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Sample records for apoplastic protein involved

  1. Uncovering plant-pathogen crosstalk through apoplastic proteomic studies.

    Science.gov (United States)

    Delaunois, Bertrand; Jeandet, Philippe; Clément, Christophe; Baillieul, Fabienne; Dorey, Stéphan; Cordelier, Sylvain

    2014-01-01

    Plant pathogens have evolved by developing different strategies to infect their host, which in turn have elaborated immune responses to counter the pathogen invasion. The apoplast, including the cell wall and extracellular space outside the plasma membrane, is one of the first compartments where pathogen-host interaction occurs. The plant cell wall is composed of a complex network of polysaccharides polymers and glycoproteins and serves as a natural physical barrier against pathogen invasion. The apoplastic fluid, circulating through the cell wall and intercellular spaces, provides a means for delivering molecules and facilitating intercellular communications. Some plant-pathogen interactions lead to plant cell wall degradation allowing pathogens to penetrate into the cells. In turn, the plant immune system recognizes microbial- or damage-associated molecular patterns (MAMPs or DAMPs) and initiates a set of basal immune responses, including the strengthening of the plant cell wall. The establishment of defense requires the regulation of a wide variety of proteins that are involved at different levels, from receptor perception of the pathogen via signaling mechanisms to the strengthening of the cell wall or degradation of the pathogen itself. A fine regulation of apoplastic proteins is therefore essential for rapid and effective pathogen perception and for maintaining cell wall integrity. This review aims to provide insight into analyses using proteomic approaches of the apoplast to highlight the modulation of the apoplastic protein patterns during pathogen infection and to unravel the key players involved in plant-pathogen interaction.

  2. Protein profile of Beta vulgaris leaf apoplastic fluid and changes induced by Fe deficiency and Fe resupply

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    Laura eCeballos-Laita

    2015-03-01

    Full Text Available The fluid collected by direct leaf centrifugation has been used to study the proteome of the sugar beet apoplastic fluid as well as the changes induced by Fe deficiency and Fe resupply to Fe-deficient plants in the protein profile. Plants were grown in Fe-sufficient and Fe-deficient conditions, and Fe resupply was carried out with 45 μM Fe(III-EDTA for 24 h. Protein extracts of leaf apoplastic fluid were analyzed by two-dimensional isoelectric focusing-SDS-PAGE electrophoresis. Gel image analysis revealed 203 consistent spots, and proteins in 81% of them (164 were identified by nLC-MS/MS using a custom made reference repository of beet protein sequences. When redundant UniProt entries were deleted, a non-redundant leaf apoplastic proteome consisting of 109 proteins was obtained. TargetP and SecretomeP algorithms predicted that 63% of them were secretory proteins. Functional classification of the non-redundant proteins indicated that stress and defense, protein metabolism, cell wall and C metabolism accounted for approximately 75% of the identified proteome. The effects of Fe-deficiency on the leaf apoplast proteome were limited, with only five spots (2.5% changing in relative abundance, thus suggesting that protein homeostasis in the leaf apoplast fluid is well maintained upon Fe shortage. The identification of three chitinase isoforms among proteins increasing in relative abundance with Fe-deficiency suggests that one of the few effects of Fe deficiency in the leaf apoplast proteome includes cell wall modifications. Iron resupply to Fe deficient plants changed the relative abundance of 16 spots when compared to either Fe-sufficient or Fe-deficient samples. Proteins identified in these spots can be broadly classified as those responding to Fe-resupply, which included defense and cell wall related proteins, and non-responsive, which are mainly protein metabolism related proteins and whose changes in relative abundance followed the same trend as

  3. The Apoplastic Secretome of Trichoderma virens During Interaction With Maize Roots Shows an Inhibition of Plant Defence and Scavenging Oxidative Stress Secreted Proteins

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    Guillermo Nogueira-Lopez

    2018-04-01

    Full Text Available In Nature, almost every plant is colonized by fungi. Trichoderma virens is a biocontrol fungus which has the capacity to behave as an opportunistic plant endophyte. Even though many plants are colonized by this symbiont, the exact mechanisms by which Trichoderma masks its entrance into its plant host remain unknown, but likely involve the secretion of different families of proteins into the apoplast that may play crucial roles in the suppression of plant immune responses. In this study, we investigated T. virens colonization of maize roots under hydroponic conditions, evidencing inter- and intracellular colonization by the fungus and modifications in root morphology and coloration. Moreover, we show that upon host penetration, T. virens secretes into the apoplast an arsenal of proteins to facilitate inter- and intracellular colonization of maize root tissues. Using a gel-free shotgun proteomics approach, 95 and 43 secretory proteins were identified from maize and T. virens, respectively. A reduction in the maize secretome (36% was induced by T. virens, including two major groups, glycosyl hydrolases and peroxidases. Furthermore, T. virens secreted proteins were mainly involved in cell wall hydrolysis, scavenging of reactive oxygen species and secondary metabolism, as well as putative effector-like proteins. Levels of peroxidase activity were reduced in the inoculated roots, suggesting a strategy used by T. virens to manipulate host immune responses. The results provide an insight into the crosstalk in the apoplast which is essential to maintain the T. virens-plant interaction.

  4. Localizatlon of expansin-like protein in apoplast of pea (Pisum sativum L. root nodules during interaction with Rhizobium leguminosarum bv. viciae

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    Marzena Sujkowska

    2011-01-01

    Full Text Available During nodule development on pea roots, apoplast undergoes changes in activity of plant cell wall proteins such as expansins (EXPs. Because the accumulation of EXP protein has been correlated with the growth of various plant organs, we investigated using Western Blot and immunolocalization studies with antibody against PsEXP1, whether this protein was accumulated in the expanding cells of nodule. Immunoblot results indicated the presence of a 30-kDa band specific for pea root nodules. The EXP proteins content rose during growth of pea root nodules. Expansin(s protein was localized in nodule apoplast as well as in the infection thread walls. The enhanced amount of expansin-like proteins in meristematic part of nodule, root and shoot was shown. The localization of this protein in the meristematic cell walls can be related to the loosening of plant cell wall before cell enlargement. Both, plant cell enlargement and infection thread growth require activity of expansin(s. Possible involvement of EXPs in the process of pea root nodule development is also discussed.

  5. Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses.

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    Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Côté, Olivier; Stare, Barbara Gerič; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bélair, Guy; Moffett, Peter

    2015-01-01

    The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12) and an expansin-like protein (GrEXPB2), suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.

  6. Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses.

    Directory of Open Access Journals (Sweden)

    Shawkat Ali

    Full Text Available The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12 and an expansin-like protein (GrEXPB2, suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.

  7. IDENTIFICATION AND FUNCTIONAL ANNOTATION OF APOPLASTIC PHOSPHOPROTEINS OF HIPPOPHAE RHAMNOIDES SEEDLINGS

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    Ravi Gupta

    2016-12-01

    Full Text Available Phosphorylation is a reversible switch that regulates the biological activities of the proteins. Although there are ample of reports on the plant phosphoproteome analysis, phosphorylation status of apoplastic proteins has not been investigated profoundly. Here a shotgun proteomics approach was used to identify the phosphoproteins from the apoplast of the Hippophae rhamnoides (Seabuckthorn. A total of 123 phosphoproteins were identified using an SYNAPT G2 quadrupole time-of-flight mass spectrometer (Q-ToF-MS. Functional annotation of the identified phosphoproteins using PANTHER, Gene ontology, and KEGG programs showed that the majority of proteins were associated with the transporter, nucleic acid binding and amino acid metabolic activities. Prediction of secretory nature of the identified proteins using SignalP and SecretomeP servers showed that 56 % of the proteins were secretory, while rest of the 44 % of the proteins were non-secretory. PhosPhAt 4.0 detected 534 putative phosphorylation sites in the 75 unique Arabidopsis annotated proteins, wherein 195 (36% were on the serine residue, 196 (37% were on the threonine residue and 143 (27% were detected on the tyrosine residue. Taken together, our results provide the first insight into the phosphorylation-mediated regulation of apoplastic proteins by cellular processes, which would be helpful in an in-depth understanding of the apoplastic signaling

  8. Effect of greenhouse conditions on the leaf apoplastic proteome of Coffea arabica plants.

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    Guerra-Guimarães, Leonor; Vieira, Ana; Chaves, Inês; Pinheiro, Carla; Queiroz, Vagner; Renaut, Jenny; Ricardo, Cândido P

    2014-06-02

    This work describes the coffee leaf apoplastic proteome and its modulation by the greenhouse conditions. The apoplastic fluid (APF) was obtained by leaf vacuum infiltration, and the recovered proteins were separated by 2-DE and subsequently identified by matrix assisted laser desorption/ionization time of flight-mass spectrometry, followed by homology search in EST coffee databases. Prediction tools revealed that the majority of the 195 identified proteins are involved in cell wall metabolism and in stress/defense responses. Although most of the proteins follow the classical secretory mechanism, a low percentage of them seem to result from unconventional secretion (leaderless secreted proteins). Principal components analysis revealed that the APF samples formed two distinct groups, with the temperature amplitude mostly contributing for this separation (higher or lower than 10°C, respectively). Sixty one polypeptide spots allowed defining these two groups and 28 proteins were identified, belonging to carbohydrate metabolism, cell wall modification and proteolysis. Interestingly stress/defense proteins appeared as more abundant in Group I which is associated with a higher temperature amplitude. It seems that the proteins in the coffee leaf APF might be implicated in structural modifications in the extracellular space that are crucial for plant development/adaptation to the conditions of the prevailing environment. This is the first detailed proteomic study of the coffee leaf apoplastic fluid (APF) and of its modulation by the greenhouse conditions. The comprehensive overview of the most abundant proteins present in the extra-cellular compartment is particularly important for the understanding of coffee responses to abiotic/biotic stress. This article is part of a Special Issue entitled: Environmental and structural proteomics. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Collection of apoplastic fluids from Arabidopsis thaliana leaves

    DEFF Research Database (Denmark)

    Madsen, Svend Roesen; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    The leaf apoplast comprises the extracellular continuum outside cell membranes. A broad range of processes take place in the apoplast, including intercellular signaling, metabolite transport, and plant-microbe interactions. To study these processes, it is essential to analyze the metabolite conte...... in apoplastic fluids. Due to the fragile nature of leaf tissues, it is a challenge to obtain apoplastic fluids from leaves. Here, methods to collect apoplastic washing fluid and guttation fluid from Arabidopsis thaliana leaves are described....

  10. A genetic screen reveals Arabidopsis stomatal and/or apoplastic defenses against Pseudomonas syringae pv. tomato DC3000.

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    Weiqing Zeng

    2011-10-01

    Full Text Available Bacterial infection of plants often begins with colonization of the plant surface, followed by entry into the plant through wounds and natural openings (such as stomata, multiplication in the intercellular space (apoplast of the infected tissues, and dissemination of bacteria to other plants. Historically, most studies assess bacterial infection based on final outcomes of disease and/or pathogen growth using whole infected tissues; few studies have genetically distinguished the contribution of different host cell types in response to an infection. The phytotoxin coronatine (COR is produced by several pathovars of Pseudomonas syringae. COR-deficient mutants of P. s. tomato (Pst DC3000 are severely compromised in virulence, especially when inoculated onto the plant surface. We report here a genetic screen to identify Arabidopsis mutants that could rescue the virulence of COR-deficient mutant bacteria. Among the susceptible to coronatine-deficient Pst DC3000 (scord mutants were two that were defective in stomatal closure response, two that were defective in apoplast defense, and four that were defective in both stomatal and apoplast defense. Isolation of these three classes of mutants suggests that stomatal and apoplastic defenses are integrated in plants, but are genetically separable, and that COR is important for Pst DC3000 to overcome both stomatal guard cell- and apoplastic mesophyll cell-based defenses. Of the six mutants defective in bacterium-triggered stomatal closure, three are defective in salicylic acid (SA-induced stomatal closure, but exhibit normal stomatal closure in response to abscisic acid (ABA, and scord7 is compromised in both SA- and ABA-induced stomatal closure. We have cloned SCORD3, which is required for salicylic acid (SA biosynthesis, and SCORD5, which encodes an ATP-binding cassette (ABC protein, AtGCN20/AtABCF3, predicted to be involved in stress-associated protein translation control. Identification of SCORD5 begins to

  11. A paralogous decoy protects Phytophthora sojae apoplastic effector PsXEG1 from a host inhibitor.

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    Ma, Zhenchuan; Zhu, Lin; Song, Tianqiao; Wang, Yang; Zhang, Qi; Xia, Yeqiang; Qiu, Min; Lin, Yachun; Li, Haiyang; Kong, Liang; Fang, Yufeng; Ye, Wenwu; Wang, Yan; Dong, Suomeng; Zheng, Xiaobo; Tyler, Brett M; Wang, Yuanchao

    2017-02-17

    The extracellular space (apoplast) of plant tissue represents a critical battleground between plants and attacking microbes. Here we show that a pathogen-secreted apoplastic xyloglucan-specific endoglucanase, PsXEG1, is a focus of this struggle in the Phytophthora sojae -soybean interaction. We show that soybean produces an apoplastic glucanase inhibitor protein, GmGIP1, that binds to PsXEG1 to block its contribution to virulence. P. sojae , however, secretes a paralogous PsXEG1-like protein, PsXLP1, that has lost enzyme activity but binds to GmGIP1 more tightly than does PsXEG1, thus freeing PsXEG1 to support P. sojae infection. The gene pair encoding PsXEG1 and PsXLP1 is conserved in many Phytophthora species, and the P. parasitica orthologs PpXEG1 and PpXLP1 have similar functions. Thus, this apoplastic decoy strategy may be widely used in Phytophthora pathosystems. Copyright © 2017, American Association for the Advancement of Science.

  12. Salicylic acid signaling inhibits apoplastic reactive oxygen species signaling.

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    Xu, Enjun; Brosché, Mikael

    2014-06-04

    Reactive oxygen species (ROS) are used by plants as signaling molecules during stress and development. Given the amount of possible challenges a plant face from their environment, plants need to activate and prioritize between potentially conflicting defense signaling pathways. Until recently, most studies on signal interactions have focused on phytohormone interaction, such as the antagonistic relationship between salicylic acid (SA)-jasmonic acid and cytokinin-auxin. In this study, we report an antagonistic interaction between SA signaling and apoplastic ROS signaling. Treatment with ozone (O3) leads to a ROS burst in the apoplast and induces extensive changes in gene expression and elevation of defense hormones. However, Arabidopsis thaliana dnd1 (defense no death1) exhibited an attenuated response to O3. In addition, the dnd1 mutant displayed constitutive expression of defense genes and spontaneous cell death. To determine the exact process which blocks the apoplastic ROS signaling, double and triple mutants involved in various signaling pathway were generated in dnd1 background. Simultaneous elimination of SA-dependent and SA-independent signaling components from dnd1 restored its responsiveness to O3. Conversely, pre-treatment of plants with SA or using mutants that constitutively activate SA signaling led to an attenuation of changes in gene expression elicited by O3. Based upon these findings, we conclude that plants are able to prioritize the response between ROS and SA via an antagonistic action of SA and SA signaling on apoplastic ROS signaling.

  13. Apoplastic domains and sub-domains in the shoots of etiolated corn seedlings

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    Epel, B. L.; Bandurski, R. S.

    1990-01-01

    Light Green, an apoplastic probe, was applied to the cut mesocotyl base or to the cut coleoptile apex of etiolated seedlings of Zea mays L. cv. Silver Queen. Probe transport was measured and its tissue distribution determined. In the mesocotyl, there is an apoplastic barrier between cortex and stele. This barrier creates two apoplastic domains which are non-communicating. A kinetic barrier exists between the apoplast of the mesocotyl stele and that of the coleoptile. This kinetic barrier is not absolute and there is limited communication between the apoplasts of the two regions. This kinetic barrier effectively creates two sub-domains. In the coleoptile, there is communication between the apoplast of the vascular strands and that of the surrounding cortical tissue. No apoplastic communication was observed between the coleoptile cortex and the mesocotyl cortex. Thus, the apoplastic space of the coleoptile cortex is a sub-domain of the integrated coleoptile domain and is separate from that of the apoplastic domain of the mesocotyl cortex.

  14. Controlled free radical attack in the apoplast: a hypothesis for roles of O, N and S species in regulatory and polysaccharide cleavage events during rapid abscission by Azolla.

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    Cohen, Michael F; Gurung, Sushma; Fukuto, Jon M; Yamasaki, Hideo

    2014-03-01

    Shedding of organs by abscission is a key terminal step in plant development and stress responses. Cell wall (CW) loosening at the abscission zone can occur through a combination chain breakage of apoplastic polysaccharides and tension release of cellulose microfibrils. Two distinctly regulated abscission cleavage events are amenable to study in small water ferns of the genus Azolla; one is a rapid abscission induced by environmental stimuli such as heat or chemicals, and the other is an ethylene-induced process occurring more slowly through the action of hydrolytic enzymes. Although free radicals are suggested to be involved in the induction of rapid root abscission, its mechanism is not fully understood. The apoplast contains peroxidases, metal-binding proteins and phenolic compounds that potentially generate free radicals from H2O2 to cleave polysaccharides in the CW and middle lamella. Effects of various thiol-reactive agents implicate the action of apoplastic peroxidases having accessible cysteine thiols in rapid abscission. The Ca(2+) dependency of rapid abscission may reflect the stabilization Ca(2+) confers to peroxidase structure and binding to pectin. To spur further investigation, we present a hypothetical model for small signaling molecules H2O2 and NO and their derivatives in regulating, via modification of putative protein thiols, free radical attack of apoplastic polysaccharides. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  15. Apoplastic interactions between plants and plant root intruders

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    Kanako eMitsumasu

    2015-08-01

    Full Text Available Numerous pathogenic or parasitic organisms attack plant roots to obtain nutrients, and the apoplast including the plant cell wall is where the plant cell meets such organisms. Root-parasitic angiosperms and nematodes are two distinct types of plant root parasites but share some common features in their strategies for breaking into plant roots. Striga and Orobanche are obligate root parasitic angiosperms that cause devastating agricultural problems worldwide. Parasitic plants form an invasion organ called a haustorium, where plant cell wall degrading enzymes (PCWDEs are highly expressed. Plant-parasitic nematodes are another type of agriculturally important plant root parasite. These nematodes breach the plant cell walls by protruding a sclerotized stylet from which PCWDEs are secreted. Responding to such parasitic invasion, host plants activate their own defense responses against parasites. Endoparasitic nematodes secrete apoplastic effectors to modulate host immune responses and to facilitate the formation of a feeding site. Apoplastic communication between hosts and parasitic plants also contributes to their interaction. Parasitic plant germination stimulants, strigolactones (SLs, are recently identified apoplastic signals that are transmitted over long distances from biosynthetic sites to functioning sites. Here, we discuss recent advances in understanding the importance of apoplastic signals and cell walls for plant-parasite interactions.

  16. Apoplastic interactions between plants and plant root intruders.

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    Mitsumasu, Kanako; Seto, Yoshiya; Yoshida, Satoko

    2015-01-01

    Numerous pathogenic or parasitic organisms attack plant roots to obtain nutrients, and the apoplast including the plant cell wall is where the plant cell meets such organisms. Root parasitic angiosperms and nematodes are two distinct types of plant root parasites but share some common features in their strategies for breaking into plant roots. Striga and Orobanche are obligate root parasitic angiosperms that cause devastating agricultural problems worldwide. Parasitic plants form an invasion organ called a haustorium, where plant cell wall degrading enzymes (PCWDEs) are highly expressed. Plant-parasitic nematodes are another type of agriculturally important plant root parasite. These nematodes breach the plant cell walls by protruding a sclerotized stylet from which PCWDEs are secreted. Responding to such parasitic invasion, host plants activate their own defense responses against parasites. Endoparasitic nematodes secrete apoplastic effectors to modulate host immune responses and to facilitate the formation of a feeding site. Apoplastic communication between hosts and parasitic plants also contributes to their interaction. Parasitic plant germination stimulants, strigolactones, are recently identified apoplastic signals that are transmitted over long distances from biosynthetic sites to functioning sites. Here, we discuss recent advances in understanding the importance of apoplastic signals and cell walls for plant-parasite interactions.

  17. Accumulation and localization of extensin protein in apoplast of pea root nodule under aluminum stress.

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    Sujkowska-Rybkowska, Marzena; Borucki, Wojciech

    2014-12-01

    Cell wall components such as hydroxyproline-rich glycoproteins (HRGPs, extensins) have been proposed to be involved in aluminum (Al) resistance mechanisms in plants. We have characterized the distribution of extensin in pea (Pisum sativum L.) root nodules apoplast under short (for 2 and 24h) Al stress. Monoclonal antibodie LM1 have been used to locate extensin protein epitope by immunofluorescence and immunogold labeling. The nodules were shown to respond to Al stress by thickening of plant and infection thread (IT) walls and disturbances in threads growth and bacteria endocytosis. Immunoblot results indicated the presence of a 17-kDa band specific for LM1. Irrespective of the time of Al stress, extensin content increased in root nodules. Further observation utilizing fluorescence and transmission electron microscope showed that LM1 epitope was localized in walls and intercellular spaces of nodule cortex tissues and in the infection threads matrix. Al stress in nodules appears to be associated with higher extensin accumulation in matrix of enlarged thick-walled ITs. In addition to ITs, thickened walls and intercellular spaces of nodule cortex were also associated with intense extensin accumulation. These data suggest that Al-induced extensin accumulation in plant cell walls and ITs matrix may have influence on the process of IT growth and tissue and cell colonization by Rhizobium bacteria. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Unraveling incompatibility between wheat and the fungal pathogen Zymoseptoria tritici through apoplastic proteomics.

