic Fe/S protein assembly (CIA) machinery participating in a step after Nbp35 and Nar1. PMID:16314508

  4. ZO-2, a tight junction scaffold protein involved in the regulation of cell proliferation and apoptosis.

    PubMed

    Gonzalez-Mariscal, Lorenza; Bautista, Pablo; Lechuga, Susana; Quiros, Miguel

    2012-06-01

    ZO-2 is a membrane-associated guanylate kinase homologue (MAGUK) tight protein associated with the cytoplasmic surface of tight junctions. Here, we describe how ZO-2 is a multidomain molecule that binds to a variety of cell signaling proteins, to the actin cytoskeleton, and to gap, tight, and adherens junction proteins. In sparse cultures, ZO-2 is present at the nucleus and associates with molecules active in gene transcription and pre-mRNA processing. ZO-2 inhibits the Wnt signaling pathway, reduces cell proliferation, and promotes apoptosis; its absence, mutation, or overexpression is present in various human diseases, including deafness and cancer. © 2012 New York Academy of Sciences.

  5. Interactions with RNA/DNA of proteins involved in the regulation of transcription, translation and telomere elongation.

    PubMed

    Ohyama, Takako; Furukawa, Ayako; Miyoshi, Tatsuya; Takada, Yuusuke; Ohgara, Shouta; Hiratsuka, Kazuyuki; Imai, Takao; Okano, Hideyuki; Nakagama, Hitoshi; Nagata, Takashi; Katahira, Masato

    2007-01-01

    Interactions with DNA and RNA of three different proteins involved in the regulation of (1) transcription, (2) translation, and (3) telomere elongation were examined by NMR. In the first case, the combination of structural determination, dynamical analysis on the basis of relaxation data and identification of interactive surface for wild and phosphorylation-mimicking mutant proteins has given the insight on the increase of DNA-binding affinity through phosphorylation of the protein. In the second case, the arrangement of two tandem domains interacting with RNA has been determined with residual dipolar couplings and paramagnetic relaxation enhancement, which has given the idea on how the two tandem domains recognize the target RNA. In the third case, simultaneous binding of the other two tandem domains to both DNA and RNA has been analyzed with chemical shift perturbation analysis. The result has suggested that the protein composed of two tandem domains can recruit telomerase to telomere DNA.

  6. Identification of surface proteins involved in the adhesion of a probiotic Bacillus cereus strain to mucin and fibronectin.

    PubMed

    Sánchez, B; Arias, S; Chaignepain, S; Denayrolles, M; Schmitter, J M; Bressollier, P; Urdaci, M C

    2009-05-01

    Several Bacillus strains isolated from commercial probiotic preparations were identified at the species level, and their adhesion capabilities to three different model intestinal surfaces (mucin, Matrigel and Caco-2 cells) were assessed. In general, adhesion of spores was higher than that of vegetative cells to the three matrices, and overall strain Bacillus cereus(CH) displayed the best adhesion. Different biochemical treatments revealed that surface proteins of B. cereus(CH) were involved in the adhesion properties of the strain. Surface-associated proteins from vegetative cells and spores of B. cereus(CH) were extracted and identified, and some proteins such as S-layer components, flagellin and cell-bound proteases were found to bind to mucin or fibronectin. These facts suggest that those proteins might play important roles in the interaction of this probiotic Bacillus strain within the human gastrointestinal tract.

  7. The LspB protein is involved in the secretion of the LspA1 and LspA2 proteins by Haemophilus ducreyi.

    PubMed

    Ward, Christine K; Mock, Jason R; Hansen, Eric J

    2004-04-01

    The LspA1 and LspA2 proteins of Haemophilus ducreyi 35000 are two very large macromolecules that can be detected in concentrated culture supernatant fluid. Both of these proteins exhibit homology with the N-terminal region of the Bordetella pertussis filamentous hemagglutinin (FHA), which is involved in secretion of the latter macromolecule. The lspA2 open reading frame is flanked upstream by a gene, lspB, that encodes a predicted protein with homology to the B. pertussis FhaC outer membrane protein that is involved in secretion of FHA across the outer membrane. The H. ducreyi lspB gene encodes a protein with a predicted molecular mass of 66,573 Da. Reverse transcription-PCR analysis suggested that the lspB gene was transcribed together with the lspA2 gene on a single mRNA transcript. Polyclonal H. ducreyi LspB antiserum reacted with a 64-kDa antigen present in the Sarkosyl-insoluble cell envelope fraction of H. ducreyi 35000, which indicated that the LspB protein is likely an outer membrane protein. Concentrated culture supernatant fluids from H. ducreyi lspB and lspA1 lspB mutants did not contain detectable LspA1 and detectable LspA2, respectively. However, complementation of the lspB mutant with the wild-type lspB gene on a plasmid restored LspB protein expression and resulted in release of detectable amounts of the LspA1 protein into culture supernatant fluid. When evaluated in the temperature-dependent rabbit model of infection, the lspB mutant was attenuated in the ability to cause lesions and was never recovered in a viable form from lesions. These results indicated that the H. ducreyi LspB protein is involved in secretion of the LspA1 and LspA2 proteins across the outer membrane.