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    Yang, Fen; Li, Wanshun; Derbyshire, Mark; Larsen, Martin R; Rudd, Jason J; Palmisano, Giuseppe

    2015-05-08

    Hemibiotrophic fungal pathogen Zymoseptoria tritici causes severe foliar disease in wheat. However, current knowledge of molecular mechanisms involved in plant resistance to Z. tritici and Z. tritici virulence factors is far from being complete. The present work investigated the proteome of leaf apoplastic fluid with emphasis on both host wheat and Z. tritici during the compatible and incompatible interactions. The proteomics analysis revealed rapid host responses to the biotrophic growth, including enhanced carbohydrate metabolism, apoplastic defenses and stress, and cell wall reinforcement, might contribute to resistance. Compatibility between the host and the pathogen was associated with inactivated plant apoplastic responses as well as fungal defenses to oxidative stress and perturbation of plant cell wall during the initial biotrophic stage, followed by the strong induction of plant defenses during the necrotrophic stage. To study the role of anti-oxidative stress in Z. tritici pathogenicity in depth, a YAP1 transcription factor regulating antioxidant expression was deleted and showed the contribution to anti-oxidative stress in Z. tritici, but was not required for pathogenicity. This result suggests the functional redundancy of antioxidants in the fungus. The data demonstrate that incompatibility is probably resulted from the proteome-level activation of host apoplastic defenses as well as fungal incapability to adapt to stress and interfere with host cell at the biotrophic stage of the interaction.

  19. Recovery of active pathogenesis-related enzymes from the apoplast ...

    African Journals Online (AJOL)

    Overall protease activity intensity was higher in the symplast. Maximum symplast contamination of the apoplast was 2% as estimated by glucose 6-phosphate dehydrogenase activity, a biochemical marker for symplast. Accumulation of pathogenesis-related enzymatic activities in the apoplast of M. acuminata leaf tissue was ...

  20. Apoplastic Sugar Extraction and Quantification from Wheat Leaves Infected with Biotrophic Fungi.

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    Roman-Reyna, Veronica; Rathjen, John P

    2017-01-01

    Biotrophic fungi such as rusts modify the nutrient status of their hosts by extracting sugars. Hemibiotrophic and biotrophic fungi obtain nutrients from the cytoplasm of host cells and/or the apoplastic spaces. Uptake of nutrients from the cytoplasm is via intracellular hyphae or more complex structures such as haustoria. Apoplastic nutrients are taken up by intercellular hyphae. Overall the infection creates a sink causing remobilization of nutrients from local and distal tissues. The main mobile sugar in plants is sucrose which is absorbed via plant or fungal transporters once unloaded into the cytoplasm or the apoplast. Infection by fungal pathogens alters the apoplastic sugar contents and stimulates the influx of nutrients towards the site of infection as the host tissue transitions to sink. Quantification of solutes in the apoplast can help to understand the allocation of nutrients during infection. However, separation of apoplastic fluids from whole tissue is not straightforward and leakage from damaged cells can alter the results of the extraction. Here, we describe how variation in cytoplasmic contamination and infiltrated leaf volumes must be controlled when extracting apoplastic fluids from healthy and rust-infected wheat leaves. We show the importance of correcting the data for these parameters to measure sugar concentrations accurately.

  1. Specific Hypersensitive Response–Associated Recognition of New Apoplastic Effectors from Cladosporium fulvum in Wild Tomato

    NARCIS (Netherlands)

    Mesarich, Carl H.; Ӧkmen, Bilal; Rovenich, Hanna; Griffiths, Scott A.; Wang, Changchun; Karimi Jashni, Mansoor; Mihajlovski, Aleksandar; Collemare, Jérôme; Hunziker, Lukas; Deng, Cecilia H.; Burgt, Van Der Ate; Beenen, Henriek G.; Templeton, Matthew D.; Bradshaw, Rosie E.; Wit, De Pierre J.G.M.

    2018-01-01

    Tomato leaf mold disease is caused by the biotrophic fungus Cladosporium fulvum. During infection, C. fulvum produces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed by Cf immune receptor genes

  2. Bacterially produced Pt-GFP as ratiometric dual-excitation sensor for in planta mapping of leaf apoplastic pH in intact Avena sativa and Vicia faba.

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    Geilfus, Christoph-Martin; Mühling, Karl H; Kaiser, Hartmut; Plieth, Christoph

    2014-01-01

    Ratiometric analysis with H(+)-sensitive fluorescent sensors is a suitable approach for monitoring apoplastic pH dynamics. For the acidic range, the acidotropic dual-excitation dye Oregon Green 488 is an excellent pH sensor. Long lasting (hours) recordings of apoplastic pH in the near neutral range, however, are more problematic because suitable pH indicators that combine a good pH responsiveness at a near neutral pH with a high photostability are lacking. The fluorescent pH reporter protein from Ptilosarcus gurneyi (Pt-GFP) comprises both properties. But, as a genetically encoded indicator and expressed by the plant itself, it can be used almost exclusively in readily transformed plants. In this study we present a novel approach and use purified recombinant indicators for measuring ion concentrations in the apoplast of crop plants such as Vicia faba L. and Avena sativa L. Pt-GFP was purified using a bacterial expression system and subsequently loaded through stomata into the leaf apoplast of intact plants. Imaging verified the apoplastic localization of Pt-GFP and excluded its presence in the symplast. The pH-dependent emission signal stood out clearly from the background. PtGFP is highly photostable, allowing ratiometric measurements over hours. By using this approach, a chloride-induced alkalinizations of the apoplast was demonstrated for the first in oat. Pt-GFP appears to be an excellent sensor for the quantification of leaf apoplastic pH in the neutral range. The presented approach encourages to also use other genetically encoded biosensors for spatiotemporal mapping of apoplastic ion dynamics.

  3. Pathogen-Induced Changes in the Antioxidant Status of the Apoplast in Barley Leaves

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    Vanacker, Hélène; Carver, Tim L.W.; Foyer, Christine H.

    1998-01-01

    Leaves of two barley (Hordeum vulgare L.) isolines, Alg-R, which has the dominant Mla1 allele conferring hypersensitive race-specific resistance to avirulent races of Blumeria graminis, and Alg-S, which has the recessive mla1 allele for susceptibility to attack, were inoculated with B. graminis f. sp. hordei. Total leaf and apoplastic antioxidants were measured 24 h after inoculation when maximum numbers of attacked cells showed hypersensitive death in Alg-R. Cytoplasmic contamination of the apoplastic extracts, judged by the marker enzyme glucose-6-phosphate dehydrogenase, was very low (less than 2%) even in inoculated plants. Dehydroascorbate, glutathione, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were present in the apoplast. Inoculation had no effect on the total foliar ascorbate pool size or the redox state. The glutathione content of Alg-S leaves and apoplast decreased, whereas that of Alg-R leaves and apoplast increased after pathogen attack, but the redox state was unchanged in both cases. Large increases in foliar catalase activity were observed in Alg-S but not in Alg-R leaves. Pathogen-induced increases in the apoplastic antioxidant enzyme activities were observed. We conclude that sustained oxidation does not occur and that differential strategies of antioxidant response in Alg-S and Alg-R may contribute to pathogen sensitivity. PMID:9662553

  4. Movement of Abscisic Acid into the Apoplast in Response to Water Stress in Xanthium strumarium L.

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    Cornish, K; Zeevaart, J A

    1985-07-01

    The effect of water stress on the redistribution of abcisic acid (ABA) in mature leaves of Xanthium strumarium L. was investigated using a pressure dehydration technique. In both turgid and stressed leaves, the ABA in the xylem exudate, the ;apoplastic' ABA, increased before ;bulk leaf' stress-induced ABA accumulation began. In the initially turgid leaves, the ABA level remained constant in both the apoplast and the leaf as a whole until wilting symptoms appeared. Following turgor loss, sufficient quantities of ABA moved into the apoplast to stimulate stomatal closure. Thus, the initial increase of apoplastic ABA may be relevant to the rapid stomatal closure seen in stressed leaves before their bulk leaf ABA levels rise.Following recovery from water stress, elevated levels of ABA remained in the apoplast after the bulk leaf contents had returned to their prestress values. This apoplastic ABA may retard stomatal reopening during the initial recovery period.

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

    Science.gov (United States)

    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.

  6. Movement of Abscisic Acid into the Apoplast in Response to Water Stress in Xanthium strumarium L. 1

    Science.gov (United States)

    Cornish, Katrina; Zeevaart, Jan A. D.

    1985-01-01

    The effect of water stress on the redistribution of abcisic acid (ABA) in mature leaves of Xanthium strumarium L. was investigated using a pressure dehydration technique. In both turgid and stressed leaves, the ABA in the xylem exudate, the `apoplastic' ABA, increased before `bulk leaf' stress-induced ABA accumulation began. In the initially turgid leaves, the ABA level remained constant in both the apoplast and the leaf as a whole until wilting symptoms appeared. Following turgor loss, sufficient quantities of ABA moved into the apoplast to stimulate stomatal closure. Thus, the initial increase of apoplastic ABA may be relevant to the rapid stomatal closure seen in stressed leaves before their bulk leaf ABA levels rise. Following recovery from water stress, elevated levels of ABA remained in the apoplast after the bulk leaf contents had returned to their prestress values. This apoplastic ABA may retard stomatal reopening during the initial recovery period. PMID:16664294

  7. Ascorbate oxidase-dependent changes in the redox state of the apoplast modulate gene transcript accumulation leading to modified hormone signaling and orchestration of defense processes in tobacco.

    Science.gov (United States)

    Pignocchi, Cristina; Kiddle, Guy; Hernández, Iker; Foster, Simon J; Asensi, Amparo; Taybi, Tahar; Barnes, Jeremy; Foyer, Christine H

    2006-06-01

    The role of the redox state of the apoplast in hormone responses, signaling cascades, and gene expression was studied in transgenic tobacco (Nicotiana tabacum) plants with modified cell wall-localized ascorbate oxidase (AO). High AO activity specifically decreased the ascorbic acid (AA) content of the apoplast and altered plant growth responses triggered by hormones. Auxin stimulated shoot growth only when the apoplastic AA pool was reduced in wild-type or AO antisense lines. Oxidation of apoplastic AA in AO sense lines was associated with loss of the auxin response, higher mitogen-activated protein kinase activities, and susceptibility to a virulent strain of the pathogen Pseudomonas syringae. The total leaf glutathione pool, the ratio of reduced glutathione to glutathione disulfide, and glutathione reductase activities were similar in the leaves of all lines. However, AO sense leaves exhibited significantly lower dehydroascorbate reductase and ascorbate peroxidase activities than wild-type and antisense leaves. The abundance of mRNAs encoding antioxidant enzymes was similar in all lines. However, the day/night rhythms in the abundance of transcripts encoding the three catalase isoforms were changed in response to the AA content of the apoplast. Other transcripts influenced by AO included photorespiratory genes and a plasma membrane Ca(2+) channel-associated gene. We conclude that the redox state of the apoplast modulates plant growth and defense responses by regulating signal transduction cascades and gene expression patterns. Hence, AO activity, which modulates the redox state of the apoplastic AA pool, strongly influences the responses of plant cells to external and internal stimuli.

  8. Apoplastic pH in corn root gravitropism: a laser scanning confocal microscopy measurement

    International Nuclear Information System (INIS)

    Taylor, D.P.; Slattery, J.; Leopold, A.C.

    1996-01-01

    The ability to measure the pH of the apoplast in situ is of special interest as a test of the cell wall acidification theory. Optical sectioning of living seedlings of corn roots using the laser scanning confocal microscope (LSCM) permits us to make pH measurements in living tissue. The pH of the apoplast of corn roots was measured by this method after infiltration with CI-NERF, a pH-sensitive dye, along with Texas Red Dextran 3000, a pH-insensitive dye, as an internal standard. In the elongation zone of corn roots, the mean apoplastic pH was 4.9. Upon gravitropic stimulation, the pH on the convex side of actively bending roots was 4.5. The lowering of the apoplastic pH by 0.4 units appears to be sufficient to account for the increased growth on that side. This technique provides site-specific evidence for the acid growth theory of cell elongation. The LSCM permits measurements of the pH of living tissues, and has a sensitivity of approximately 0.2 pH units. (author)

  9. Proteomic analysis of apoplastic fluid of Coffea arabica leaves highlights novel biomarkers for resistance against Hemileia vastatrix

    Directory of Open Access Journals (Sweden)

    Leonor eGuerra-Guimarães

    2015-06-01

    Full Text Available A proteomic analysis of the apoplastic fluid (APF of coffee leaves was conducted to investigate the cellular processes associated with incompatible (resistant and compatible (susceptible Coffea arabica-Hemileia vastatrix interactions, during the 24-96 hai period. The APF proteins were extracted by leaf vacuum infiltration and protein profiles were obtained by 2-DE. The comparative analysis of the gels revealed 210 polypeptide spots whose volume changed in abundance between samples (control, resistant and susceptible during the 24-96 hai period. The proteins identified were involved mainly in protein degradation, cell wall metabolism and stress/defense responses, most of them being hydrolases (around 70%, particularly sugar hydrolases and peptidases/proteases. The changes in the APF proteome along the infection process revealed two distinct phases of defense responses, an initial/basal one (24-48 hai and a late/specific one (72-96 hai. Compared to susceptibility, resistance was associated with a higher number of proteins, which was more evident in the late/specific phase. Proteins involved in the resistance response were mainly, glycohydrolases of the cell wall, serine proteases and pathogen related-like proteins (PR-proteins, suggesting that some of these proteins could be putative candidates for resistant markers of coffee to H. vastatrix. Antibodies were produced against chitinase, pectin methylesterase, serine carboxypeptidase, reticuline oxidase and subtilase and by an immunodetection assay it was observed an increase of these proteins in the resistant sample. With this methodology we have identified proteins that are candidate markers of resistance and that will be useful in coffee breeding programs to assist in the selection of cultivars with resistance to H. vastatrix.

  10. Autophagy-Related Direct Membrane Import from ER/Cytoplasm into the Vacuole or Apoplast: A Hidden Gateway also for Secondary Metabolites and Phytohormones?

    Directory of Open Access Journals (Sweden)

    Ivan Kulich

    2014-04-01

    Full Text Available Transportation of low molecular weight cargoes into the plant vacuole represents an essential plant cell function. Several lines of evidence indicate that autophagy-related direct endoplasmic reticulum (ER to vacuole (and also, apoplast transport plays here a more general role than expected. This route is regulated by autophagy proteins, including recently discovered involvement of the exocyst subcomplex. Traffic from ER into the vacuole bypassing Golgi apparatus (GA acts not only in stress-related cytoplasm recycling or detoxification, but also in developmentally-regulated biopolymer and secondary metabolite import into the vacuole (or apoplast, exemplified by storage proteins and anthocyanins. We propose that this pathway is relevant also for some phytohormones’ (e.g., auxin, abscisic acid (ABA and salicylic acid (SA degradation. We hypothesize that SA is not only an autophagy inducer, but also a cargo for autophagy-related ER to vacuole membrane container delivery and catabolism. ER membrane localized enzymes will potentially enhance the area of biosynthetic reactive surfaces, and also, abundant ER localized membrane importers (e.g., ABC transporters will internalize specific molecular species into the autophagosome biogenesis domain of ER. Such active ER domains may create tubular invaginations of tonoplast into the vacuoles as import intermediates. Packaging of cargos into the ER-derived autophagosome-like containers might be an important mechanism of vacuole and exosome biogenesis and cytoplasm protection against toxic metabolites. A new perspective on metabolic transformations intimately linked to membrane trafficking in plants is emerging.

  11. Phenotypic analysis of apoplastic effectors from the phytopathogenic nematode, Globodera rostochiensis demonstrates that an expansin can induce and suppress host defenses

    Science.gov (United States)

    The potato cyst nematode Globodera rostochiensis (Woll.) is an important pest of potato. Like other biotrophic pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm to successfully infect their hosts. We have identifie...

  12. POLYGALACTURONASE INVOLVED IN EXPANSION1 functions in cell elongation and flower development in Arabidopsis.

    Science.gov (United States)

    Xiao, Chaowen; Somerville, Chris; Anderson, Charles T

    2014-03-01

    Pectins are acidic carbohydrates that comprise a significant fraction of the primary walls of eudicotyledonous plant cells. They influence wall porosity and extensibility, thus controlling cell and organ growth during plant development. The regulated degradation of pectins is required for many cell separation events in plants, but the role of pectin degradation in cell expansion is poorly defined. Using an activation tag screen designed to isolate genes involved in wall expansion, we identified a gene encoding a putative polygalacturonase that, when overexpressed, resulted in enhanced hypocotyl elongation in etiolated Arabidopsis thaliana seedlings. We named this gene POLYGALACTURONASE INVOLVED IN EXPANSION1 (PGX1). Plants lacking PGX1 display reduced hypocotyl elongation that is complemented by transgenic PGX1 expression. PGX1 is expressed in expanding tissues throughout development, including seedlings, roots, leaves, and flowers. PGX1-GFP (green fluorescent protein) localizes to the apoplast, and heterologously expressed PGX1 displays in vitro polygalacturonase activity, supporting a function for this protein in apoplastic pectin degradation. Plants either overexpressing or lacking PGX1 display alterations in total polygalacturonase activity, pectin molecular mass, and wall composition and also display higher proportions of flowers with extra petals, suggesting PGX1's involvement in floral organ patterning. These results reveal new roles for polygalacturonases in plant development.

  13. Protein Dynamics in the Plant Extracellular Space

    Directory of Open Access Journals (Sweden)

    Leonor Guerra-Guimarães

    2016-07-01

    Full Text Available The extracellular space (ECS or apoplast is the plant cell compartment external to the plasma membrane, which includes the cell walls, the intercellular space and the apoplastic fluid (APF. The present review is focused on APF proteomics papers and intends to draw information on the metabolic processes occurring in the ECS under abiotic and biotic stresses, as well as under non-challenged conditions. The large majority of the proteins detected are involved in “cell wall organization and biogenesis”, “response to stimulus” and “protein metabolism”. It becomes apparent that some proteins are always detected, irrespective of the experimental conditions, although with different relative contribution. This fact suggests that non-challenged plants have intrinsic constitutive metabolic processes of stress/defense in the ECS. In addition to the multiple functions ascribed to the ECS proteins, should be considered the interactions established between themselves and with the plasma membrane and its components. These interactions are crucial in connecting exterior and interior of the cell, and even simple protein actions in the ECS can have profound effects on plant performance. The proteins of the ECS are permanently contributing to the high dynamic nature of this plant compartment, which seems fundamental to plant development and adaptation to the environmental conditions.

  14. The Physiological and Biochemical Mechanisms Providing the Increased Constitutive Cold Resistance in the Potato Plants, Expressing the Yeast SUC2 Gene Encoding Apoplastic Invertase

    Directory of Open Access Journals (Sweden)

    A.N. Deryabin

    2016-05-01

    Full Text Available The expression of heterologous genes in plants is an effective method to improve our understanding of plant resistance mechanisms. The purpose of this work was to investigate the involvement of cell-wall invertase and apoplastic sugars into constitutive cold resistance of potato (Solanum tuberosum L., cv. Dйsirйe plants, which expressed the yeast SUC2 gene encoding apoplastic invertase. WT-plants of a potato served as the control. The increase in the essential cell-wall invertase activity in the leaves of transformed plants indicates significant changes in the cellular carbohydrate metabolism and regulatory function of this enzyme. The activity of yeast invertase changed the composition of intracellular sugars in the leaves of the transformed potato plant. The total content of sugars (sucrose, glucose, fructose in the leaves and apoplast was higher in the transformants, in comparison by WT-plants. Our data indicate higher constitutive resistance of transformants to severe hypothermia conditions compared to WT-plants. This fact allows us to consider cell-wall invertase as a enzyme of carbohydrate metabolism playing an important regulatory role in the metabolic signaling upon forming increased plant resistance to low temperature. Thus, the potato line with the integrated SUC2 gene is a convenient tool to study the role of the apoplastic invertase and the products of its activity during growth, development and formation constitutive resistance to hypothermia.

  15. DspA/E contributes to apoplastic accumulation of ROS in nonhost A. thaliana

    Directory of Open Access Journals (Sweden)

    Alban eLaunay

    2016-04-01

    Full Text Available The bacterium Erwinia amylovora is responsible for the fire blight disease of Maleae, which provokes necrotic symptoms on aerial parts. The pathogenicity of this bacterium in hosts relies on its type three-secretion system (T3SS, a molecular syringe that allows the bacterium to inject effectors into the plant cell. E. amylovora-triggered disease in host plants is associated with the T3SS-dependent production of reactive oxygen species (ROS, although ROS are generally associated with resistance in other pathosystems. We showed previously that E. amylovora can multiply transiently in the nonhost plant Arabidopsis thaliana and that a T3SS-dependent production of intracellular ROS occurs during this interaction. In the present work we characterize the localization and source of hydrogen peroxide accumulation following E. amylovora infection. Transmission electron microscope (TEM analysis of infected tissues showed that hydrogen peroxide accumulation occurs in the cytosol, plastids, peroxisomes, and mitochondria as well as in the apoplast. Furthermore, TEM analysis showed that an E. amylovora dspA/E-deficient strain does not induce hydrogen peroxide accumulation in the apoplast. Consistently, a transgenic line expressing DspA/E accumulated ROS in the apoplast. The NADPH oxidase-deficient rbohD mutant showed a very strong reduction in hydrogen peroxide accumulation in response to E. amylovora inoculation. However, we did not find an increase in bacterial titers of E. amylovora in the rbohD mutant and the rbohD mutation did not suppress the toxicity of DspA/E when introgressed into a DspA/E-expressing transgenic line. Co-inoculation of E. amylovora with cycloheximide (CHX, which we found previously to suppress callose deposition and allow strong multiplication of E. amylovora in A. thaliana leaves, led to a strong reduction of apoplastic ROS accumulation but did not affect intracellular ROS. Our data strongly suggest that apoplastic ROS accumulation is

  16. Apoplastic and intracellular plant sugars regulate developmental transitions in witches’ broom disease of cacao

    Science.gov (United States)

    Barau, Joan; Grandis, Adriana; Carvalho, Vinicius Miessler de Andrade; Teixeira, Gleidson Silva; Zaparoli, Gustavo Henrique Alcalá; do Rio, Maria Carolina Scatolin; Rincones, Johana; Buckeridge, Marcos Silveira; Pereira, Gonçalo Amarante Guimarães

    2015-01-01

    Witches’ broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant–fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation. PMID:25540440

  17. Ozone tolerance in snap bean is associated with elevated ascorbic acid in the leaf apoplast

    Energy Technology Data Exchange (ETDEWEB)

    Burkey, K.O. [North Carolina State Univ., United States Dept. of Agriculture-Agricultural Research Service, and Dept. of Crop Science, Raleigh, NC (United States); Eason, G. [North Carolina, State Univ., United States Dept. of Plant Pathology, Raleigh, NC (United States)

    2002-03-01

    Ascorbic acid (AA) in the leaf apoplast has the potential to limit ozone injury by participating in reactions that detoxify ozone and reactive oxygen intermediates and thus prevent plasma membrane damage. Genotypes of snap bean (Phaseolus vulgaris L) were compared in controlled environments and in open-top field chambers to assess the relationship between extracellular AA content and ozone tolerance. Vacuum infiltration methods were employed to separate leaf AA into extracellular and intracellular fractions. For plants grown in controlled environments at low ozone concentration (4 nmol mol{sup -1} ozone), leaf apoplast AA was significantly higher in tolerant genotypes (300-400 nmol g{sup -1} FW) compared with sensitive genotypes (approximately 50 nmol g{sup -1} FW), evidence that ozone tolerance is associated with elevated extracellular AA. For the open top chamber study, plants were grown in pots under charcoal-filtered air (CF) conditions and then either maintained under CF conditions (29 nmol mol{sup -1} ozone) or exposed to elevated ozone (67 nmol mol{sup -1} ozone). Following an 8-day treatment period, leaf apoplast AA was in the range of 100-190 nmol g{sup -1} FW for all genotypes, but no relationship was observed between apoplast AA content and ozone tolerance. The contrasting results in the two studies demonstrated a potential limitation in the interpretation of extracellular AA data. Apoplast AA levels presumably reflect the steady-state condition between supply from the cytoplasm and utilization within the cell wall. The capacity to detoxify ozone in the extracellular space may be underestimated under elevated ozone conditions where the dynamics of AA supply and utilization are not adequately represented by a steady-state measurement. (au)

  18. Apoplastic ascorbate contributes to the differential ozone sensitivity in two varieties of winter wheat under fully open-air field conditions

    International Nuclear Information System (INIS)

    Feng Zhaozhong; Pang Jing; Nouchi, Isamu; Kobayashi, Kazuhiko; Yamakawa, Takashi; Zhu Jianguo

    2010-01-01

    We studied leaf apoplastic ascorbates in relation to ozone (O 3 ) sensitivity in two winter wheat (Triticum aestivum L.) varieties: Yangfumai 2 (Y2) and Yangmai 16 (Y16). The plants were exposed to elevated O 3 concentration 27% higher than the ambient O 3 concentration in a fully open-air field from tillering stage until final maturity. The less sensitive variety Y16 had higher concentration of reduced ascorbate in the apoplast and leaf tissue by 33.5% and 12.0%, respectively, than those in the more sensitive variety Y2, whereas no varietal difference was detected in the decline of reduced ascorbate concentration in response to elevated O 3 . No effects of O 3 or variety were detected in either oxidized ascorbate or the redox state of ascorbate in the apoplast and leaf tissue. The lower ascorbate concentrations in both apoplast and leaf tissue should have contributed to the higher O 3 sensitivity in variety Y2. - Apoplastic ascorbate contributes to varietal difference in wheat tolerance to O 3 .

  19. Apoplastic and intracellular plant sugars regulate developmental transitions in witches' broom disease of cacao.

    Science.gov (United States)

    Barau, Joan; Grandis, Adriana; Carvalho, Vinicius Miessler de Andrade; Teixeira, Gleidson Silva; Zaparoli, Gustavo Henrique Alcalá; do Rio, Maria Carolina Scatolin; Rincones, Johana; Buckeridge, Marcos Silveira; Pereira, Gonçalo Amarante Guimarães

    2015-03-01

    Witches' broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant-fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  20. The Arabidopsis homolog of human G3BP1 is a key regulator of stomatal and apoplastic immunity

    KAUST Repository

    Abulfaraj, Aala A.; Mariappan, Kiruthiga; Bigeard, Jean; Manickam, Prabhu; Blilou, Ikram; Guo, Xiujie; Al-Babili, Salim; Pflieger, Delphine; Hirt, Heribert; Rayapuram, Naganand

    2018-01-01

    Mammalian Ras-GTPase–activating protein SH3-domain–binding proteins (G3BPs) are a highly conserved family of RNA-binding proteins that link kinase receptor-mediated signaling to RNA metabolism. Mammalian G3BP1 is a multifunctional protein that functions in viral immunity. Here, we show that the Arabidopsis thaliana homolog of human G3BP1 negatively regulates plant immunity. Arabidopsis g3bp1 mutants showed enhanced resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato. Pathogen resistance was mediated in Atg3bp1 mutants by altered stomatal and apoplastic immunity. Atg3bp1 mutants restricted pathogen entry into stomates showing insensitivity to bacterial coronatine–mediated stomatal reopening. AtG3BP1 was identified as a negative regulator of defense responses, which correlated with moderate up-regulation of salicylic acid biosynthesis and signaling without growth penalty.

  1. The Arabidopsis homolog of human G3BP1 is a key regulator of stomatal and apoplastic immunity

    KAUST Repository

    Abulfaraj, Aala Abdulaziz Hussien

    2018-05-31

    Mammalian Ras-GTPase–activating protein SH3-domain–binding proteins (G3BPs) are a highly conserved family of RNA-binding proteins that link kinase receptor-mediated signaling to RNA metabolism. Mammalian G3BP1 is a multifunctional protein that functions in viral immunity. Here, we show that the Arabidopsis thaliana homolog of human G3BP1 negatively regulates plant immunity. Arabidopsis g3bp1 mutants showed enhanced resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato. Pathogen resistance was mediated in Atg3bp1 mutants by altered stomatal and apoplastic immunity. Atg3bp1 mutants restricted pathogen entry into stomates showing insensitivity to bacterial coronatine–mediated stomatal reopening. AtG3BP1 was identified as a negative regulator of defense responses, which correlated with moderate up-regulation of salicylic acid biosynthesis and signaling without growth penalty.

  2. Early changes of the pH of the apoplast are different in leaves, stem and roots of Vicia faba L. under declining water availability.

    Science.gov (United States)

    Karuppanapandian, T; Geilfus, C-M; Mühling, K-H; Novák, O; Gloser, V

    2017-02-01

    Changes in pH of the apoplast have recently been discussed as an important factor in adjusting transpiration and water relations under conditions of drought via modulatory effect on abscisic acid (ABA) concentration. Using Vicia faba L., we investigated whether changes in the root, shoot and leaf apoplastic pH correlated with (1) a drought-induced reduction in transpiration and with (2) changes in ABA concentration. Transpiration, leaf water potential and ABA in leaves were measured and correlated with root and shoot xylem pH, determined by a pH microelectrode, and pH of leaf apoplast quantified by microscopy-based in vivo ratiometric analysis. Results revealed that a reduction in transpiration rate in the early phase of soil drying could not be linked with changes in the apoplastic pH via effects on the stomata-regulating hormone ABA. Moreover, drought-induced increase in pH of xylem or leaf apoplast was not the remote effect of an acropetal transport of alkaline sap from root, because root xylem acidified during progressive soil drying, whereas the shoot apoplast alkalized. We reason that other, yet unknown signalling mechanism was responsible for reduction of transpiration rate in the early phase of soil drying. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. The Craterostigma plantagineum glycine-rich protein CpGRP1 interacts with a cell wall-associated protein kinase 1 (CpWAK1) and accumulates in leaf cell walls during dehydration.

    Science.gov (United States)

    Giarola, Valentino; Krey, Stephanie; von den Driesch, Barbara; Bartels, Dorothea

    2016-04-01

    Craterostigma plantagineum tolerates extreme desiccation. Leaves of this plant shrink and extensively fold during dehydration and expand again during rehydration, preserving their structural integrity. Genes were analysed that may participate in the reversible folding mechanism. Analysis of transcripts abundantly expressed in desiccated leaves identified a gene putatively coding for an apoplastic glycine-rich protein (CpGRP1). We studied the expression, regulation and subcellular localization of CpGRP1 and its ability to interact with a cell wall-associated protein kinase (CpWAK1) to understand the role of CpGRP1 in the cell wall during dehydration. The CpGRP1 protein accumulates in the apoplast of desiccated leaves. Analysis of the promoter revealed that the gene expression is mainly regulated at the transcriptional level, is independent of abscisic acid (ABA) and involves a drought-responsive cis-element (DRE). CpGRP1 interacts with CpWAK1 which is down-regulated in response to dehydration. Our data suggest a role of the CpGRP1-CpWAK1 complex in dehydration-induced morphological changes in the cell wall during dehydration in C. plantagineum. Cell wall pectins and dehydration-induced pectin modifications are predicted to be involved in the activity of the CpGRP1-CpWAK1 complex. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  4. The potato-specific apyrase is apoplastically localized and has influence on gene expression, growth, and development.

    Science.gov (United States)

    Riewe, David; Grosman, Lukasz; Fernie, Alisdair R; Wucke, Cornelia; Geigenberger, Peter

    2008-07-01

    Apyrases hydrolyze nucleoside triphosphates and diphosphates and are found in all eukaryotes and a few prokaryotes. Although their enzymatic properties have been well characterized, relatively little is known regarding their subcellular localization and physiological function in plants. In this study, we used reverse genetic and biochemical approaches to investigate the role of potato (Solanum tuberosum)-specific apyrase. Silencing of the apyrase gene family with RNA interference constructs under the control of the constitutive 35S promoter led to a strong decrease in apyrase activity to below 10% of the wild-type level. This decreased activity led to phenotypic changes in the transgenic lines, including a general retardation in growth, an increase in tuber number per plant, and differences in tuber morphology. Silencing of apyrase under the control of a tuber-specific promoter led to similar changes in tuber morphology; however, there were no direct effects of apyrase inhibition on tuber metabolism. DNA microarrays revealed that decreased expression of apyrase leads to increased levels of transcripts coding for cell wall proteins involved in growth and genes involved in energy transfer and starch synthesis. To place these results in context, we determined the subcellular localization of the potato-specific apyrase. Using a combination of approaches, we were able to demonstrate that this enzyme is localized to the apoplast. We describe the evidence that underlies both this fact and that potato-specific apyrase has a crucial role in regulating growth and development.

  5. Cotton Ascorbate Oxidase Promotes Cell Growth in Cultured Tobacco Bright Yellow-2 Cells through Generation of Apoplast Oxidation

    Science.gov (United States)

    Li, Rong; Xin, Shan; Tao, Chengcheng; Jin, Xiang; Li, Hongbin

    2017-01-01

    Ascorbate oxidase (AO) plays an important role in cell growth through the modulation of reduction/oxidation (redox) control of the apoplast. Here, a cotton (Gossypium hirsutum) apoplastic ascorbate oxidase gene (GhAO1) was obtained from fast elongating fiber tissues. GhAO1 belongs to the multicopper oxidase (MCO) family and includes a signal peptide and several transmembrane regions. Analyses of quantitative real-time polymerase chain reaction (QRT-PCR) and enzyme activity showed that GhAO1 was expressed abundantly in 15-day post-anthesis (dpa) wild-type (WT) fibers in comparison with fuzzless-lintless (fl) mutant ovules. Subcellular distribution analysis in onion cells demonstrated that GhAO1 is localized in the cell wall. In transgenic tobacco bright yellow-2 (BY-2) cells with ectopic overexpression of GhAO1, the enhancement of cell growth with 1.52-fold increase in length versus controls was indicated, as well as the enrichment of both total ascorbate in whole-cells and dehydroascorbate acid (DHA) in apoplasts. In addition, promoted activities of AO and monodehydroascorbate reductase (MDAR) in apoplasts and dehydroascorbate reductase (DHAR) in whole-cells were displayed in transgenic tobacco BY-2 cells. Accumulation of H2O2, and influenced expressions of Ca2+ channel genes with the activation of NtMPK9 and NtCPK5 and the suppression of NtTPC1B were also demonstrated in transgenic tobacco BY-2 cells. Finally, significant induced expression of the tobacco NtAO gene in WT BY-2 cells under indole-3-acetic acid (IAA) treatment appeared; however, the sensitivity of the NtAO gene expression to IAA disappeared in transgenic BY-2 cells, revealing that the regulated expression of the AO gene is under the control of IAA. Taken together, these results provide evidence that GhAO1 plays an important role in fiber cell elongation and may promote cell growth by generating the oxidation of apoplasts, via the auxin-mediated signaling pathway. PMID:28644407

  6. Arabinogalactan Proteins Are Involved in Salt-Adaptation and Vesicle Trafficking in Tobacco by-2 Cell Cultures.

    Science.gov (United States)

    Olmos, Enrique; García De La Garma, Jesús; Gomez-Jimenez, Maria C; Fernandez-Garcia, Nieves

    2017-01-01

    Arabinogalactan proteins (AGPs) are a highly diverse family of glycoproteins that are commonly found in most plant species. However, little is known about the physiological and molecular mechanisms of their function. AGPs are involved in different biological processes such as cell differentiation, cell expansion, tissue development and somatic embryogenesis. AGPs are also involved in abiotic stress response such as salinity modulating cell wall expansion. In this study, we describe how salt-adaptation in tobacco BY-2 cell cultures induces important changes in arabinogalactan proteins distribution and contents. Using the immuno-dot blot technique with different anti-AGP antibodies (JIM13, JIM15, and others), we observed that AGPs were highly accumulated in the culture medium of salt-adapted tobacco cells, probably due to the action of phospholipases. We located these AGP epitopes using immunogold labeling in the cytoplasm associated to the endoplasmic reticulum, the golgi apparatus, and vesicles, plasma membrane and tonoplast. Our results show that salt-adaptation induced a significant reduction of the cytoplasm, plasma membrane and tonoplast content of these epitopes. Yariv reagent was added to the control and salt-adapted tobacco cell cultures, leading to cell death induction in control cells but not in salt-adapted cells. Ultrastructural and immunogold labeling revealed that cell death induced by Yariv reagent in control cells was due to the interaction of Yariv reagent with the AGPs linked to the plasma membranes. Finally, we propose a new function of AGPs as a possible sodium carrier through the mechanism of vesicle trafficking from the apoplast to the vacuoles in salt-adapted tobacco BY-2 cells. This mechanism may contribute to sodium homeostasis during salt-adaptation to high saline concentrations.

  7. A peroxidase-dependent apoplastic oxidative burst in cultured Arabidopsis cells functions in MAMP-elicited defense.

    Science.gov (United States)

    O'Brien, Jose A; Daudi, Arsalan; Finch, Paul; Butt, Vernon S; Whitelegge, Julian P; Souda, Puneet; Ausubel, Frederick M; Bolwell, G Paul

    2012-04-01

    Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). We previously identified two cell wall peroxidases, PRX33 and PRX34, involved in apoplastic hydrogen peroxide (H2O2) production in Arabidopsis (Arabidopsis thaliana). Here, we describe the generation of Arabidopsis tissue culture lines in which the expression of PRX33 and PRX34 is knocked down by antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase cDNA construct. Using these tissue culture lines and two inhibitors of ROS generation, azide and diphenylene iodonium, we found that perxoxidases generate about half of the H2O2 that accumulated in response to MAMP treatment and that NADPH oxidases and other sources such as mitochondria account for the remainder of the ROS. Knockdown of PRX33/PRX34 resulted in decreased expression of several MAMP-elicited genes, including MYB51, CYP79B2, and CYP81F2. Similarly, proteomic analysis showed that knockdown of PRX33/PRX34 led to the depletion of various MAMP-elicited defense-related proteins, including the two cysteine-rich peptides PDF2.2 and PDF2.3. Knockdown of PRX33/PRX34 also led to changes in the cell wall proteome, including increases in enzymes involved in cell wall remodeling, which may reflect enhanced cell wall expansion as a consequence of reduced H2O2-mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars. Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of pattern-triggered immunity in tissue culture cells.

  8. Cell-specific vacuolar calcium storage mediated by CAX1 regulates apoplastic calcium concentration, gas exchange, and plant productivity in Arabidopsis.

    Science.gov (United States)

    Conn, Simon J; Gilliham, Matthew; Athman, Asmini; Schreiber, Andreas W; Baumann, Ute; Moller, Isabel; Cheng, Ning-Hui; Stancombe, Matthew A; Hirschi, Kendal D; Webb, Alex A R; Burton, Rachel; Kaiser, Brent N; Tyerman, Stephen D; Leigh, Roger A

    2011-01-01

    The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis 60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca(2+) transporters, CAX1 (Ca(2+)/H(+)-antiporter), ACA4, and ACA11 (Ca(2+)-ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO(2) assimilation, and leaf growth rate; increased transcript abundance of other Ca(2+) transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca(2+)], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca(2+)] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.

  9. Separation of abscission zone cells in detached Azolla roots depends on apoplastic pH.

    Science.gov (United States)

    Fukuda, Kazuma; Yamada, Yoshiya; Miyamoto, Kensuke; Ueda, Junichi; Uheda, Eiji

    2013-01-01

    In studies on the mechanism of cell separation during abscission, little attention has been paid to the apoplastic environment. We found that the apoplastic pH surrounding abscission zone cells in detached roots of the water fern Azolla plays a major role in cell separation. Abscission zone cells of detached Azolla roots were separated rapidly in a buffer at neutral pH and slowly in a buffer at pH below 4.0. However, cell separation rarely occurred at pH 5.0-5.5. Light and electron microscopy revealed that cell separation was caused by a degradation of the middle lamella between abscission zone cells at both pH values, neutral and below 4.0. Low temperature and papain treatment inhibited cell separation. Enzyme(s) in the cell wall of the abscission zone cells might be involved in the degradation of the pectin of the middle lamella and the resultant, pH-dependent cell separation. By contrast, in Phaseolus leaf petioles, unlike Azolla roots, cell separation was slow and increased only at acidic pH. The rapid cell separation, as observed in Azolla roots at neutral pH, did not occur. Indirect immunofluorescence microscopy, using anti-pectin monoclonal antibodies, revealed that the cell wall pectins of the abscission zone cells of Azolla roots and Phaseolus leaf petioles looked similar and changed similarly during cell separation. Thus, the pH-related differences in cell separation mechanisms of Azolla and Phaseolus might not be due to differences in cell wall pectin, but to differences in cell wall-located enzymatic activities responsible for the degradation of pectic substances. A possible enzyme system is discussed. Copyright © 2012 Elsevier GmbH. All rights reserved.

  10. Silicon alleviates iron deficiency in cucumber by promoting mobilization of iron in the root apoplast

    DEFF Research Database (Denmark)

    Pavlovic, Jelena; Samardzic, Jelena; Maksimović, Vuk

    2013-01-01

    Root responses to lack of iron (Fe) have mainly been studied in nutrient solution experiments devoid of silicon (Si). Here we investigated how Si ameliorates Fe deficiency in cucumber (Cucumis sativus) with focus on the storage and utilization of Fe in the root apoplast. A combined approach...

  11. Plant natriuretic peptides are apoplastic and paracrine stress response molecules

    KAUST Repository

    Wang, Yuhua

    2011-04-07

    Higher plants contain biologically active proteins that are recognized by antibodies against human atrial natriuretic peptide (ANP). We identified and isolated two Arabidopsis thaliana immunoreactive plant natriuretic peptide (PNP)-encoding genes, AtPNP-A and AtPNP-B, which are distantly related members of the expansin superfamily and have a role in the regulation of homeostasis in abiotic and biotic stresses, and have shown that AtPNP-A modulates the effects of ABA on stomata. Arabidopsis PNP (PNP-A) is mainly expressed in leaf mesophyll cells, and in protoplast assays we demonstrate that it is secreted using AtPNP-A:green fluorescent protein (GFP) reporter constructs and flow cytometry. Transient reporter assays provide evidence that AtPNP-A expression is enhanced by heat, osmotica and salt, and that AtPNP-A itself can enhance its own expression, thereby generating a response signature diagnostic for paracrine action and potentially also autocrine effects. Expression of native AtPNP-A is enhanced by osmotica and transiently by salt. Although AtPNP-A expression is induced by salt and osmotica, ABA does not significantly modulate AtPNP-A levels nor does recombinant AtPNP-A affect reporter expression of the ABA-responsive RD29A gene. Together, these results provide experimental evidence that AtPNP-A is stress responsive, secreted into the apoplastic space and can enhance its own expression. Furthermore, our findings support the idea that AtPNP-A, together with ABA, is an important component in complex plant stress responses and that, much like in animals, peptide signaling molecules can create diverse and modular signals essential for growth, development and defense under rapidly changing environmental conditions. © 2011 The Author.

  12. Association between photosynthesis and contrasting features of minor veins in leaves of summer annuals loading phloem via symplastic versus apoplastic routes.