  8. A Protein Involved in the Peptidyltransferase Activity of Escherichia coli Ribosomes*

    PubMed Central

    Nierhaus, Knud H.; Montejo, Vicente

    1973-01-01

    Cores were prepared from 50S ribosomal subunits by incubation with 0.4 M LiCl/Mg++ (0.4c cores); 0.8c cores and corresponding SP0.4-0.8 split proteins were obtained from 0.4c cores. In the fragment reaction 0.4c cores were active, but 0.8c cores were not. Activity of the 0.8c cores could be restored by reconstitution with the SP0.4-0.8 fraction. The split proteins were separated by DEAE-cellulose chromatography and Sephadex gel filtration. The peptidyltransferase activity is correlated with the amount of protein L11 added to the 0.8c core under reconstitution conditions. Whether protein L11 displays the enzymatic activity itself or is part of the enzymatic center is discussed. Images PMID:4579005

  9. Nitrile-specifier Proteins Involved in Glucosinolate Hydrolysis in Arabidopsis thaliana*S⃞

    PubMed Central

    Kissen, Ralph; Bones, Atle M.

    2009-01-01

    Glucosinolates are plant secondary metabolites present in Brassicaceae plants such as the model plant Arabidopsis thaliana. Intact glucosinolates are believed to be biologically inactive, whereas degradation products after hydrolysis have multiple roles in growth regulation and defense. The degradation of glucosinolates is catalyzed by thioglucosidases called myrosinases and leads by default to the formation of isothiocyanates. The interaction of a protein called epithiospecifier protein (ESP) with myrosinase diverts the reaction toward the production of epithionitriles or nitriles depending on the glucosinolate structure. Here we report the identification of a new group of nitrile-specifier proteins (AtNSPs) in A. thaliana able to generate nitriles in conjunction with myrosinase and a more detailed characterization of one member (AtNSP2). Recombinant AtNSP2 expressed in Escherichia coli was used to test its impact on the outcome of glucosinolate hydrolysis using a gas chromatography-mass spectrometry approach. AtNSP proteins share 30–45% sequence homology with A. thaliana ESP. Although AtESP and AtNSP proteins can switch myrosinase-catalyzed degradation of 2-propenylglucosinolate from isothiocyanate to nitrile, only AtESP generates the corresponding epithionitrile. Using the aromatic benzylglucosinolate, recombinant AtNSP2 is also able to direct product formation to the nitrile. Analysis of glucosinolate hydrolysis profiles of transgenic A. thaliana plants overexpressing AtNSP2 confirms its nitrile-specifier activity in planta. In silico expression analysis reveals distinctive expression patterns of AtNSPs, which supports a biological role for these proteins. In conclusion, we show that AtNSPs belonging to a new family of A. thaliana proteins structurally related to AtESP divert product formation from myrosinase-catalyzed glucosinolate hydrolysis and, thereby, likely affect the biological consequences of glucosinolate degradation. We discuss similarities and

  10. iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness

    PubMed Central

    Wu, Guiqin; Shi, Fengying; Liu, Aiqiao; Yang, Ning

    2016-01-01

    Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57) would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5 (SLC25A5) and down-regulated translocator protein (TSPO) would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF). In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation. PMID:28006025

  11. 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.

  12. Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles.

    PubMed

    Neuman, Benjamin W

    2016-11-01

    Replication of eukaryotic positive-stranded RNA viruses is usually linked to the presence of membrane-associated replicative organelles. The purpose of this review is to discuss the function of proteins responsible for formation of the coronavirus replicative organelle. This will be done by identifying domains that are conserved across the order Nidovirales, and by summarizing what is known about function and structure at the level of protein domains.

  13. iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness.

    PubMed

    Li, Guangqi; Sun, Congjiao; Wu, Guiqin; Shi, Fengying; Liu, Aiqiao; Yang, Ning

    2016-01-01

    Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57) would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5 (SLC25A5) and down-regulated translocator protein (TSPO) would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF). In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation.

  14. Is Peripheral Benzodiazepine Receptor (PBR) Gene Expression Involved in Breast Cancer Suppression by Dietary Soybean Protein?

    DTIC Science & Technology

    2006-06-01

    elucidate the molecular mechanism(s) by which dietary soy protein may offer its tumor suppressing effect. We developed a breast cancer model in female rats ...30, 2006) APPROVED STATEMENT OF WORK Task 1. To develop a breast cancer model by administration of DMBA to female rats fed a diet containing...for PBRs from breast epithelial cells (Specific Aim 6) Study 1. Beneficial Effects of Soy Protein in Breast Cancer Development in Female Rats