    Science.gov (United States)

    Muller, Onno; Cohu, Christopher M; Stewart, Jared J; Protheroe, Johanna A; Demmig-Adams, Barbara; Adams, William W

    2014-09-01

    Foliar vascular anatomy and photosynthesis were evaluated for a number of summer annual species that either load sugars into the phloem via a symplastic route (Cucumis sativus L. cv. Straight Eight; Cucurbita pepo L. cv. Italian Zucchini Romanesco; Citrullus lanatus L. cv. Faerie Hybrid; Cucurbita pepo L. cv. Autumn Gold) or an apoplastic route (Nicotiana tabacum L.; Solanum lycopersicum L. cv. Brandywine; Gossypium hirsutum L.; Helianthus annuus L. cv. Soraya), as well as winter annual apoplastic loaders (Spinacia oleracea L. cv. Giant Nobel; Arabidopsis thaliana (L.) Heynhold Col-0, Swedish and Italian ecotypes). For all summer annuals, minor vein cross-sectional xylem area and tracheid number as well as the ratio of phloem loading cells to phloem sieve elements, each when normalized for foliar vein density (VD), was correlated with photosynthesis. These links presumably reflect (1) the xylem's role in providing water to meet foliar transpirational demand supporting photosynthesis and (2) the importance of the driving force of phloem loading as well as the cross-sectional area for phloem sap flux to match foliar photosynthate production. While photosynthesis correlated with the product of VD and cross-sectional phloem cell area among symplastic loaders, photosynthesis correlated with the product of VD and phloem cell number per vein among summer annual apoplastic loaders. Phloem cell size has thus apparently been a target of selection among symplastic loaders (where loading depends on enzyme concentration within loading cells) versus phloem cell number among apoplastic loaders (where loading depends on membrane transporter numbers). © 2014 Scandinavian Plant Physiology Society.

  13. The redox state of the apoplast influences the acclimation of photosynthesis and leaf metabolism to changing irradiance.

    Science.gov (United States)

    Karpinska, Barbara; Zhang, Kaiming; Rasool, Brwa; Pastok, Daria; Morris, Jenny; Verrall, Susan R; Hedley, Pete E; Hancock, Robert D; Foyer, Christine H

    2018-05-01

    The redox state of the apoplast is largely determined by ascorbate oxidase (AO) activity. The influence of AO activity on leaf acclimation to changing irradiance was explored in wild-type (WT) and transgenic tobacco (Nicotiana tobaccum) lines containing either high [pumpkin AO (PAO)] or low [tobacco AO (TAO)] AO activity at low [low light (LL); 250 μmol m -2  s -1 ] and high [high light (HL); 1600 μmol m -2  s -1 ] irradiance and following the transition from HL to LL. AO activities changed over the photoperiod, particularly in the PAO plants. AO activity had little effect on leaf ascorbate, which was significantly higher under HL than under LL. Apoplastic ascorbate/dehydroascorbate (DHA) ratios and threonate levels were modified by AO activity. Despite decreased levels of transcripts encoding ascorbate synthesis enzymes, leaf ascorbate increased over the first photoperiod following the transition from HL to LL, to much higher levels than LL-grown plants. Photosynthesis rates were significantly higher in the TAO leaves than in WT or PAO plants grown under HL but not under LL. Sub-sets of amino acids and fatty acids were lower in TAO and WT leaves than in the PAO plants under HL, and following the transition to LL. Light acclimation processes are therefore influenced by the apoplastic as well as chloroplastic redox state. © 2017 John Wiley & Sons Ltd.

  14. Localization of acid phosphatase activity in the apoplast of root nodules of pea (Pisum sativum

    Directory of Open Access Journals (Sweden)

    Marzena Sujkowska

    2011-01-01

    Full Text Available Changes in the activity of acid phosphatase (AcPase in the apoplast of pea root nodule were investigated. The activity was determined using lead and cerium methods. The results indicated a following sequence of AcPase activity appearance during the development of the infection thread: 1 low AcPase activity appears in the outer part of cells of symbiotic bacteria; 2 bacteria show increased AcPase activity, and the enzyme activity appears in the thread walls; 3 activity exhibits also matrix of the infection thread; 4 bacteria just before their release from the infection threads show high AcPase activity; 5 AcPase activity ceases after bacteria transformation into bacteroids. The increase in bacterial AcPase activity may reflect a higher demand for inorganic phosphorus necessary for propagation of the bacteria within the infection threads and/or involved in bacteria release from the infection threads.

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

    Science.gov (United States)

    Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins that are delivered to the apoplast, as well as...

  16. Abnormal expression of leiomyoma cytoskeletal proteins involved in cell migration.

    Science.gov (United States)

    Ura, Blendi; Scrimin, Federica; Arrigoni, Giorgio; Athanasakis, Emmanouil; Aloisio, Michelangelo; Monasta, Lorenzo; Ricci, Giuseppe

    2016-05-01

    Uterine leiomyomas are monoclonal tumors. Several factors are involved in the neoplastic transformation of the myometrium. In our study we focused on dysregulated cytoskeletal proteins in the leiomyoma as compared to the myometrium. Paired tissue samples of ten leiomyomas and adjacent myometria were obtained and analyzed by two‑dimensional gel electrophoresis (2-DE). Mass spectrometry was used for protein identification, and western blotting for 2-DE data validation. The values of ten cytoskeletal proteins were found to be significantly different: eight proteins were upregulated in the leiomyoma and two proteins were downregulated. Three of the upregulated proteins (myosin regulatory light polypeptide 9, four and a half LIM domains protein 1 and LIM and SH3 domain protein 1) are involved in cell migration, while downregulated protein transgelin is involved in replicative senescence. Myosin regulatory light polypeptide 9 (MYL9) was further validated by western blotting because it is considered to be a cell migration marker in several cancers and could play a key role in leiomyoma development. Our data demonstrate significant alterations in the expression of cytoskeletal proteins involved in leiomyoma growth. A better understanding of the involvement of cytoskeletal proteins in leiomyoma pathogenesis may contribute to the identification of new therapeutic targets and the development of new pharmacological approaches.

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

    Directory of Open Access Journals (Sweden)

    Daniela Marasco

    2015-04-01

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

  18. Autohydrolysis of plant xylans by apoplastic expression of thermophilic bacterial endo-xylanases

    DEFF Research Database (Denmark)

    Borkhardt, Bernhard; Harholt, Jesper; Ulvskov, Peter Bjarne

    2010-01-01

    The genes encoding the two endo-xylanases XynA and XynB from the thermophilic bacterium Dictyoglomus thermophilum were codon optimized for expression in plants. Both xylanases were designed to be constitutively expressed under the control of the CaMV 35S promoter and targeted to the apoplast....... Transient expression in tobacco and stable expression in transgenic Arabidopsis showed that both enzymes were expressed in an active form with temperature optima at 85 °C. Transgenic Arabidopsis accumulating heterologous endo-xylanases appeared phenotypically normal and were fully fertile. The highest...... xylanase activity in Arabidopsis was found in dry stems indicating that the enzymes were not degraded during stem senescence. High levels of enzyme activity were maintained in cell-free extracts from dry transgenic stems during incubation at 85 °C for 24 h. Analysis of cell wall polysaccharides after heat...

  19. Papaya pulp gelling: is it premature ripening or problems of water accumulation in the apoplast?

    Directory of Open Access Journals (Sweden)

    Jurandi Gonçalves de Oliveira

    Full Text Available Gelled aspect in papaya fruit is typically confused with premature ripening. This research reports the characterization of this physiological disorder in the pulp of papaya fruit by measuring electrolyte leakage, Pi content, lipid peroxidation, pulp firmness, mineral contents (Ca, Mg and K - in pulp and seed tissues, and histological analysis of pulp tissue. The results showed that the gelled aspect of the papaya fruit pulp is not associated with tissue premature ripening. Data indicate a reduction of the vacuole water intake as the principal cause of the loss of cellular turgor; while the waterlogged aspect of the tissue may be due to water accumulation in the apoplast.

  20. Urea retranslocation from senescing Arabidopsis leaves is promoted by DUR3-mediated urea retrieval from leaf apoplast

    Science.gov (United States)

    Bohner, Anne; Kojima, Soichi; Hajirezaei, Mohammad; Melzer, Michael; von Wirén, Nicolaus

    2015-01-01

    In plants, urea derives either from root uptake or protein degradation. Although large quantities of urea are released during senescence, urea is mainly seen as a short-lived nitrogen (N) catabolite serving urease-mediated hydrolysis to ammonium. Here, we investigated the roles of DUR3 and of urea in N remobilization. During natural leaf senescence urea concentrations and DUR3 transcript levels showed a parallel increase with senescence markers like ORE1 in a plant age- and leaf age-dependent manner. Deletion of DUR3 decreased urea accumulation in leaves, whereas the fraction of urea lost to the leaf apoplast was enhanced. Under natural and N deficiency-induced senescence DUR3 promoter activity was highest in the vasculature, but was also found in surrounding bundle sheath and mesophyll cells. An analysis of petiole exudates from wild-type leaves revealed that N from urea accounted for >13% of amino acid N. Urea export from senescent leaves further increased in ureG-2 deletion mutants lacking urease activity. In the dur3 ureG double insertion line the absence of DUR3 reduced urea export from leaf petioles. These results indicate that urea can serve as an early metabolic marker for leaf senescence, and that DUR3-mediated urea retrieval contributes to the retranslocation of N from urea during leaf senescence. PMID:25440717

  1. Apoplastic Venom Allergen-like Proteins of Cyst Nematodes Modulate the Activation of Basal Plant Innate Immunity by Cell Surface Receptors

    Science.gov (United States)

    Lozano-Torres, Jose L.; Wilbers, Ruud H. P.; Warmerdam, Sonja; Finkers-Tomczak, Anna; Diaz-Granados, Amalia; van Schaik, Casper C.; Helder, Johannes; Bakker, Jaap; Goverse, Aska; Schots, Arjen; Smant, Geert

    2014-01-01

    Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of the host. However, the nature and mode of action of most immunomodulatory compounds in nematode secretions are not well understood. Here, we show that venom allergen-like proteins of plant-parasitic nematodes selectively suppress host immunity mediated by surface-localized immune receptors. Venom allergen-like proteins are uniquely conserved in secretions of all animal- and plant-parasitic nematodes studied to date, but their role during the onset of parasitism has thus far remained elusive. Knocking-down the expression of the venom allergen-like protein Gr-VAP1 severely hampered the infectivity of the potato cyst nematode Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and two other venom allergen-like proteins from the beet cyst nematode Heterodera schachtii in plants resulted in the loss of basal immunity to multiple unrelated pathogens. The modulation of basal immunity by ectopic venom allergen-like proteins in Arabidopsis thaliana involved extracellular protease-based host defenses and non-photochemical quenching in chloroplasts. Non-photochemical quenching regulates the initiation of the defense-related programmed cell death, the onset of which was commonly suppressed by venom allergen-like proteins from G. rostochiensis, H. schachtii, and the root-knot nematode Meloidogyne incognita. Surprisingly, these venom allergen-like proteins only affected the programmed cell death mediated by surface-localized immune receptors. Furthermore, the delivery of venom allergen-like proteins into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilize

  2. Apoplastic venom allergen-like proteins of cyst nematodes modulate the activation of basal plant innate immunity by cell surface receptors.

    Science.gov (United States)

    Lozano-Torres, Jose L; Wilbers, Ruud H P; Warmerdam, Sonja; Finkers-Tomczak, Anna; Diaz-Granados, Amalia; van Schaik, Casper C; Helder, Johannes; Bakker, Jaap; Goverse, Aska; Schots, Arjen; Smant, Geert

    2014-12-01

    Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of the host. However, the nature and mode of action of most immunomodulatory compounds in nematode secretions are not well understood. Here, we show that venom allergen-like proteins of plant-parasitic nematodes selectively suppress host immunity mediated by surface-localized immune receptors. Venom allergen-like proteins are uniquely conserved in secretions of all animal- and plant-parasitic nematodes studied to date, but their role during the onset of parasitism has thus far remained elusive. Knocking-down the expression of the venom allergen-like protein Gr-VAP1 severely hampered the infectivity of the potato cyst nematode Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and two other venom allergen-like proteins from the beet cyst nematode Heterodera schachtii in plants resulted in the loss of basal immunity to multiple unrelated pathogens. The modulation of basal immunity by ectopic venom allergen-like proteins in Arabidopsis thaliana involved extracellular protease-based host defenses and non-photochemical quenching in chloroplasts. Non-photochemical quenching regulates the initiation of the defense-related programmed cell death, the onset of which was commonly suppressed by venom allergen-like proteins from G. rostochiensis, H. schachtii, and the root-knot nematode Meloidogyne incognita. Surprisingly, these venom allergen-like proteins only affected the programmed cell death mediated by surface-localized immune receptors. Furthermore, the delivery of venom allergen-like proteins into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilize

  3. Apoplastic venom allergen-like proteins of cyst nematodes modulate the activation of basal plant innate immunity by cell surface receptors.

    Directory of Open Access Journals (Sweden)

    Jose L Lozano-Torres

    2014-12-01

    Full Text Available Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of the host. However, the nature and mode of action of most immunomodulatory compounds in nematode secretions are not well understood. Here, we show that venom allergen-like proteins of plant-parasitic nematodes selectively suppress host immunity mediated by surface-localized immune receptors. Venom allergen-like proteins are uniquely conserved in secretions of all animal- and plant-parasitic nematodes studied to date, but their role during the onset of parasitism has thus far remained elusive. Knocking-down the expression of the venom allergen-like protein Gr-VAP1 severely hampered the infectivity of the potato cyst nematode Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and two other venom allergen-like proteins from the beet cyst nematode Heterodera schachtii in plants resulted in the loss of basal immunity to multiple unrelated pathogens. The modulation of basal immunity by ectopic venom allergen-like proteins in Arabidopsis thaliana involved extracellular protease-based host defenses and non-photochemical quenching in chloroplasts. Non-photochemical quenching regulates the initiation of the defense-related programmed cell death, the onset of which was commonly suppressed by venom allergen-like proteins from G. rostochiensis, H. schachtii, and the root-knot nematode Meloidogyne incognita. Surprisingly, these venom allergen-like proteins only affected the programmed cell death mediated by surface-localized immune receptors. Furthermore, the delivery of venom allergen-like proteins into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes

  4. Molecular signaling involving intrinsically disordered proteins in prostate cancer

    Directory of Open Access Journals (Sweden)

    Anna Russo

    2016-01-01

    Full Text Available 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.

  5. The endoplasmic reticulum is a hub to sort proteins toward unconventional traffic pathways and endosymbiotic organelles.

    Science.gov (United States)

    Bellucci, Michele; De Marchis, Francesca; Pompa, Andrea

    2017-12-18

    The discovery that much of the extracellular proteome in eukaryotic cells consists of proteins lacking a signal peptide, which cannot therefore enter the secretory pathway, has led to the identification of alternative protein secretion routes bypassing the Golgi apparatus. However, proteins harboring a signal peptide for translocation into the endoplasmic reticulum can also be transported along these alternative routes, which are still far from being well elucidated in terms of the molecular machineries and subcellular/intermediate compartments involved. In this review, we first try to provide a definition of all the unconventional protein secretion pathways in eukaryotic cells, as those pathways followed by proteins directed to an 'external space' bypassing the Golgi, where 'external space' refers to the extracellular space plus the lumen of the secretory route compartments and the inner space of mitochondria and plastids. Then, we discuss the role of the endoplasmic reticulum in sorting proteins toward unconventional traffic pathways in plants. In this regard, various unconventional pathways exporting proteins from the endoplasmic reticulum to the vacuole, plasma membrane, apoplast, mitochondria, and plastids are described, including the short routes followed by the proteins resident in the endoplasmic reticulum. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  6. Multiple proteins of White spot syndrome virus involved in ...

    Indian Academy of Sciences (India)

    The recognition and attachment of virus to its host cell surface is a critical step for viral infection. Recent research revealed that -integrin was involved in White spot syndrome virus (WSSV) infection. In this study, the interaction of -integrin with structure proteins of WSSV and motifs involved in WSSV infection was ...

  7. Protein function prediction involved on radio-resistant bacteria

    International Nuclear Information System (INIS)

    Mezhoud, Karim; Mankai, Houda; Sghaier, Haitham; Barkallah, Insaf

    2009-01-01

    Previously, we identified 58 proteins under positive selection in ionizing-radiation-resistant bacteria (IRRB) but absent in all ionizing-radiation-sensitive bacteria (IRSB). These are good reasons to believe these 58 proteins with their interactions with other proteins (interactomes) are a part of the answer to the question as to how IRRB resist to radiation, because our knowledge of interactomes of positively selected orphan proteins in IRRB might allow us to define cellular pathways important to ionizing-radiation resistance. Using the Database of Interacting Proteins and the PSIbase, we have predicted interactions of orthologs of the 58 proteins under positive selection in IRRB but absent in all IRSB. We used integrate experimental data sets with molecular interaction networks and protein structure prediction from databases. Among these, 18 proteins with their interactomes were identified in Deinococcus radiodurans R1. DNA checkpoint and repair, kinases pathways, energetic and nucleotide metabolisms were the important biological process that found. We predicted the interactomes of 58 proteins under positive selection in IRRB. It is hoped our data will provide new clues as to the cellular pathways that are important for ionizing-radiation resistance. We have identified news proteins involved on DNA management which were not previously mentioned. It is an important input in addition to protein that studied. It does still work to deepen our study on these new proteins

  8. Apoplastic peroxidases are required for salicylic acid-mediated defense against Pseudomonas syringae.

    Science.gov (United States)

    Mammarella, Nicole D; Cheng, Zhenyu; Fu, Zheng Qing; Daudi, Arsalan; Bolwell, G Paul; Dong, Xinnian; Ausubel, Frederick M

    2015-04-01

    Reactive oxygen species (ROS) generated by NADPH oxidases or apoplastic peroxidases play an important role in the plant defense response. Diminished expression of at least two Arabidopsis thaliana peroxidase encoding genes, PRX33 (At3g49110) and PRX34 (At3g49120), as a consequence of anti-sense expression of a heterologous French bean peroxidase gene (asFBP1.1), were previously shown to result in reduced levels of ROS following pathogen attack, enhanced susceptibility to a variety of bacterial and fungal pathogens, and reduced levels of callose production and defense-related gene expression in response to the microbe associated molecular pattern (MAMP) molecules flg22 and elf26. These data demonstrated that the peroxidase-dependent oxidative burst plays an important role in the elicitation of pattern-triggered immunity (PTI). Further work reported in this paper, however, shows that asFBP1.1 antisense plants are not impaired in all PTI-associated responses. For example, some but not all flg22-elicited genes are induced to lower levels by flg22 in asFPB1.1, and callose deposition in asFPB1.1 is similar to wild-type following infiltration with a Pseudomonas syringae hrcC mutant or with non-host P. syringae pathovars. Moreover, asFPB1.1 plants did not exhibit any apparent defect in their ability to mount a hypersensitive response (HR). On the other hand, salicylic acid (SA)-mediated activation of PR1 was dramatically impaired in asFPB1.1 plants. In addition, P. syringae-elicited expression of many genes known to be SA-dependent was significantly reduced in asFBP1.1 plants. Consistent with this latter result, in asFBP1.1 plants the key regulator of SA-mediated responses, NPR1, showed both dramatically decreased total protein abundance and a failure to monomerize, which is required for its translocation into the nucleus. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Elke eVan Assche

    2015-03-01

    Full Text Available 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 towards the role of small RNAs in bacterial post-transcriptional regulation. However, small RNAs are not the only molecules controlling gene expression at this level, RNA-binding proteins 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 RNA-binding proteins, 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.

  10. Early changes of the pH of the apoplast are different in leaves, stem and roots of Vicia faba L. under declining water availability

    Czech Academy of Sciences Publication Activity Database

    Karuppanapandian, T.; Geilfus, C.M.; Muehling, K.H.; Novák, Ondřej; Gloser, V.

    2017-01-01

    Roč. 255, FEB (2017), s. 51-58 ISSN 0168-9452 Institutional support: RVO:61389030 Keywords : xylem sap constituents * abscisic-acid * stomatal conductance * leaf apoplast * helianthus-annuus * plant-responses * intact plants * nacl stress * drying soil * guard-cells * Drought stress * Abscisic acid * Soil drying * Xylem sap * Osmolality * Water relations * Leaf water potential Subject RIV: EF - Botanics OBOR OECD: Plant sciences, botany Impact factor: 3.437, year: 2016

  11. A cell wall-bound adenosine nucleosidase is involved in the salvage of extracellular ATP in Solanum tuberosum.

    Science.gov (United States)

    Riewe, David; Grosman, Lukasz; Fernie, Alisdair R; Zauber, Henrik; Wucke, Cornelia; Geigenberger, Peter

    2008-10-01

    Extracellular ATP (eATP) has recently been demonstrated to play a crucial role in plant development and growth. To investigate the fate of eATP within the apoplast, we used intact potato (Solanum tuberosum) tuber slices as an experimental system enabling access to the apoplast without interference of cytosolic contamination. (i) Incubation of intact tuber slices with ATP led to the formation of ADP, AMP, adenosine, adenine and ribose, indicating operation of apyrase, 5'-nucleotidase and nucleosidase. (ii) Measurement of apyrase, 5'-nucleotidase and nucleosidase activities in fractionated tuber tissue confirmed the apoplastic localization for apyrase and phosphatase in potato and led to the identification of a novel cell wall-bound adenosine nucleosidase activity. (iii) When intact tuber slices were incubated with saturating concentrations of adenosine, the conversion of adenosine into adenine was much higher than adenosine import into the cell, suggesting a potential bypass of adenosine import. Consistent with this, import of radiolabeled adenine into tuber slices was inhibited when ATP, ADP or AMP were added to the slices. (iv) In wild-type plants, apyrase and adenosine nucleosidase activities were found to be co-regulated, indicating functional linkage of these enzymes in a shared pathway. (v) Moreover, adenosine nucleosidase activity was reduced in transgenic lines with strongly reduced apoplastic apyrase activity. When taken together, these results suggest that a complete ATP salvage pathway is present in the apoplast of plant cells.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Yasuko Kitagishi

    2015-01-01

    Full Text Available 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.

  15. Polyamines, peroxidase and proteins involved in the senescence ...

    African Journals Online (AJOL)

    Senescence is the natural aging process at the cellular level or range of phenomena associated with this process. The objective of this review was to show the involvement of substances that may be related to senescence in plants, such as polyamines, peroxidase and proteins. These substances were related with the ...

  16. Involvement of lipid-protein complexes in plant-microorganism interactions

    Directory of Open Access Journals (Sweden)

    Blein Jean-Pierre

    2002-01-01

    Full Text Available Increasing concerns about the environmental impact of modern agricultural have prompted research for alternate practices to pesticide treatments, notably using plant defense mechanisms. Thus, isolation and characterization of plant defense elicitors have been the main step of studies in many groups. Moreover, in the global concept of interactions between organisms and their environment, a major concern is to discriminate recognition between exogenous and endogenous signals, notably during pathogenic or allergenic interactions involving small proteins, such as elicitins or lipid transfer proteins (LTPs. Elicitins and lipid transfer proteins (LTP are both able to load and transfer lipidic molecules and share some structural and functional properties. While elicitins are known as elicitors of plant defense mechanisms, the biological function of LTPs is still an enigma. They are ubiquitous plant proteins able to load and transfer hydrophobic molecules such as fatty acids or phospholipids. Among them, LTPs1 (type 1 lipid transfer proteins constitute a multigenic family of secreted plant lipid binding proteins that are constitutively expressed in specific tissues and/or induced in response to biotic and abiotic stress (for reviews [1-4]. Their biological function is still unknown, even if some data provide arguments for a role of these proteins in the assembly of extracellular hydrophobic polymers (i.e., cutin and suberin [2, 4] and/or in plant defense against fungal pathogens [1, 3]. Beside their involvement in plant defense, LTPs1 are also known to be pan-allergens of plant-derived foods [5]. Finally, the discovery of the sterol carrier-properties of elicitins has opened new perspectives dealing with the relationship between this function and the elicitor activity of these small cystein-rich proteins. Nevertheless, this elicitor activity is restrained to few plant species, and thus does not appear in accordance with a universal lipid transfer

  17. Multiple proteins of White spot syndrome virus involved in ...

    Indian Academy of Sciences (India)

    2014-03-20

    Mar 20, 2014 ... β-integrin with structure proteins of WSSV and motifs involved in WSSV infection was examined. The results showed ... Introduction. White spot ... denatured conditions and renatured by successive 12 h incu- bations with 6, 4, ...

  18. Cerium oxide nanoparticles alter the salt stress tolerance of Brassica napus L. by modifying the formation of root apoplastic barriers.

    Science.gov (United States)

    Rossi, Lorenzo; Zhang, Weilan; Ma, Xingmao

    2017-10-01

    Rapidly growing global population adds significant strains on the fresh water resources. Consequently, saline water is increasingly tapped for crop irrigation. Meanwhile, rapid advancement of nanotechnology is introducing more and more engineered nanoparticles into the environment and in agricultural soils. While some negative effects of ENPs on plant health at very high concentrations have been reported, more beneficial effects of ENPs at relatively low concentrations are increasingly noticed, opening doors for potential applications of nanotechnology in agriculture. In particular, we found that cerium oxide nanoparticles (CeO 2 NPs) improved plant photosynthesis in salt stressed plants. Due to the close connections between salt stress tolerance and the root anatomical structures, we postulated that CeO 2 NPs could modify plant root anatomy and improve plant salt stress tolerance. This study aimed at testing the hypothesis with Brassica napus in the presence of CeO 2 NPs (0, 500 mg kg -1 dry sand) and/or NaCl (0, 50 mM) in a growth chamber. Free hand sections of fresh roots were taken every seven days for three weeks and the suberin lamellae development was examined under a fluorescence microscope. The results confirmed the hypothesis that CeO 2 NPs modified the formation of the apoplastic barriers in Brassica roots. In salt stressed plants, CeO 2 NPs shortened the root apoplastic barriers which allowed more Na + transport to shoots and less accumulation of Na + in plant roots. The altered Na + fluxes and transport led to better physiological performance of Brassica and may lead to new applications of nanotechnology in agriculture. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Manipulation of the apoplastic pH of intact plants mimics stomatal and growth responses to water availability and microclimatic variation.

    Science.gov (United States)

    Wilkinson, Sally; Davies, William J

    2008-01-01

    The apoplastic pH of intact Forsythiaxintermedia (cv. Lynwood) and tomato (Solanum lycopersicum) plants has been manipulated using buffered foliar sprays, and thereby stomatal conductance (g(s)), leaf growth rate, and plant water loss have been controlled. The more alkaline the pH of the foliar spray, the lower the g(s) and/or leaf growth rate subsequently measured. The most alkaline pH that was applied corresponds to that measured in sap extracted from shoots of tomato and Forsythia plants experiencing, respectively, soil drying or a relatively high photon flux density (PFD), vapour pressure deficit (VPD), and temperature in the leaf microclimate. The negative correlation between PFD/VPD/temperature and g(s) determined in well-watered Forsythia plants exposed to a naturally varying summer microclimate was eliminated by spraying the plants with relatively alkaline but not acidic buffers, providing evidence for a novel pH-based signalling mechanism linking the aerial microclimate with stomatal aperture. Increasing the pH of the foliar spray only reduced g(s) in plants of the abscisic acid (ABA)-deficient flacca mutant of tomato when ABA was simultaneously sprayed onto leaves or injected into stems. In well-watered Forsythia plants exposed to a naturally varying summer microclimate (variable PFD, VPD, and temperature), xylem pH and leaf ABA concentration fluctuated but were positively correlated. Manipulation of foliar apoplastic pH also affected the response of g(s) and leaf growth to ABA injected into stems of intact Forsythia plants. The techniques used here to control physiology and water use in intact growing plants could easily be applied in a horticultural context.

  20. Increased de novo riboflavin synthesis and hydrolysis of FMN are involved in riboflavin secretion from Hyoscyamus albus hairy roots under iron deficiency

    OpenAIRE

    Higa, Ataru; Khandakar, Jebunnahar; Mori, Yuko; Kitamura, Yoshie

    2012-01-01

    Riboflavin secretion by Hyoscyamus albus hairy roots under Fe deficiency was examined to determine where riboflavin is produced and whether production occurs via an enhancement of riboflavin biosynthesis or a stimulation of flavin mononucleotide (FMN) hydrolysis. Confocal fluorescent microscopy showed that riboflavin was mainly localized in the epidermis and cortex of the root tip and, at the cellular level, in the apoplast. The expressions of three genes involved in the de novo biosynthesis ...

  1. DUF581 is plant specific FCS-like zinc finger involved in protein-protein interaction.

    Directory of Open Access Journals (Sweden)

    Muhammed Jamsheer K

    Full Text Available 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.

  2. Identification of Protein-Protein Interactions Involved in Pectin Biosynthesis in the golgi Apparatus

    DEFF Research Database (Denmark)

    Lund, Christian Have

    for instance as food additives, nutraceutical, for paper and energy production. Pectin is a cell wall glycan that crucial for every plant growing on land. Pectin is said to be one of the most complex glycans on earth and it is hypothesized that at least 67 enzymatic reactions are involved in its biosynthesis......The plant cell wall surrounds every plant cell and is an essential component that is involved in diverse functions including plant development, morphology, resistance towards plant pathogens etc. The plant cell wall is not only important for the plant. The cell wall has many industrial applications...... the diverse pectin structures for industrial, agronomic and biomedical uses. Increasing evidence suggests that complex formation is important in governing functional coordination of proteins involved in cell wall biosynthesis. In Arabidopsis thaliana, a homogalacturonan (HG) synthase core complex between...

  3. FOP is a centriolar satellite protein involved in ciliogenesis.

    Directory of Open Access Journals (Sweden)

    Joanna Y Lee

    Full Text Available Centriolar satellites are proteinaceous granules that are often clustered around the centrosome. Although centriolar satellites have been implicated in protein trafficking in relation to the centrosome and cilium, the details of their function and composition remain unknown. FOP (FGFR1 Oncogene Partner is a known centrosome protein with homology to the centriolar satellite proteins FOR20 and OFD1. We find that FOP partially co-localizes with the satellite component PCM1 in a cell cycle-dependent manner, similarly to the satellite and cilium component BBS4. As for BBS4, FOP localization to satellites is cell cycle dependent, with few satellites labeled in G1, when FOP protein levels are lowest, and most labeled in G2. FOP-FGFR1, an oncogenic fusion that causes a form of leukemia called myeloproliferative neoplasm, also localizes to centriolar satellites where it increases tyrosine phosphorylation. Depletion of FOP strongly inhibits primary cilium formation in human RPE-1 cells. These results suggest that FOP is a centriolar satellite cargo protein and, as for several other satellite-associated proteins, is involved in ciliogenesis. Localization of the FOP-FGFR1 fusion kinase to centriolar satellites may be relevant to myeloproliferative neoplasm disease progression.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Stephan B Jekat

    2013-07-01

    Full Text Available Structural phloem proteins (P-proteins are characteristic components of the sieve elements in all dicotyledonous and many monocotyledonous angiosperms. Tobacco P-proteins were recently evidenced to be encoded by the widespread 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. Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

    Directory of Open Access Journals (Sweden)

    Rom William N

    2005-08-01

    Full Text Available Abstract Background Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Methods Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD. Two-color rolling-circle amplification was used to measure protein abundance. Results Seven of the 84 antibodies gave a significant difference (p Conclusion Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer.

  7. Secretomics identifies Fusarium graminearum proteins involved in the interaction with barley and wheat

    DEFF Research Database (Denmark)

    Yang, Fen; Jensen, Jens D.; Svensson, Birte

    2012-01-01

    Fusarium graminearum is a phytopathogenic fungus primarily infecting small grain cereals, including barley and wheat. Secreted enzymes play important roles in the pathogenicity of many fungi. In order to access the secretome of F. graminearum, the fungus was grown in liquid culture with barley...... or wheat flour as the sole nutrient source to mimic the host–pathogen interaction. A gel‐based proteomics approach was employed to identify the proteins secreted into the culture medium. Sixty‐nine unique fungal proteins were identified in 154 protein spots, including enzymes involved in the degradation...... between wheat and barley flour medium were mainly involved in fungal cell wall remodelling and the degradation of plant cell walls, starch and proteins. The in planta expression of corresponding F. graminearum genes was confirmed by quantitative reverse transcriptase‐polymerase chain reaction in barley...

  8. MEMBRANE-FUSION OF SEMLIKI FOREST VIRUS INVOLVES HOMOTRIMERS OF THE FUSION PROTEIN

    NARCIS (Netherlands)

    WAHLBERG, JM; WILSCHUT, J; GAROFF, H

    1992-01-01

    Infection of cells with enveloped viruses is accomplished through membrane fusion. The binding and fusion Processes are mediated by the spike proteins in the envelope of the virus particle and usually involve a series of conformational changes in these proteins. We have studied the low-pH-mediated

  9. Intact long-type DupA protein in Helicobacter pylori is an ATPase involved in multifunctional biological activities.

    Science.gov (United States)

    Wang, Ming-yi; Chen, Cheng; Shao, Chen; Wang, Shao-bo; Wang, Ai-chu; Yang, Ya-chao; Yuan, Xiao-yan; Shao, Shi-he

    2015-04-01

    The function of intact long-type DupA protein in Helicobacter pylori was analyzed using immunoblotting and molecular biology techniques in the study. After cloning, expression and purification, ATPase activity of DupA protein was detected. Antibody was produced for localization and interaction proteins analysis. The dupA-deleted mutant was generated for adhesion and CagA protein translocation assay, susceptibility to different pH, IL-8 secretion assay, cytotoxicity to MKN-45 cells and proteins-involved apoptosis analysis. DupA protein exhibited an ATPase activity (129.5±17.8 U/mgprot) and located in bacterial membrane, while it did not involve the adhesion and CagA protein delivery of H. pylori. DupA protein involved the urease secretion as the interaction proteins. The wild type strain had a stronger growth in low pH than the dupA-deleted mutant (p DupA protein located in membrane as ATPase is a true virulence factor associated with duodenal ulcer development involving the IL-8 induction and urease secretion, while it inhibits gastric cancer cell growth in vitro by activating the mitochondria-mediated apoptotic pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Repeat-containing protein effectors of plant-associated organisms

    Directory of Open Access Journals (Sweden)

    Carl H. Mesarich

    2015-10-01

    Full Text Available Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.

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

    Directory of Open Access Journals (Sweden)

    Miroslav Arambasic

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  13. Analysis of proteins involved in biodegradation of crop biomass

    Science.gov (United States)

    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. Proteomes and Ubiquitylomes Analysis Reveals the Involvement of Ubiquitination in Protein Degradation in Petunias1

    Science.gov (United States)

    Liu, Juanxu; Wei, Qian; Wang, Rongmin; Yang, Weiyuan; Ma, Yueyue; Chen, Guoju

    2017-01-01

    Petal senescence is a complex programmed process. It has been demonstrated previously that treatment with ethylene, a plant hormone involved in senescence, can extensively alter transcriptome and proteome profiles in plants. However, little is known regarding the impact of ethylene on posttranslational modification (PTM) or the association between PTM and the proteome. Protein degradation is one of the hallmarks of senescence, and ubiquitination, a major PTM in eukaryotes, plays important roles in protein degradation. In this study, we first obtained reference petunia (Petunia hybrida) transcriptome data via RNA sequencing. Next, we quantitatively investigated the petunia proteome and ubiquitylome and the association between them in petunia corollas following ethylene treatment. In total, 51,799 unigenes, 3,606 proteins, and 2,270 ubiquitination sites were quantified 16 h after ethylene treatment. Treatment with ethylene resulted in 14,448 down-regulated and 6,303 up-regulated unigenes (absolute log2 fold change > 1 and false discovery rate petunia. Several putative ubiquitin ligases were up-regulated at the protein and transcription levels. Our results showed that the global proteome and ubiquitylome were negatively correlated and that ubiquitination could be involved in the degradation of proteins during ethylene-mediated corolla senescence in petunia. Ethylene regulates hormone signaling transduction pathways at both the protein and ubiquitination levels in petunia corollas. In addition, our results revealed that ethylene increases the ubiquitination levels of proteins involved in endoplasmic reticulum-associated degradation. PMID:27810942

  15. Dual localized AtHscB involved in iron sulfur protein biogenesis in Arabidopsis.

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    Xiang Ming Xu

    2009-10-01

    Full Text Available Iron-sulfur clusters are ubiquitous structures which act as prosthetic groups for numerous proteins involved in several fundamental biological processes including respiration and photosynthesis. Although simple in structure both the assembly and insertion of clusters into apoproteins requires complex biochemical pathways involving a diverse set of proteins. In yeast, the J-type chaperone Jac1 plays a key role in the biogenesis of iron sulfur clusters in mitochondria.In this study we demonstrate that AtHscB from Arabidopsis can rescue the Jac1 yeast knockout mutant suggesting a role for AtHscB in iron sulfur protein biogenesis in plants. In contrast to mitochondrial Jac1, AtHscB localizes to both mitochondria and the cytosol. AtHscB interacts with AtIscU1, an Isu-like scaffold protein involved in iron-sulfur cluster biogenesis, and through this interaction AtIscU1 is most probably retained in the cytosol. The chaperone AtHscA can functionally complement the yeast Ssq1knockout mutant and its ATPase activity is enhanced by AtHscB and AtIscU1. Interestingly, AtHscA is also localized in both mitochondria and the cytosol. Furthermore, AtHscB is highly expressed in anthers and trichomes and an AtHscB T-DNA insertion mutant shows reduced seed set, a waxless phenotype and inappropriate trichome development as well as dramatically reduced activities of the iron-sulfur enzymes aconitase and succinate dehydrogenase.Our data suggest that AtHscB together with AtHscA and AtIscU1 plays an important role in the biogenesis of iron-sulfur proteins in both mitochondria and the cytosol.

  16. A Deg-protease family protein in marine Synechococcus is involved in outer membrane protein organization

    Directory of Open Access Journals (Sweden)

    Rhona Kayra Stuart

    2014-06-01

    Full Text Available Deg-family proteases are a periplasm-associated group of proteins that are known to be involved in envelope stress responses and are found in most microorganisms. Orthologous genes SYNW2176 (in strain WH8102 and sync_2523 (strain CC9311 are predicted members of the Deg-protease family and are among the few genes induced by copper stress in both open ocean and coastal marine Synechococcus strains. In contrast to the lack of a phenotype in a similar knockout in Synechocystis PCC6803, a SYNW2176 knockout mutant in strain WH8102 was much more resistant to copper than the wild-type. The mutant also exhibited a significantly altered outer membrane protein composition which may contribute to copper resistance, longer lag phase after transfer, low-level consistent alkaline phosphatase activity, and an inability to induce high alkaline phosphatase activity in response to phosphate stress. This phenotype suggests a protein-quality-control role for SYNW2176, the absence of which leads to a constitutively activated stress response. Deg-protease family proteins in this ecologically important cyanobacterial group thus help to determine outer membrane responses to both nutrients and toxins.

  17. Oxidative defence reactions in sunflower roots induced by methyl-jasmonate and methyl-salicylate and their relation with calcium signalling.

    Science.gov (United States)

    Garrido, Inmaculada; Espinosa, Francisco; Alvarez-Tinaut, M Carmen

    2009-10-01

    Ca(2+) plays a critical role as second messenger in the signal-response coupling of plant defence responses, and methyl-jasmonate and methyl-salicylate are important components of signal transduction cascades activating plant defences. When intact axenic non-induced seedling roots of sunflower were treated with different Ca(2+) concentrations up to 1 mM, there was no significant increase in O(2)(*-) generation or DMAB-MBTH peroxidase (extracellular, ECPOX) activities in the apoplast, probably because these roots had enough Ca(2+) in their exo- and endocellular reservoirs. Both activities were strongly inhibited by the RBOH-NADPH oxidase inhibitor DPI and by the Ca(2+) surrogate antagonist La(3+), but the voltage-dependent Ca(2+) channel blocker verapamil was only inhibitory at concentrations higher than those active on animal L-type Ca(2+) channels. Concentrations >5 mM EGTA (chelating Ca(2+) in the apoplast) and Li(+) (inhibiting PI cycle dependent endogenous Ca(2+) fluxes) also inhibited both activities. W7, inhibitor of binding of Ca-CaM to its target protein, enhanced both activities, but the inactive analogue W5 showed a similar effect. Our data suggest that Ca(2+) from exocellular and, to a lesser extent, from endocellular stores is involved in oxidative activities, and that RBOH-NADPH oxidase is the main system supporting them. Ca(2+) activation of the PM cytosolic side of RBOH-NADPH oxidase is probably the key to Ca(2+) involvement in these processes. Roots induced by MeJA or MeSA showed significant enhancement of both oxidative activities, as corresponding to the oxidative burst evoked by the two phytohormones in the root apoplast. But while ECPOX activity showed a response to the effectors similar to that described above for non-induced roots, O(2)(*-) generation activity in the apoplast of induced roots was insensitive to EGTA, verapamil and Li(+), the inhibitors of exogenous and endogenous Ca(2+) fluxes; only DPI and La(3+) were inhibitory. As

  18. Identification of a Stelar-Localized Transport Protein That Facilitates Root-to-Shoot Transfer of Chloride in Arabidopsis

    KAUST Repository

    Li, Bo; Byrt, Caitlin; Qiu, Jiaen; Baumann, Ute; Hrmova, Maria; Evrard, Aurelie; Johnson, Alexander A T; Birnbaum, Kenneth D.; Mayo, Gwenda M.; Jha, Deepa; Henderson, Sam W.; Tester, Mark A.; Gilliham, Mathew; Roy, Stuart J.

    2015-01-01

    Under saline conditions, higher plants restrict the accumulation of chloride ions (Cl–) in the shoot by regulating their transfer from the root symplast into the xylem-associated apoplast. To identify molecular mechanisms underpinning this phenomenon, we undertook a transcriptional screen of salt stressed Arabidopsis (Arabidopsis thaliana) roots. Microarrays, quantitative RT-PCR, and promoter-GUS fusions identified a candidate gene involved in Cl– xylem loading from the Nitrate transporter 1/Peptide Transporter family (NPF2.4). This gene was highly expressed in the root stele compared to the cortex, and its expression decreased after exposure to NaCl or abscisic acid. NPF2.4 fused to fluorescent proteins, expressed either transiently or stably, was targeted to the plasma membrane. Electrophysiological analysis of NPF2.4 in Xenopus laevis oocytes suggested that NPF2.4 catalyzed passive Cl– efflux out of cells and was much less permeable to NO3−. Shoot Cl– accumulation was decreased following NPF2.4 artificial microRNA knockdown, whereas it was increased by overexpression of NPF2.4. Taken together, these results suggest that NPF2.4 is involved in long-distance transport of Cl– in plants, playing a role in the loading and the regulation of Cl– loading into the xylem of Arabidopsis roots during salinity stress.