  15. A conserved motif in S-layer proteins is involved in peptidoglycan binding in Thermus thermophilus.

    PubMed Central

    Olabarría, G; Carrascosa, J L; de Pedro, M A; Berenguer, J

    1996-01-01

    There is experimental evidence to suggest that the 100-kDa S-layer protein from Thermus thermophilus HB8 binds to the peptidoglycan cell wall. This property could be related to the presence of a region (SLH) of homology with other S-layer proteins and extracellular enzymes (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224-1233, 1994). By using specific monoclonal antibodies, we show that similar regions are present in different members of the Deinococcus-Thermus phylogenetic group. To analyze the role that the SLH domain plays in vivo and in vitro in T. thermophilus, we have obtained a mutant form (slpA.X) of the S-layer gene (slpA) in which the SLH domain was deleted. The slpA.X gene was inserted into the chromosome of the thermophile by gene replacement, resulting in a mutant which expressed a major membrane protein with the size expected from the construction (90 kDa). This protein was identified as the product of slpA.X by its differential reaction with monoclonal antibodies. Mutants expressing the SlpA.X protein grow as groups of cells, surrounded by a common external envelope of trigonal symmetry that contains the SlpA.X protein as a main component, thus showing the inability of the SLH-defective protein to attach to the underlying material in vivo. In addition, averaged images of SlpA.X-rich fractions showed a regular arrangement, identical to that built up by the wild-type (SlpA) protein in the absence of peptidoglycan. Finally, we demonstrate by Western blotting (immunoblotting) the direct role of the SLH domain in the binding of the S-layer of T. thermophilus HB8 to the peptidoglycan layer. PMID:8759836

  16. Dietary fish protein alters blood lipid concentrations and hepatic genes involved in cholesterol homeostasis in the rat model.

    PubMed

    Shukla, Anjali; Bettzieche, Anja; Hirche, Frank; Brandsch, Corinna; Stangl, Gabriele I; Eder, Klaus

    2006-10-01

    It is known that various dietary plant proteins are capable of influencing the lipid metabolism of human subjects and animals when compared with casein. Less, however, is known about the effects of fish protein on the cholesterol and triacylglycerol metabolism. Therefore, two experiments were conducted in which rats were fed diets containing 200 g of either fish protein, prepared from Alaska pollack fillets, or casein, which served as control, per kilogram, over 20 and 22 d, respectively. As parameters of lipid metabolism, the concentrations of cholesterol and triacylglycerols in the plasma and liver, the faecal excretion of bile acids and the hepatic expression of genes encoding proteins involved in lipid homeostasis were determined. In both experiments, rats fed fish protein had higher concentrations of cholesteryl esters in the liver, a lower concentration of cholesterol in the HDL fraction (rho > 1.063 kg/l) and lower plasma triacylglycerol concentrations than rats fed casein (P < 0.05). The gene expression analysis performed in experiment 2 showed that rats fed fish protein had higher relative mRNA concentrations of sterol regulatory element-binding protein (SREBP)-2, 3-hydroxy-3-methylglutaryl coenzyme A reductase, LDL receptor, apo AI, scavenger receptor B1 and lecithin-cholesterol-acyltransferase in their liver than did rats fed casein (P < 0.05). The faecal excretion of bile acids and the mRNA concentrations of cholesterol 7alpha-hydroxylase, SREBP-1c and corresponding target genes were not altered. These findings show that fish protein had multiple effects on plasma and liver lipids that were at least in part caused by an altered expression of the hepatic genes involved in lipid homeostasis.

  17. Mining the human complexome database identifies RBM14 as an XPO1-associated protein involved in HIV-1 Rev function.

    PubMed

    Budhiraja, Sona; Liu, Hongbing; Couturier, Jacob; Malovannaya, Anna; Qin, Jun; Lewis, Dorothy E; Rice, Andrew P

    2015-04-01

    By recruiting the host protein XPO1 (CRM1), the HIV-1 Rev protein mediates the nuclear export of incompletely spliced viral transcripts. We mined data from the recently described human nuclear complexome to identify a host protein, RBM14, which associates with XPO1 and Rev and is involved in Rev function. Using a Rev-dependent p24 reporter plasmid, we found that RBM14 depletion decreased Rev activity and Rev-mediated enhancement of the cytoplasmic levels of unspliced viral transcripts. RBM14 depletion also reduced p24 expression during viral infection, indicating that RBM14 is limiting for Rev function. RBM14 has previously been shown to localize to nuclear paraspeckles, a structure implicated in retaining unspliced HIV-1 transcripts for either Rev-mediated nuclear export or degradation. We found that depletion of NEAT1 RNA, a long noncoding RNA required for paraspeckle integrity, abolished the ability of overexpressed RBM14 to enhance Rev function, indicating the dependence of RBM14 function on paraspeckle integrity. Our study extends the known host cell interactome of Rev and XPO1 and further substantiates a critical role for paraspeckles in the mechanism of action of Rev. Our study also validates the nuclear complexome as a database from which viral cofactors can be mined. This study mined a database of nuclear protein complexes to identify a cellular protein named RBM14 that is associated with XPO1 (CRM1), a nuclear protein that binds to the HIV-1 Rev protein and mediates nuclear export of incompletely spliced viral RNAs. Functional assays demonstrated that RBM14, a protein found in paraspeckle structures in the nucleus, is involved in HIV-1 Rev function. This study va