  19. Identification of a Stelar-Localized Transport Protein That Facilitates Root-to-Shoot Transfer of Chloride in Arabidopsis

    KAUST Repository

    Li, Bo

    2015-12-11

    Under saline conditions, higher plants restrict the accumulation of chloride ions (Cl–) in the shoot by regulating their transfer from the root symplast into the xylem-associated apoplast. To identify molecular mechanisms underpinning this phenomenon, we undertook a transcriptional screen of salt stressed Arabidopsis (Arabidopsis thaliana) roots. Microarrays, quantitative RT-PCR, and promoter-GUS fusions identified a candidate gene involved in Cl– xylem loading from the Nitrate transporter 1/Peptide Transporter family (NPF2.4). This gene was highly expressed in the root stele compared to the cortex, and its expression decreased after exposure to NaCl or abscisic acid. NPF2.4 fused to fluorescent proteins, expressed either transiently or stably, was targeted to the plasma membrane. Electrophysiological analysis of NPF2.4 in Xenopus laevis oocytes suggested that NPF2.4 catalyzed passive Cl– efflux out of cells and was much less permeable to NO3−. Shoot Cl– accumulation was decreased following NPF2.4 artificial microRNA knockdown, whereas it was increased by overexpression of NPF2.4. Taken together, these results suggest that NPF2.4 is involved in long-distance transport of Cl– in plants, playing a role in the loading and the regulation of Cl– loading into the xylem of Arabidopsis roots during salinity stress.

  20. Accumulation of cynaropicrin in globe artichoke and localization of enzymes involved in its biosynthesis.

    Science.gov (United States)

    Eljounaidi, K; Comino, C; Moglia, A; Cankar, K; Genre, A; Hehn, A; Bourgaud, F; Beekwilder, J; Lanteri, S

    2015-10-01

    Globe artichoke (Cynara cardunculus var. scolymus) belongs to the Asteraceae family, in which one of the most biologically significant class of secondary metabolites are sesquiterpene lactones (STLs). In globe artichoke the principal STL is the cynaropicrin, which contributes to approximately 80% of its characteristic bitter taste. Cynaropicrin content was assessed in globe artichoke tissues and was observed to accumulate in leaves of different developmental stages. In the receptacle, a progressive decrease was observed during inflorescence development, while the STL could not be detected in the inflorescence bracts. Almost undetectable amounts were found in the roots and inflorescence stems at the commercial stage. Cynaropicrin content was found to correlate with expression of genes encoding CcGAS, CcGAO and CcCOS, which are involved in the STL biosynthesis. A more detailed study of leaf material revealed that cynaropicrin predominantly accumulates in the trichomes, and not in the apoplastic cavity fluids. Analysis of the promoter regions of CcGAO and CcCOS revealed the presence of L1-box motifs, which confers trichome-specific expression in Arabidopsis, suggesting that cynaropicrin is not only stored but also synthesized in trichomes. A transient expression of GFP fusion proteins was performed in Nicotiana benthamiana plants: the CcGAS fluorescence signal was located in the cytoplasm while the CcGAO and CcCOS localized to the endoplasmatic reticulum. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    XIMENA ORTEGA

    2005-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  3. Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

    International Nuclear Information System (INIS)

    Gao, Wei-Min; Haab, Brian B; Hanash, Samir M; Kuick, Rork; Orchekowski, Randal P; Misek, David E; Qiu, Ji; Greenberg, Alissa K; Rom, William N; Brenner, Dean E; Omenn, Gilbert S

    2005-01-01

    Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance. Seven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and α-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified. Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer

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

    Science.gov (United States)

    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.

  5. Gemin5: A Multitasking RNA-Binding Protein Involved in Translation Control

    Directory of Open Access Journals (Sweden)

    David Piñeiro

    2015-04-01

    Full Text Available Gemin5 is a RNA-binding protein (RBP that was first identified as a peripheral component of the survival of motor neurons (SMN complex. This predominantly cytoplasmic protein recognises the small nuclear RNAs (snRNAs through its WD repeat domains, allowing assembly of the SMN complex into small nuclear ribonucleoproteins (snRNPs. Additionally, the amino-terminal end of the protein has been reported to possess cap-binding capacity and to interact with the eukaryotic initiation factor 4E (eIF4E. Gemin5 was also shown to downregulate translation, to be a substrate of the picornavirus L protease and to interact with viral internal ribosome entry site (IRES elements via a bipartite non-canonical RNA-binding site located at its carboxy-terminal end. These features link Gemin5 with translation control events. Thus, beyond its role in snRNPs biogenesis, Gemin5 appears to be a multitasking protein cooperating in various RNA-guided processes. In this review, we will summarise current knowledge of Gemin5 functions. We will discuss the involvement of the protein on translation control and propose a model to explain how the proteolysis fragments of this RBP in picornavirus-infected cells could modulate protein synthesis.

  6. Search for Partner Proteins of A. thaliana Immunophilins Involved in the Control of Plant Immunity

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    Inna A. Abdeeva

    2018-04-01

    Full Text Available The involvement of plant immunophilins in multiple essential processes such as development, various ways of adapting to biotic and abiotic stresses, and photosynthesis has already been established. Previously, research has demonstrated the involvement of three immunophilin genes (AtCYP19-1/ROC3, AtFKBP65/ROF2, and AtCYP57 in the control of plant response to invasion by various pathogens. Current research attempts to identify host target proteins for each of the selected immunophilins. As a result, candidate interactors have been determined and confirmed using a yeast 2-hybrid (Y2H system for protein–protein interaction assays. The generation of mutant isoforms of ROC3 and AtCYP57 harboring substituted amino acids in the in silico-predicted active sites became essential to achieving significant binding to its target partners. This data shows that ROF2 targets calcium-dependent lipid-binding domain-containing protein (At1g70790; AT1 and putative protein phosphatase (At2g30020; АТ2, whereas ROC3 interacts with GTP-binding protein (At1g30580; ENGD-1 and RmlC-like cupin (At5g39120. The immunophilin AtCYP57 binds to putative pyruvate decarboxylase-1 (Pdc1 and clathrin adaptor complex-related protein (At5g05010. Identified interactors confirm our previous findings that immunophilins ROC3, ROF2, and AtCYP57 are directly involved with stress response control. Further, these findings extend our understanding of the molecular functional pathways of these immunophilins.

  7. Expression of apoplast-targeted plant defensin MtDef4.2 confers resistance to leaf rust pathogen Puccinia triticina but does not affect mycorrhizal symbiosis in transgenic wheat.

    Science.gov (United States)

    Kaur, Jagdeep; Fellers, John; Adholeya, Alok; Velivelli, Siva L S; El-Mounadi, Kaoutar; Nersesian, Natalya; Clemente, Thomas; Shah, Dilip

    2017-02-01

    Rust fungi of the order Pucciniales are destructive pathogens of wheat worldwide. Leaf rust caused by the obligate, biotrophic basidiomycete fungus Puccinia triticina (Pt) is an economically important disease capable of causing up to 50 % yield losses. Historically, resistant wheat cultivars have been used to control leaf rust, but genetic resistance is ephemeral and breaks down with the emergence of new virulent Pt races. There is a need to develop alternative measures for control of leaf rust in wheat. Development of transgenic wheat expressing an antifungal defensin offers a promising approach to complement the endogenous resistance genes within the wheat germplasm for durable resistance to Pt. To that end, two different wheat genotypes, Bobwhite and Xin Chun 9 were transformed with a chimeric gene encoding an apoplast-targeted antifungal plant defensin MtDEF4.2 from Medicago truncatula. Transgenic lines from four independent events were further characterized. Homozygous transgenic wheat lines expressing MtDEF4.2 displayed resistance to Pt race MCPSS relative to the non-transgenic controls in growth chamber bioassays. Histopathological analysis suggested the presence of both pre- and posthaustorial resistance to leaf rust in these transgenic lines. MtDEF4.2 did not, however, affect the root colonization of a beneficial arbuscular mycorrhizal fungus Rhizophagus irregularis. This study demonstrates that the expression of apoplast-targeted plant defensin MtDEF4.2 can provide substantial resistance to an economically important leaf rust disease in transgenic wheat without negatively impacting its symbiotic relationship with the beneficial mycorrhizal fungus.

  8. 3DSwap: Curated knowledgebase of proteins involved in 3D domain swapping

    KAUST Repository

    Shameer, Khader

    2011-09-29

    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. The Author(s) 2011.

  9. SCR96, a small cysteine-rich secretory protein of Phytophthora cactorum, can trigger cell death in the Solanaceae and is important for pathogenicity and oxidative stress tolerance.

    Science.gov (United States)

    Chen, Xiao-Ren; Li, Yan-Peng; Li, Qi-Yuan; Xing, Yu-Ping; Liu, Bei-Bei; Tong, Yun-Hui; Xu, Jing-You

    2016-05-01

    Peptides and small molecules produced by both the plant pathogen Phytophthora and host plants in the apoplastic space mediate the relationship between the interplaying organisms. Various Phytophthora apoplastic effectors, including small cysteine-rich (SCR) secretory proteins, have been identified, but their roles during interaction remain to be determined. Here, we identified an SCR effector encoded by scr96, one of three novel genes encoding SCR proteins in P. cactorum with similarity to the P. cactorum phytotoxic protein PcF. Together with the other two genes, scr96 was transcriptionally induced throughout the developmental and infection stages of the pathogen. These genes triggered plant cell death (PCD) in the Solanaceae, including Nicotiana benthamiana and tomato. The scr96 gene did not show single nucleotide polymorphisms in a collection of P. cactorum isolates from different countries and host plants, suggesting that its role is essential and non-redundant during infection. Homologues of SCR96 were identified only in oomycetes, but not in fungi and other organisms. A stable protoplast transformation protocol was adapted for P. cactorum using green fluorescent protein as a marker. The silencing of scr96 in P. cactorum caused gene-silenced transformants to lose their pathogenicity on host plants and these transformants were significantly more sensitive to oxidative stress. Transient expression of scr96 partially recovered the virulence of gene-silenced transformants on plants. Overall, our results indicate that the P. cactorum scr96 gene encodes an important virulence factor that not only causes PCD in host plants, but is also important for pathogenicity and oxidative stress tolerance. © 2015 BSPP AND JOHN WILEY & SONS LTD.

  10. Extraction and Characterization of Extracellular Proteins and Their Post-Translational Modifications from Arabidopsis thaliana Suspension Cell Cultures and Seedlings: A Critical Review

    Directory of Open Access Journals (Sweden)

    Mina Ghahremani

    2016-09-01

    Full Text Available Proteins secreted by plant cells into the extracellular space, consisting of the cell wall, apoplastic fluid, and rhizosphere, play crucial roles during development, nutrient acquisition, and stress acclimation. However, isolating the full range of secreted proteins has proven difficult, and new strategies are constantly evolving to increase the number of proteins that can be detected and identified. In addition, the dynamic nature of the extracellular proteome presents the further challenge of identifying and characterizing the post-translational modifications (PTMs of secreted proteins, particularly glycosylation and phosphorylation. Such PTMs are common and important regulatory modifications of proteins, playing a key role in many biological processes. This review explores the most recent methods in isolating and characterizing the plant extracellular proteome with a focus on the model plant Arabidopsis thaliana, highlighting the current challenges yet to be overcome. Moreover, the crucial role of protein PTMs in cell wall signalling, development, and plant responses to biotic and abiotic stress is discussed.

  11. Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS

    Directory of Open Access Journals (Sweden)

    Dritan Siliqi

    2018-02-01

    Full Text Available The dynamism of proteins is central to their function, and several proteins have been described as flexible, as consisting of multiple domains joined by flexible linkers, and even as intrinsically disordered. Several techniques exist to study protein structures, but small angle X-ray scattering (SAXS has proven to be particularly powerful for the quantitative analysis of such flexible systems. In the present report, we have used SAXS in combination with X-ray crystallography to highlight their usefulness at characterizing flexible proteins, using as examples two proteins involved in different steps of ribosome biogenesis. The yeast BRCA2 and CDKN1A-interactig protein, Bcp1, is a chaperone for Rpl23 of unknown structure. We showed that it consists of a rigid, slightly elongated protein, with a secondary structure comprising a mixture of alpha helices and beta sheets. As an example of a flexible molecule, we studied the SBDS (Shwachman-Bodian-Diamond Syndrome protein that is involved in the cytoplasmic maturation of the 60S subunit and constitutes the mutated target in the Shwachman-Diamond Syndrome. In solution, this protein coexists in an ensemble of three main conformations, with the N- and C-terminal ends adopting different orientations with respect to the central domain. The structure observed in the protein crystal corresponds to an average of those predicted by the SAXS flexibility analysis.

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

    Science.gov (United States)

    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

  13. Structure of Mycobacterium tuberculosis RuvA, a protein involved in recombination

    International Nuclear Information System (INIS)

    Prabu, J. Rajan; Thamotharan, S.; Khanduja, Jasbeer Singh; Alipio, Emily Zabala; Kim, Chang-Yub; Waldo, Geoffrey S.; Terwilliger, Thomas C.; Segelke, Brent; Lekin, Tim; Toppani, Dominique; Hung, Li-Wei; Yu, Minmin; Bursey, Evan; Muniyappa, K.; Chandra, Nagasuma R.; Vijayan, M.

    2006-01-01

    RuvA, a protein from M. tuberculosis H37Rv involved in recombination, has been cloned, expressed, purified and analysed by X-ray crystallography. The process of recombinational repair is crucial for maintaining genomic integrity and generating biological diversity. In association with RuvB and RuvC, RuvA plays a central role in processing and resolving Holliday junctions, which are a critical intermediate in homologous recombination. Here, the cloning, purification and structure determination of the RuvA protein from Mycobacterium tuberculosis (MtRuvA) are reported. Analysis of the structure and comparison with other known RuvA proteins reveal an octameric state with conserved subunit–subunit interaction surfaces, indicating the requirement of octamer formation for biological activity. A detailed analysis of plasticity in the RuvA molecules has led to insights into the invariant and variable regions, thus providing a framework for understanding regional flexibility in various aspects of RuvA function

  14. Coexpression of multidrug resistance involve proteins: a flow cytometric analysis.

    Science.gov (United States)

    Boutonnat, J; Bonnefoix, T; Mousseau, M; Seigneurin, D; Ronot, X

    1998-01-01

    Cross resistance to multiple natural cytotoxic products represents a major obstacle in myeloblastic acute leukaemia (AML). Multidrug resistance (MDR) often involves overexpression of plasma membrane drug transporter P-glycoprotein (PGP) or the resistance associated protein (MRP). Recently, a protein overexpressed in a non-PGP MDR lung cancer cell line and termed lung resistance related protein (LRP) was identified. These proteins are known to be associated with a bad prognosis in AML. We have developed a triple indirect labelling analysed by flow cytometry to detect the coexpression of these proteins. Since no cell line expressing all three antigens is known, we mixed K562 cells (resistant to Adriblastine, PGP+, MRP-, LRP-) with GLC4 cells (resistant to Adriblastine, PGP-, MRP+, LRP+) to create a model system to test the method. The antibodies used were UIC2 for PGP, MRPm6 for MRP and LRP56 for LRP. They were revealed by Fab'2 coupled with Fluoresceine-isothiocyanate, Phycoerythrin or Tricolor with isotype specificity. Cells were fixed and permeabilized after PGP labelling because MRPm6 and LRP56 recognize intracellular epitopes. PGP and LRP were easily detected. MRP is expressed at relatively low levels and was more difficult to detect because in the triple labelling the non specific staining was higher than in a single labelling. Despite the increased background in the triple labelling we were able to detect coexpression of PGP, MRP, LRP by flow cytometry. This method appears to be very useful to detect coexpression of markers in AML. Such coexpression could modify the therapeutic approach with revertants.

  15. On the involvement of host proteins in Cowpea mosaic virus intercellular spread

    NARCIS (Netherlands)

    Hollander, den P.W.

    2014-01-01

    Abstract of thesis Paulus den Hollander entitled “On the involvement of host proteins in Cowpea mosaic virus intercellular spread”.

    Defence: 18th of November 13.30 h

    Abstract

    Intercellular spread of Cowpea mosaic virus (CPMV) occurs via movement

  16. Xylem diameter changes during osmotic stress, desiccation and freezing in Pinus sylvestris and Populus tremula.

    Science.gov (United States)

    Lintunen, Anna; Lindfors, Lauri; Nikinmaa, Eero; Hölttä, Teemu

    2017-04-01

    Trees experience low apoplastic water potential frequently in most environments. Low apoplastic water potential increases the risk of embolism formation in xylem conduits and creates dehydration stress for the living cells. We studied the magnitude and rate of xylem diameter change in response to decreasing apoplastic water potential and the role of living parenchyma cells in it to better understand xylem diameter changes in different environmental conditions. We compared responses of control and heat-injured xylem of Pinus sylvestris (L.) and Populus tremula (L.) branches to decreasing apoplastic water potential created by osmotic stress, desiccation and freezing. It was shown that xylem in control branches shrank more in response to decreasing apoplastic water potential in comparison with the samples that were preheated to damage living xylem parenchyma. By manipulating the osmotic pressure of the xylem sap, we observed xylem shrinkage due to decreasing apoplastic water potential even in the absence of water tension within the conduits. These results indicate that decreasing apoplastic water potential led to withdrawal of intracellular water from the xylem parenchyma, causing tissue shrinkage. The amount of xylem shrinkage per decrease in apoplastic water potential was higher during osmotic stress or desiccation compared with freezing. During desiccation, xylem diameter shrinkage involved both dehydration-related shrinkage of xylem parenchyma and water tension-induced shrinkage of conduits, whereas dehydration-related shrinkage of xylem parenchyma was accompanied by swelling of apoplastic ice during freezing. It was also shown that the exchange of water between symplast and apoplast within xylem is clearly faster than previously reported between the phloem and the xylem. Time constant of xylem shrinkage was 40 and 2 times higher during osmotic stress than during freezing stress in P. sylvestris and P. tremula, respectively. Finally, it was concluded that the

  17. Involvement of MAPK proteins in bystander effects induced by chemicals and ionizing radiation

    International Nuclear Information System (INIS)

    Asur, Rajalakshmi; Balasubramaniam, Mamtha; Marples, Brian; Thomas, Robert A.; Tucker, James D.

    2010-01-01

    Many studies have examined bystander effects induced by ionizing radiation, however few have evaluated the ability of chemicals to induce similar effects. We previously reported the ability of two chemicals, mitomycin C (MMC) and phleomycin (PHL) to induce bystander effects in normal human lymphoblastoid cell lines. The focus of the current study was to determine the involvement of the MAPK proteins in bystander effects induced by physical and chemical DNA damaging agents and to evaluate the effects of MAPK inhibition on bystander-induced caspase 3/7 activation. The phosphorylation levels of the MAPK proteins ERK1/2, JNK, and p38, were measured from 1 to 24 h following direct or bystander exposure to MMC, PHL or radiation. We observed transient phosphorylation, at early time points, of all 3 proteins in bystander cells. We also evaluated the effect of MAPK inhibition on bystander-induced caspase 3/7 activity to determine the role of MAPK proteins in bystander-induced apoptosis. We observed bystander-induced activation of caspase 3/7 in bystander cells. Inhibition of MAPK proteins resulted in a decrease in caspase 3/7 activity at the early time points, and the caspase activity increased (in the case of ERK inhibition) or returned to basal levels (in the case of JNK or p38 inhibition) between 12 and 24 h. PHL is considered to be a radiomimetic agent, however in the present study PHL behaved more like a chemical and not like radiation in terms of MAPK phosphorylation. These results point to the involvement of MAPK proteins in the bystander effect induced by radiation and chemicals and provide additional evidence that this response is not limited to radiation but is a generalized stress response in cells.

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

    Science.gov (United States)

    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

  19. The Zinc-Finger Thylakoid-Membrane Protein FIP Is Involved With Abiotic Stress Response in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Karina L. Lopes

    2018-04-01

    Full Text Available Many plant genes have their expression modulated by stress conditions. Here, we used Arabidopsis FtsH5 protease, which expression is regulated by light stress, as bait in a yeast two-hybrid screen to search for new proteins involved in the stress response. As a result, we found FIP (FtsH5 Interacting Protein, which possesses an amino proximal cleavable transit peptide, a hydrophobic membrane-anchoring region, and a carboxyl proximal C4-type zinc-finger domain. In vivo experiments using FIP fused to green fluorescent protein (GFP showed a plastid localization. This finding was corroborated by chloroplast import assays that showed FIP inserted in the thylakoid membrane. FIP expression was down-regulated in plants exposed to high light intensity, oxidative, salt, and osmotic stresses, whereas mutant plants expressing low levels of FIP were more tolerant to these abiotic stresses. Our data shows a new thylakoid-membrane protein involved with abiotic stress response in Arabidopsis thaliana.

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

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Feng-Hou [NO.3 People' s Hospital affiliated to Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 201900 (China); The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Wu, Ying-Li [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Zhao, Meng [Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China); Liu, Chuan-Xu; Wang, Li-Shun [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Chen, Guo-Qiang, E-mail: chengq@shsmu.edu.cn [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China)

    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.

  1. Which Plant Proteins Are Involved in Antiviral Defense? Review on In Vivo and In Vitro Activities of Selected Plant Proteins against Viruses

    Directory of Open Access Journals (Sweden)

    Oskar Musidlak

    2017-11-01

    Full Text Available Plants have evolved a variety of defense mechanisms to tackle virus attack. Endogenous plant proteins can function as virus suppressors. Different types of proteins mediate defense responses against plant viruses. Pathogenesis-related (PR proteins are activated upon pathogen infections or in different stress situations and their production is one of many components in plant defense. Ribosome-inactivating proteins (RIPs suppress translation by enzymatically damaging ribosomes and they have been found to have antiviral activity. RNA-binding proteins (RBPs bind to target RNAs via specialized RNA-binding domain and can directly or indirectly function in plant defense system against RNA viruses. Proteins involved in silencing machinery, namely Dicer-like (DCL proteins, Argonaute (AGO proteins, and RNA-dependent RNA polymerases (RDRs confer innate antiviral defense in plants as they are able to degrade foreign RNA of viral origin. This review aims to provide a comprehensive and up-to-date picture of plant proteins participating in antiviral defense. As a result we discuss proteins conferring plant antiviral resistance and their potential future applications in different fields of life including agriculture and medicine.

  2. Identification of genes and proteins involved in excision repair of human cells

    International Nuclear Information System (INIS)

    Hoeijmakers, J.H.J.; Westerveld, A.; Van Duin, M.; Vermeulen, W.; Odijk, H.; De Wit, J.; Bootsma, D.

    1986-01-01

    The autosomal, recessive disorder xeroderma pigmentosum (XP) is characterized by extreme sensitivity of the skin to sun exposure and prediposition to skin cancer. The basic defect in most XP patients is thought to reside in an inefficient removal of UV-induced lesions in the DNA by excision repair. The biochemical complexity of this process is amply illustrated by the fact that so far nine complementary groups within this syndrome have been identified. Despite extensive research, none of these genes or proteins involved have been isolated. Using a microinjection assay system the authors identified components in crude cell extracts that transiently correct the defect in (injected) fibroblasts of all excision-deficient XP complementation groups, as indicated by temporary restoration of UV-induced unscheduled DNA synthesis. This correction is complementation group specific, since it is only found when extracts from complementing XP cells are injected. After incubation of extracts with proteinase K the XP-A and KP-G correcting activities were lost, indicating that the complementation is due to proteins. The XP-A correcting protein was found to precipitate between 30 and 60% ammonium sulfate saturation. Furthermore this protein binds to DEAE-cellulose and to (UV-irradiated) double-strand (ds) DNA attached to cellulose. The latter affinity chromatography step allows a considerable purification, since less than 1% of the proteins applied to such columns is retained. It has to be established whether the XP-A correcting proteins binds by itself or via other proteins to the UV-irradiated DNA and whether it also binds to nonirradiated (ds or ss) DNA. Similar experiments with the XP-G correcting protein are in progress

  3. The Prenylated Rab GTPase Receptor PRA1.F4 Contributes to Protein Exit from the Golgi Apparatus.

    Science.gov (United States)

    Lee, Myoung Hui; Yoo, Yun-Joo; Kim, Dae Heon; Hanh, Nguyen Hong; Kwon, Yun; Hwang, Inhwan

    2017-07-01

    Prenylated Rab acceptor1 (PRA1) functions in the recruitment of prenylated Rab proteins to their cognate organelles. Arabidopsis ( Arabidopsis thaliana ) contains a large number of proteins belonging to the AtPRA1 family. However, their physiological roles remain largely unknown. Here, we investigated the physiological role of AtPRA1.F4, a member of the AtPRA1 family. A T-DNA insertion knockdown mutant of AtPRA1.F4 , atpra1.f4 , was smaller in stature than parent plants and possessed shorter roots, whereas transgenic plants overexpressing HA:AtPRA1.F4 showed enhanced development of secondary roots and root hairs. However, both overexpression and knockdown plants exhibited increased sensitivity to high-salt stress, lower vacuolar Na + /K + -ATPase and plasma membrane ATPase activities, lower and higher pH in the vacuole and apoplast, respectively, and highly vesiculated Golgi apparatus. HA:AtPRA1.F4 localized to the Golgi apparatus and assembled into high-molecular-weight complexes. atpra1.f4 plants displayed a defect in vacuolar trafficking, which was complemented by low but not high levels of HA : AtPRA1.F4 Overexpression of HA:AtPRA1.F4 also inhibited protein trafficking at the Golgi apparatus, albeit differentially depending on the final destination or type of protein: trafficking of vacuolar proteins, plasma membrane proteins, and trans-Golgi network (TGN)-localized SYP61 was strongly inhibited; trafficking of TGN-localized SYP51 was slightly inhibited; and trafficking of secretory proteins and TGN-localized SYP41 was negligibly or not significantly inhibited. Based on these results, we propose that Golgi-localized AtPRA1.F4 is involved in the exit of many but not all types of post-Golgi proteins from the Golgi apparatus. Additionally, an appropriate level of AtPRA1.F4 is crucial for its function at the Golgi apparatus. © 2017 American Society of Plant Biologists. All Rights Reserved.

  4. In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism

    Directory of Open Access Journals (Sweden)

    Donohue-Rolfe Arthur

    2011-06-01

    Full Text Available Abstract Background Shigella dysenteriae serotype 1 (SD1 causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1 in vitro (derived from LB cell cultures and in vivo (derived from gnotobiotic piglets was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology. Results Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate, including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM proteins (38% of in silico predicted SD1 membrane proteome contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA and mixed acid fermentation (PflA/PflB indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB were increased, while β-barrel OM proteins (OmpA, OM lipoproteins (NlpD, chaperones involved in OM protein folding pathways (YraP, NlpB and lipopolysaccharide biosynthesis (Imp were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins required for invasion of colonic epithelial cells, and release

  5. IBT-based quantitative proteomics identifies potential regulatory proteins involved in pigmentation of purple sea cucumber, Apostichopus japonicus.

    Science.gov (United States)

    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.

  6. Comparative Proteomic Analysis Reveals Proteins Putatively Involved in Toxin Biosynthesis in the Marine Dinoflagellate Alexandrium catenella

    Directory of Open Access Journals (Sweden)

    Da-Zhi Wang

    2013-01-01

    Full Text Available Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P < 0.05, and 53 proteins were identified using database searching. These proteins were involved in a variety of biological processes, i.e., protein modification and biosynthesis, metabolism, cell division, oxidative stress, transport, signal transduction, and translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to, alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

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

    Directory of Open Access Journals (Sweden)

    Carlo Travaglini-Allocatelli

    2013-01-01

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

  8. Sensitive Electrochemical Detection of Native and Aggregated x-Synuclein Protein Involved in Parkinson's Disease

    NARCIS (Netherlands)

    Masarik, Michal; Stobiecka, Agata; Kizek, René; Jelen, Frantisek; Pechan, Zdenk; Hoyer, Wolfgang; Subramaniam, Vinod; Palecek, Emil

    2004-01-01

    The aggregation of α-synuclein, a 14 kDa protein, is involved in several human neurodegenerative disorders, including Parkinson's disease. We studied native and in vitro aggregated α-synuclein by circular dichroism (CD), atomic force microscopy (AFM) and electrochemical methods. We used constant

  9. Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor

    Science.gov (United States)

    Gjetting, Kisten Sisse Krag; Ytting, Cecilie Karkov; Schulz, Alexander; Fuglsang, Anja Thoe

    2012-01-01

    Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots. PMID:22407646

  10. Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor.

    Science.gov (United States)

    Gjetting, Kisten Sisse Krag; Ytting, Cecilie Karkov; Schulz, Alexander; Fuglsang, Anja Thoe

    2012-05-01

    Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots.

  11. The proteins of Fusobacterium spp. involved in hydrogen sulfide production from L-cysteine.

    Science.gov (United States)

    Basic, Amina; Blomqvist, Madeleine; Dahlén, Gunnar; Svensäter, Gunnel

    2017-03-14

    Hydrogen sulfide (H 2 S) is a toxic foul-smelling gas produced by subgingival biofilms in patients with periodontal disease and is suggested to be part of the pathogenesis of the disease. We studied the H 2 S-producing protein expression of bacterial strains associated with periodontal disease. Further, we examined the effect of a cysteine-rich growth environment on the synthesis of intracellular enzymes in F. nucleatum polymorphum ATCC 10953. The proteins were subjected to one-dimensional (1DE) and two-dimensional (2DE) gel electrophoresis An in-gel activity assay was used to detect the H 2 S-producing enzymes; Sulfide from H 2 S, produced by the enzymes in the gel, reacted with bismuth forming bismuth sulfide, illustrated as brown bands (1D) or spots (2D) in the gel. The discovered proteins were identified with liquid chromatography - tandem mass spectrometry (LC-MS/MS). Cysteine synthase and proteins involved in the production of the coenzyme pyridoxal 5'phosphate (that catalyzes the production of H 2 S) were frequently found among the discovered enzymes. Interestingly, a higher expression of H 2 S-producing enzymes was detected from bacteria incubated without cysteine prior to the experiment. Numerous enzymes, identified as cysteine synthase, were involved in the production of H 2 S from cysteine and the expression varied among Fusobacterium spp. and strains. No enzymes were detected with the in-gel activity assay among the other periodontitis-associated bacteria tested. The expression of the H 2 S-producing enzymes was dependent on environmental conditions such as cysteine concentration and pH but less dependent on the presence of serum and hemin.

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

    Science.gov (United States)

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

    2016-04-09

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

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

    Directory of Open Access Journals (Sweden)

    Peter eMoffett

    2015-08-01

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

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

    KAUST Repository

    Ali, Shawkat

    2015-08-11

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

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

    International Nuclear Information System (INIS)

    Gupta, S.K.; Woda, B.

    1986-01-01

    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 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

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1.

    Science.gov (United States)

    Ishikawa-Sasaki, Kumiko; Nagashima, Shigeo; Taniguchi, Koki; Sasaki, Jun

    2018-04-15

    Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs. IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of

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

    Directory of Open Access Journals (Sweden)

    Juliana Ide Aoki

    2016-09-01

    Full Text Available 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.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.This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine

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

    Science.gov (United States)

    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

    2016-09-01

    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. 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. This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine metabolism is affected

  20. A polycomb group protein, PHF1, is involved in the response to DNA double-strand breaks in human cell

    Science.gov (United States)

    Hong, Zehui; Jiang, Jie; Lan, Li; Nakajima, Satoshi; Kanno, Shin-ichiro; Koseki, Haruhiko; Yasui, Akira

    2008-01-01

    DNA double-strand breaks (DSBs) represent the most toxic DNA damage arisen from endogenous and exogenous genotoxic stresses and are known to be repaired by either homologous recombination or nonhomologous end-joining processes. Although many proteins have been identified to participate in either of the processes, the whole processes still remain elusive. Polycomb group (PcG) proteins are epigenetic chromatin modifiers involved in gene silencing, cancer development and the maintenance of embryonic and adult stem cells. By screening proteins responding to DNA damage using laser micro-irradiation, we found that PHF1, a human homolog of Drosophila polycomb-like, Pcl, protein, was recruited to DSBs immediately after irradiation and dissociated within 10 min. The accumulation at DSBs is Ku70/Ku80-dependent, and knockdown of PHF1 leads to X-ray sensitivity and increases the frequency of homologous recombination in HeLa cell. We found that PHF1 interacts physically with Ku70/Ku80, suggesting that PHF1 promotes nonhomologous end-joining processes. Furthermore, we found that PHF1 interacts with a number of proteins involved in DNA damage responses, RAD50, SMC1, DHX9 and p53, further suggesting that PHF1, besides the function in PcG, is involved in genome maintenance processes. PMID:18385154

  1. eIF4A inhibition allows translational regulation of mRNAs encoding proteins involved in Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Andrew Bottley

    2010-09-01

    Full Text Available Alzheimer's disease (AD is the main cause of dementia in our increasingly aging population. The debilitating cognitive and behavioral symptoms characteristic of AD make it an extremely distressing illness for patients and carers. Although drugs have been developed to treat AD symptoms and to slow disease progression, there is currently no cure. The incidence of AD is predicted to increase to over one hundred million by 2050, placing a heavy burden on communities and economies, and making the development of effective therapies an urgent priority. Two proteins are thought to have major contributory roles in AD: the microtubule associated protein tau, also known as MAPT; and the amyloid-beta peptide (A-beta, a cleavage product of amyloid precursor protein (APP. Oxidative stress is also implicated in AD pathology from an early stage. By targeting eIF4A, an RNA helicase involved in translation initiation, the synthesis of APP and tau, but not neuroprotective proteins, can be simultaneously and specifically reduced, representing a novel avenue for AD intervention. We also show that protection from oxidative stress is increased upon eIF4A inhibition. We demonstrate that the reduction of these proteins is not due to changes in mRNA levels or increased protein degradation, but is a consequence of translational repression conferred by inhibition of the helicase activity of eIF4A. Inhibition of eIF4A selectively and simultaneously modulates the synthesis of proteins involved in Alzheimer's disease: reducing A-beta and tau synthesis, while increasing proteins predicted to be neuroprotective.

  2. Characterization of Heme Proteins Involved in Microbial Exoelectric Activity and Small Molecule-Sensing

    KAUST Repository

    Vogler, Malvina M.

    2018-01-01

    Heme proteins, also termed cytochromes, are a widespread class of metalloproteins containing an Fe-protoporphyrin IX cofactor. They perform numerous functions in nature such as oxygen-transport by hemoglobin, monooxygenation reactions catalyzed by Cytochrome P-450, and electron transfer reactions during photosynthesis. The differences between proteincofactor binding characteristics and the cofactor environment greatly influence the extensive range of functions. In this dissertation, proteins from the Mtr pathway of Shewanella oneidensis are characterized. These c-type cytochromes contain multiple heme cofactors per protein molecule that covalently attach to the protein amino acid sequence and are involved in electron transfer to extracellular metal oxides during anaerobic conditions. Successful recombinant expression of pathway components MtrC and MtrA is achieved in Escherichia coli. Heme-dependent gel staining and UV/Vis spectroscopy show characteristic c-type cytochrome characteristics. Mass spectrometry confirms that the correct extensive post-translational modifications were performed and the ten heme groups were incorporated per protein of MtrC and MtrA and the correct lipid-anchor was attached to extracellular MtrC. Raman spectroscopy measurements of MtrA provide intriguing structural information and highlight the strong influence of the heme cofactors within the protein structure. Next, an Arabidopsis thaliana protein is analyzed. It was previously identified via a motif search of the plant genome, based on conserved residues in the H4 NOX pocket. Here, the incorporation of a heme b cofactor is confirmed. UV/Vis spectroscopy under anaerobic conditions demonstrates reversible binding of nitric oxide to the heme iron and depicts the previously published characteristic absorption maxima for other H-NOX proteins.

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

    DEFF Research Database (Denmark)

    Ü Basmanav, F Buket; Cau, Laura; Tafazzoli, Aylar

    2016-01-01

    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...... 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...

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Ladislav eDokládal

    2015-11-01

    Full Text Available 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 a 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.

  6. The p38 mitogen-activated protein kinase signaling pathway is involved in regulating low-density lipoprotein receptor-related protein 1-mediated β-amyloid protein internalization in mouse brain.

    Science.gov (United States)

    Ma, Kai-Ge; Lv, Jia; Hu, Xiao-Dan; Shi, Li-Li; Chang, Ke-Wei; Chen, Xin-Lin; Qian, Yi-Hua; Yang, Wei-Na; Qu, Qiu-Min

    2016-07-01

    Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Recently, increasing evidence suggests that intracellular β-amyloid protein (Aβ) alone plays a pivotal role in the progression of AD. Therefore, understanding the signaling pathway and proteins that control Aβ internalization may provide new insight for regulating Aβ levels. In the present study, the regulation of Aβ internalization by p38 mitogen-activated protein kinases (MAPK) through low-density lipoprotein receptor-related protein 1 (LRP1) was analyzed in vivo. The data derived from this investigation revealed that Aβ1-42 were internalized by neurons and astrocytes in mouse brain, and were largely deposited in mitochondria and lysosomes, with some also being found in the endoplasmic reticulum. Aβ1-42-LRP1 complex was formed during Aβ1-42 internalization, and the p38 MAPK signaling pathway was activated by Aβ1-42 via LRP1. Aβ1-42 and LRP1 were co- localized in the cells of parietal cortex and hippocampus. Furthermore, the level of LRP1-mRNA and LRP1 protein involved in Aβ1-42 internalization in mouse brain. The results of this investigation demonstrated that Aβ1-42 induced an LRP1-dependent pathway that related to the activation of p38 MAPK resulting in internalization of Aβ1-42. These results provide evidence supporting a key role for the p38 MAPK signaling pathway which is involved in the regulation of Aβ1-42 internalization in the parietal cortex and hippocampus of mouse through LRP1 in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Hydrogen Sulfide Alleviates Aluminum Toxicity via Decreasing Apoplast and Symplast Al Contents in Rice

    Directory of Open Access Journals (Sweden)

    Chun Q. Zhu

    2018-03-01

    Full Text Available Hydrogen sulfide (H2S plays a vital role in Al3+ stress resistance in plants, but the underlying mechanism is unclear. In the present study, pretreatment with 2 μM of the H2S donor NaHS significantly alleviated the inhibition of root elongation caused by Al toxicity in rice roots, which was accompanied by a decrease in Al contents in root tips under 50 μM Al3+ treatment. NaHS pretreatment decreased the negative charge in cell walls by reducing the activity of pectin methylesterase and decreasing the pectin and hemicellulose contents in rice roots. This treatment also masked Al-binding sites in the cell wall by upregulating the expression of OsSATR1 and OsSTAR2 in roots and reduced Al binding in the cell wall by stimulating the expression of the citrate acid exudation gene OsFRDL4 and increasing the secretion of citrate acid. In addition, NaHS pretreatment decreased the symplasmic Al content by downregulating the expression of OsNRAT1, and increasing the translocation of cytoplasmic Al to the vacuole via upregulating the expression of OsALS1. The increment of antioxidant enzyme [superoxide dismutase (SOD, ascorbate peroxidase (APX, catalase (CAT, and peroxidase (POD] activity with NaHS pretreatment significantly decreased the MDA and H2O2 content in rice roots, thereby reducing the damage of Al3+ toxicity on membrane integrity in rice. H2S exhibits crosstalk with nitric oxide (NO in response to Al toxicity, and through reducing NO content in root tips to alleviate Al toxicity. Together, this study establishes that H2S alleviates Al toxicity by decreasing the Al content in the apoplast and symplast of rice roots.

  8. MPK6 controls H2 O2-induced root elongation by mediating Ca2+ influx across the plasma membrane of root cells in Arabidopsis seedlings.

    Science.gov (United States)

    Han, Shuan; Fang, Lin; Ren, Xuejian; Wang, Wenle; Jiang, Jing

    2015-01-01

    Mitogen-activated protein kinases (MPKs) play critical roles in signalling and growth, and Ca(2+) and H2 O2 control plant growth processes associated with abscisic acid (ABA). However, it remains unclear how MPKs are involved in H2 O2 - and Ca(2+) -mediated root elongation. Root elongation in seedlings of the loss-of-function mutant Atmpk6 (Arabidopsis thaliana MPK6) was less sensitive to moderate H2 O2 or ABA than that in wild-type (WT) plants. The enhanced elongation was a result of root cell expansion. This effect disappeared when ABA-induced H2 O2 accumulation or the cytosolic Ca(2+) increase were defective. Molecular and biochemical evidence showed that increased expression of the cell wall peroxidase PRX34 in Atmpk6 root cells enhanced apoplastic H2 O2 generation; this promoted a cytosolic Ca(2+) increase and Ca(2+) influx across the plasma membrane. The plasma membrane damage caused by high levels of H2 O2 was ameliorated in a Ca(2+) -dependent manner. These results suggested that there was intensified PRX34-mediated H2 O2 generation in the apoplast and increased Ca(2+) flux into the cytosol of Atmpk6 root cells; that is, the spatial separation of apoplastic H2 O2 from cytosolic Ca(2+) in root cells prevented H2 O2 -induced inhibition of root elongation in Atmpk6 seedlings. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  9. Evidence for the involvement of 5-lipoxygenase products in ethanol-induced intestinal plasma protein loss

    International Nuclear Information System (INIS)

    Beck, I.T.; Boyd, A.J.; Dinda, P.K.

    1988-01-01

    In this study the authors investigated whether the products of 5-lipoxygenase (5-LO) were involved in the jejunal microvascular injury induced by intraluminal ethanol (ETH). A group of rabbits was given orally a selective inhibitor of 5-LO in two 10-mg doses, 24, and 2 h before the experiments. A jejunal segment was perfused with a control solution (control segment) and an adjacent segment with an ETH-containing solution (ETH-perfused segment). In a series of experiments, they measured 5-LO activity of the jejunal segments of both groups using the generation of leukotriene B 4 (LTB 4 ) as an index. In a second series of experiments, they determined the ETH-induced intraluminal protein loss, which was taken as a measure of mucosal microvascular damage. The ETH-induced increase in protein loss was significantly lower in the treated than in the untreated group. These findings suggest that products of 5-LO are involved in the ETH-induced jejunal microvascular injury

  10. Proteins Potentially Involved in Immune Evasion Strategies in Sporothrix brasiliensis Elucidated by Ultra-High-Resolution Mass Spectrometry.

    Science.gov (United States)

    Rossato, Luana; Moreno, Leandro Ferreira; Jamalian, Azadeh; Stielow, Benjamin; de Almeida, Sandro Rogério; de Hoog, Sybren; Freeke, Joanna

    2018-06-27

    Sporothrix brasiliensis is the prevalent agent of a large zoonotic outbreak in Brazil. With the involvement of several thousands of cases, this is the largest cohort of human and animal sporotrichosis on record in the world. Infections are characterized by local cutaneous dissemination in humans without underlying disease. S. brasiliensis has shown a high degree of virulence in a mouse model compared to the remaining Sporothrix species, including the ancestral species, Sporothrix schenckii The present paper investigates a genomic and expressed-proteome comparison of S. brasiliensis to S. schenckii Using bottom-up proteomics, we found 60 proteins exclusively expressed in S. brasiliensis No significant genomic differences were found among the genes coding for this protein set. A comparison with literature data identified nine proteins that are known to be involved in virulence and immune evasion in other species, several of which had not yet been reported for the Sporothrix species analyzed. IMPORTANCE Sporotrichosis is an important disease in Brazil that is caused by fungi of the genus Sporothrix and affects cats and humans. Our work investigated the proteins differentially expressed by S. brasiliensis in order to find out why this species is more virulent and pathogenic than S. schenckii We verified a set of proteins that may be related to immune escape and that can explain the high virulence. Copyright © 2018 Rossato et al.

  11. Freezing avoidance by supercooling in Olea europaea cultivars: the role of apoplastic water, solute content and cell wall rigidity.

    Science.gov (United States)

    Arias, Nadia S; Bucci, Sandra J; Scholz, Fabian G; Goldstein, Guillermo

    2015-10-01

    Plants can avoid freezing damage by preventing extracellular ice formation below the equilibrium freezing temperature (supercooling). We used Olea europaea cultivars to assess which traits contribute to avoid ice nucleation at sub-zero temperatures. Seasonal leaf water relations, non-structural carbohydrates, nitrogen and tissue damage and ice nucleation temperatures in different plant parts were determined in five cultivars growing in the Patagonian cold desert. Ice seeding in roots occurred at higher temperatures than in stems and leaves. Leaves of cold acclimated cultivars supercooled down to -13 °C, substantially lower than the minimum air temperatures observed in the study site. During winter, leaf ice nucleation and leaf freezing damage (LT50 ) occurred at similar temperatures, typical of plant tissues that supercool. Higher leaf density and cell wall rigidity were observed during winter, consistent with a substantial acclimation to sub-zero temperatures. Larger supercooling capacity and lower LT50 were observed in cold-acclimated cultivars with higher osmotically active solute content, higher tissue elastic adjustments and lower apoplastic water. Irreversible leaf damage was only observed in laboratory experiments at very low temperatures, but not in the field. A comparative analysis of closely related plants avoids phylogenetic independence bias in a comparative study of adaptations to survive low temperatures. © 2015 John Wiley & Sons Ltd.

  12. Twister Protein: a ludic tool involving protein synthesis

    Directory of Open Access Journals (Sweden)

    Aline Weyh

    2015-07-01

    Full Text Available Several studies show that students of various grade levels report the Genetics as an abstract theme and difficult to assimilate by the students, with multiple problems in the teaching-learning process and becoming necessary the development of auxiliary practices. Among the teaching tools, the game is the most currently opted playful activity by stimulating multiple intelligences, allowing greater student-teacher interaction. This work seeks the production of an innovative and dynamic educational game, Twister Protein, as a pedagogical resource for Genetics discipline. The development of the game was based on the use of easily accessible and low cost materials by teachers, allowing the knowledge of transcription, translation and protein folding. The activity was proposed and applied in the classroom with pilot undergraduate students. The fun associated with the knowledge of science not only allowed a better memorization of the content addressed, as aroused the curiosity, theme reflection, character building and collaborative spirits, as well as competitiveness through the interaction between class. This practice proved to be an effective tool in the escape from routine and fault repair of the theoretical process.

  13. Identification of New Epididymal Luminal Fluid Proteins Involved in Sperm Maturation in Infertile Rats Treated by Dutasteride Using iTRAQ

    Directory of Open Access Journals (Sweden)

    Shu-Wu Xie

    2016-05-01

    Full Text Available Background: Spermatozoa become mature and acquire fertilizing capacity during their passage through the epididymal lumen. In this study, we identified new epididymal luminal fluid proteins involved in sperm maturation in infertile rats by dutasteride, a dual 5α-reductase inhibitor, in order to provide potential epididymal targets for new contraceptives and infertility treatment. Methods: Male rats were treated with dutasteride for 28 consecutive days. We observed the protein expression profiles in the epididymal luminal fluids in infertile and normal rats using isobaric tags for relative and absolute quantitation (iTRAQ technique. The confidence of proteome data was validated by enzyme-linked immunosorbent assays. Results: 1045 proteins were tested, and 23 of them presented different expression profiling in the infertile and normal rats. The seven proteins were down-regulated, and 16 proteins were up-regulated. Among the seven proteins which were significantly down-regulated by dutasteride in the epididymal luminal fluids, there were three β-defensins (Defb2, Defb18 and Defb39, which maybe the key proteins involved in epididymal sperm maturation and male fertility. Conclusions: We report for the first time that dutasteride influences the protein expression profiling in the epididymal luminal fluids of rats, and this result provides some new epididymal targets for male contraception and infertility therapy.

  14. Determination of extra and intracellular content from some lytic enzymes related with carnation (Dianthus caryophyllus L. root cell wall

    Directory of Open Access Journals (Sweden)

    Sixta Tulia Martínez Peralta

    2016-11-01

    Full Text Available The presence of some enzymes related to cell wall (polygalacturonase, the pectate lyase, protease and xylanase in carnation (Dianthus caryophyllus L. roots as well as the activity levels were determined. These levels were analyzed in different cellular places: the intercellular fluid that is part of apoplast, the symplast, and the total level (apoplast and symplast in carnation roots. Two methods were tested to extract the intercellular fluid. To obtain the intracellular content (symplast and total extract (apoplast+symplast, three methods were tested, using as extracting solution  i phosphate buffer, ii phosphate buffer + PVPP,  iii before the extraction with phosphate buffer, the carnation roots were washed with acetone.  The results showed the effect of different extracting solutions in the enzymatic activities and in the protein content. A new only one step method is proposed to extract the four enzymes and make the comparative analysis of enzymatic activity.

  15. Kin3 protein, a NIMA-related kinase of Saccharomyces cerevisiae, is involved in DNA adduct damage response.

    Science.gov (United States)

    Moura, Dinara J; Castilhos, Bruna; Immich, Bruna F; Cañedo, Andrés D; Henriques, João A P; Lenz, Guido; Saffi, Jenifer

    2010-06-01

    Kin3 is a nonessential serine/threonine protein kinase of the budding yeast Saccharomyces cerevisiae with unknown cellular role. It is an ortholog of the Aspergillus nidulans protein kinase NIMA (Never-In Mitosis, gene A), which is involved in the regulation of G2/M phase progression, DNA damage response and mitosis. The aim of this study was to determine whether Kin3 is required for proper checkpoint activation and DNA repair. Here we show that KIN3 gene deficient cells present sensitivity and fail to arrest properly at G2/M-phase checkpoint in response to the DNA damage inducing agents MMS, cisplatin, doxorubicin and nitrogen mustard, suggesting that Kin3 can be involved in DNA strand breaks recognition or signaling. In addition, there is an increase in KIN3 gene expression in response to the mutagenic treatment, which was confirmed by the increase of Kin3 protein. We also showed that co-treatment with caffeine induces a slight increase in the susceptibility to genotoxic agents in kin3 cells and abolishes KIN3 gene expression in wild-type strain, suggesting that Kin3p can play a role in Tel1/Mec1-dependent pathway activation induced after genotoxic stress. These data provide the first evidence of the involvement of S. cerevisiae Kin3 in the DNA damage response.

  16. Elicitin-induced distal systemic resistance in plants is mediated through the protein-protein interactions influenced by selected lysine residues

    Directory of Open Access Journals (Sweden)

    Hana eUhlíková

    2016-02-01

    Full Text Available Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium spp. classified as oomycete PAMPs. Although alfa- and beta-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, beta-elicitins (possessing 6-7 lysine residues are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the alfa-isoforms (with only 1-3 lysine residues.To examine the role of lysine residues in elicitin biological activity, we employed site-directed mutagenesis to prepare a series of beta-elicitin cryptogein variants with mutations on specific lysine residues. In contrast to direct infiltration of protein into leaves, application to the stem revealed a rough correlation between protein’s charge and biological activity, resulting in protection against Phytophthora parasitica. A detailed analysis of proteins’ movement in plants showed no substantial differences in distribution through phloem indicating differences in consequent apoplastic or symplastic transport. In this process, an important role of homodimer formation together with the ability to form a heterodimer with potential partner represented by endogenous plants LTPs is suggested. Our work demonstrates a key role of selected lysine residues in these interactions and stresses the importance of processes preceding elicitin recognition responsible for induction of distal systemic resistance.

  17. Involvement of Calmodulin and Calmodulin-like Proteins in Plant Responses to Abiotic Stresses

    Directory of Open Access Journals (Sweden)

    B W Poovaiah

    2015-08-01

    Full Text Available 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 ROS 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 crops.

  18. Macrophage replication screen identifies a novel Francisella hydroperoxide resistance protein involved in virulence.

    Directory of Open Access Journals (Sweden)

    Anna C Llewellyn

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

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

    Science.gov (United States)

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

    2017-06-01

    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. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Role of the NAD(P)H quinone oxidoreductase NQR and the cytochrome b AIR12 in controlling superoxide generation at the plasma membrane.

    Science.gov (United States)

    Biniek, Catherine; Heyno, Eiri; Kruk, Jerzy; Sparla, Francesca; Trost, Paolo; Krieger-Liszkay, Anja

    2017-04-01

    The quinone reductase NQR and the b-type cytochrome AIR12 of the plasma membrane are important for the control of reactive oxygen species in the apoplast. AIR12 and NQR are two proteins attached to the plant plasma membrane which may be important for generating and controlling levels of reactive oxygen species in the apoplast. AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b. The NADPH quinone oxidoreductase NQR is a two-electron-transferring flavoenzyme that contributes to the generation of O 2 •- in isolated plasma membranes. A. thaliana double knockout plants of both NQR and AIR12 generated more O 2 •- and germinated faster than the single mutant affected in AIR12. To test whether NQR and AIR12 are able to interact functionally, recombinant purified proteins were added to plasma membranes isolated from soybean hypocotyls. In vitro NADH-dependent O 2 •- production at the plasma membrane in the presence of NQR was reduced upon addition of AIR12. Electron donation from semi-reduced menadione to AIR12 was shown to take place. Biochemical analysis showed that purified plasma membrane from soybean hypocotyls or roots contained phylloquinone and menaquinone-4 as redox carriers. This is the first report on the occurrence of menaquinone-4 in eukaryotic photosynthetic organisms. We propose that NQR and AIR12 interact via the quinone, allowing an electron transfer from cytosolic NAD(P)H to apoplastic monodehydroascorbate and control thereby the level of reactive oxygen production and the redox state of the apoplast.

  1. Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

    Science.gov (United States)

    Xin, Qi-Lin; Deng, Cheng-Lin; Chen, Xi; Wang, Jun; Wang, Shao-Bo; Wang, Wei; Deng, Fei; Zhang, Bo; Xiao, Gengfu; Zhang, Lei-Ke

    2017-06-15

    Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate

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

    International Nuclear Information System (INIS)

    Khotin, Mikhail; Turoverova, Lidia; Aksenova, Vasilisa; Barlev, Nikolai; Borutinskaite, Veronika Viktorija; Vener, Alexander; Bajenova, Olga; Magnusson, Karl-Eric; Pinaev, George P.; Tentler, Dmitri

    2010-01-01

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

  3. Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV replication.

    Directory of Open Access Journals (Sweden)

    Ji'an Pan

    Full Text Available Analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12 provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.

  4. Involvement of fractalkine and macrophage inflammatory protein-1 alpha in moderate-severe depression

    Directory of Open Access Journals (Sweden)

    Rosaria Alba Merendino

    2004-01-01

    Full Text Available MODERATE-severe depression (MSD is linked to overexpression of proinflammatory cytokines and chemokines. Fractalkine (FKN and macrophage inflammatory protein-1 alpha (MIP-1α are, respectively, members of CX3C and C-C chemokines, and both are involved in recruiting and activating mononuclear phagocytes in the central nervous system. We analysed the presence of FKN and MIP-1α in sera of untreated MSD patients and healthy donors. High FKN levels were observed in all MSD patients as compared with values only detectable in 26% of healthy donors. MIP-1α was measurable in 20% of patients, while no healthy donors showed detectable chemokine levels. In conclusion, we describe a previously unknown involvement of FKN in the pathogenesis of MSD, suggesting that FKN may represent a target for a specific immune therapy of this disease.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

    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-κB 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-κB-activating TRAF family proteins are potential cellular E3 ubiquitin ligases toward Tax

  6. DCD – a novel plant specific domain in proteins involved in development and programmed cell death

    Directory of Open Access Journals (Sweden)

    Doerks Tobias

    2005-07-01

    Full Text Available Abstract Background Recognition of microbial pathogens by plants triggers the hypersensitive reaction, a common form of programmed cell death in plants. These dying cells generate signals that activate the plant immune system and alarm the neighboring cells as well as the whole plant to activate defense responses to limit the spread of the pathogen. The molecular mechanisms behind the hypersensitive reaction are largely unknown except for the recognition process of pathogens. We delineate the NRP-gene in soybean, which is specifically induced during this programmed cell death and contains a novel protein domain, which is commonly found in different plant proteins. Results The sequence analysis of the protein, encoded by the NRP-gene from soybean, led to the identification of a novel domain, which we named DCD, because it is found in plant proteins involved in development and cell death. The domain is shared by several proteins in the Arabidopsis and the rice genomes, which otherwise show a different protein architecture. Biological studies indicate a role of these proteins in phytohormone response, embryo development and programmed cell by pathogens or ozone. Conclusion It is tempting to speculate, that the DCD domain mediates signaling in plant development and programmed cell death and could thus be used to identify interacting proteins to gain further molecular insights into these processes.

  7. Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription.

    Science.gov (United States)

    Assenberg, R; Delmas, O; Morin, B; Graham, S C; De Lamballerie, X; Laubert, C; Coutard, B; Grimes, J M; Neyts, J; Owens, R J; Brandt, B W; Gorbalenya, A; Tucker, P; Stuart, D I; Canard, B; Bourhy, H

    2010-08-01

    Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication. Copyright 2010 Elsevier B.V. All rights reserved.

  8. Mechanosensitive molecular networks involved in transducing resistance exercise-signals into muscle protein accretion

    Directory of Open Access Journals (Sweden)

    Emil Rindom

    2016-11-01

    Full Text Available 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 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 2TSC2-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 how it may implicate deformation of Notch to affect Bone Morphogenetic Protein (BMP signaling through a small mother of decapentaplegic (Smad axis.

  9. The small nucleoid protein Fis is involved in Vibrio cholerae quorum sensing.

    Science.gov (United States)

    Lenz, Derrick H; Bassler, Bonnie L

    2007-02-01

    Quorum sensing is a process of cell-cell communication that bacteria use to relay information to one another about the cell density and species composition of the bacterial community. Quorum sensing involves the production, secretion and population-wide detection of small signalling molecules called autoinducers. This process allows bacteria to synchronize group behaviours and act as multicellular units. The human pathogen, Vibrio cholerae, uses quorum sensing to co-ordinate such complex behaviours as pathogenicity and biofilm formation. The quorum-sensing circuit of V. cholerae consists of two autoinducer/sensor systems, CAI-1/CqsS and AI-2/LuxPQ, and the VarS/A-CsrA/BCD growth-phase regulatory system. Genetic analysis suggests that an additional regulatory arm involved in quorum sensing exists in V. cholerae. All of these systems channel information into the histidine phosphotransfer protein, LuxU, and/or the response regulator, LuxO. LuxO, when phosphorylated, activates the expression of four genes encoding the Qrr (quorum regulatory RNAs) small RNAs (sRNAs). The Qrr sRNAs destabilize the hapR transcript encoding the master regulator of quorum sensing, HapR. Here we identify the nucleoid protein Fis as playing a major role in the V. cholerae quorum-sensing circuit. Fis fulfils the predictions required to be the putative additional component that inputs information into the cascade: its expression is regulated in a growth phase-dependent manner; it requires LuxO but acts independently of LuxU, and it regulates all four qrr genes and, in turn, HapR by directly binding to the qrr gene promoters and modulating their expression.

  10. Peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus

    International Nuclear Information System (INIS)

    Rowland, Raymond R.R.; Schneider, Paula; Fang Ying; Wootton, Sarah; Yoo, Dongwan; Benfield, David A.

    2003-01-01

    The nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is the principal component of the viral nucleocapsid and localizes to the nucleolus. Peptide sequence analysis of the N protein of several North American isolates identified two potential nuclear localization signal (NLS) sequences located at amino acids 10-13 and 41-42, which were labeled NLS-1 and NLS-2, respectively. Peptides containing NLS-1 or NLS-2 were sufficient to accumulate enhanced green fluorescent protein (EGFP) in the nucleus. The inactivation of NLS-1 by site-directed mutagenesis or the deletion of the first 14 amino acids did not affect N protein localization to the nucleolus. The substitution of key lysine residues with uncharged amino acids in NLS-2 blocked nuclear/nucleolar localization. Site-directed mutagenesis within NLS-2 identified the sequence, KKNKK, as forming the core localization domain within NLS-2. Using an in vitro pull-down assay, the N protein was able to bind importin-α, importin-β nuclear transport proteins. The localization pattern of N-EGFP fusion peptides represented by a series of deletions from the C- and N-terminal ends of the N protein identified a region covering amino acids 41-72, which contained a nucleolar localization signal (NoLS) sequence. The 41-72 N peptide when fused to EGFP mimicked the nucleolar-cytoplasmic distribution of native N. These results identify a single NLS involved in the transport of N from the cytoplasm and into nucleus. An additional peptide sequence, overlapping NLS-2, is involved in the further targeting of N to the nucleolus

  11. RPA-Binding Protein ETAA1 Is an ATR Activator Involved in DNA Replication Stress Response.

    Science.gov (United States)

    Lee, Yuan-Cho; Zhou, Qing; Chen, Junjie; Yuan, Jingsong

    2016-12-19

    ETAA1 (Ewing tumor-associated antigen 1), also known as ETAA16, was identified as a tumor-specific antigen in the Ewing family of tumors. However, the biological function of this protein remains unknown. Here, we report the identification of ETAA1 as a DNA replication stress response protein. ETAA1 specifically interacts with RPA (Replication protein A) via two conserved RPA-binding domains and is therefore recruited to stalled replication forks. Interestingly, further analysis of ETAA1 function revealed that ETAA1 participates in the activation of ATR signaling pathway via a conserved ATR-activating domain (AAD) located near its N terminus. Importantly, we demonstrate that both RPA binding and ATR activation are required for ETAA1 function at stalled replication forks to maintain genome stability. Therefore, our data suggest that ETAA1 is a new ATR activator involved in replication checkpoint control. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1.

    Science.gov (United States)

    Bakari, Sana; Lembrouk, Mehdi; Sourd, Laura; Ousalem, Fares; André, François; Orlowski, Stéphane; Delaforge, Marcel; Frelet-Barrand, Annie

    2016-04-01

    Despite the great importance of human membrane proteins involved in detoxification mechanisms, their wide use for biochemical approaches is still hampered by several technical difficulties considering eukaryotic protein expression in order to obtain the large amounts of protein required for functional and/or structural studies. Lactococcus lactis has emerged recently as an alternative heterologous expression system to Escherichia coli for proteins that are difficult to express. The aim of this work was to check its ability to express mammalian membrane proteins involved in liver detoxification, i.e., CYP3A4 and two isoforms of MGST1 (rat and human). Genes were cloned using two different strategies, i.e., classical or Gateway-compatible cloning, and we checked the possible influence of two affinity tags (6×-His-tag and Strep-tag II). Interestingly, all proteins could be successfully expressed in L. lactis at higher yields than those previously obtained for these proteins with classical expression systems (E. coli, Saccharomyces cerevisiae) or those of other eukaryotic membrane proteins expressed in L. lactis. In addition, rMGST1 was fairly active after expression in L. lactis. This study highlights L. lactis as an attractive system for efficient expression of mammalian detoxification membrane proteins at levels compatible with further functional and structural studies.

  13. Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

    Science.gov (United States)

    Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

    2009-12-01

    A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

  14. Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

    Energy Technology Data Exchange (ETDEWEB)

    Dahms, Sven O., E-mail: sdahms@fli-leibniz.de [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany); Mayer, Magnus C. [Freie Universität Berlin, Thielallee 63, 14195 Berlin (Germany); Miltenyi Biotec GmbH, Robert-Koch-Strasse 1, 17166 Teterow (Germany); Roeser, Dirk [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany); Multhaup, Gerd [McGill University Montreal, Montreal, Quebec H3G 1Y6 (Canada); Than, Manuel E., E-mail: sdahms@fli-leibniz.de [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany)

    2015-03-01

    Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects various (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.

  15. ProFITS of maize: a database of protein families involved in the transduction of signalling in the maize genome

    Directory of Open Access Journals (Sweden)

    Zhang Zhenhai

    2010-10-01

    Full Text Available Abstract Background Maize (Zea mays ssp. mays L. is an important model for plant basic and applied research. In 2009, the B73 maize genome sequencing made a great step forward, using clone by clone strategy; however, functional annotation and gene classification of the maize genome are still limited. Thus, a well-annotated datasets and informative database will be important for further research discoveries. Signal transduction is a fundamental biological process in living cells, and many protein families participate in this process in sensing, amplifying and responding to various extracellular or internal stimuli. Therefore, it is a good starting point to integrate information on the maize functional genes involved in signal transduction. Results Here we introduce a comprehensive database 'ProFITS' (Protein Families Involved in the Transduction of Signalling, which endeavours to identify and classify protein kinases/phosphatases, transcription factors and ubiquitin-proteasome-system related genes in the B73 maize genome. Users can explore gene models, corresponding transcripts and FLcDNAs using the three abovementioned protein hierarchical categories, and visualize them using an AJAX-based genome browser (JBrowse or Generic Genome Browser (GBrowse. Functional annotations such as GO annotation, protein signatures, protein best-hits in the Arabidopsis and rice genome are provided. In addition, pre-calculated transcription factor binding sites of each gene are generated and mutant information is incorporated into ProFITS. In short, ProFITS provides a user-friendly web interface for studies in signal transduction process in maize. Conclusion ProFITS, which utilizes both the B73 maize genome and full length cDNA (FLcDNA datasets, provides users a comprehensive platform of maize annotation with specific focus on the categorization of families involved in the signal transduction process. ProFITS is designed as a user-friendly web interface and it is

  16. Receptor-like proteins involved in plant disease resistance

    NARCIS (Netherlands)

    Kruijt, M.; Kock, de M.J.D.; Wit, de P.J.G.M.

    2005-01-01

    Race-specific resistance in plants against microbial pathogens is governed by several distinct classes of resistance (R) genes. This review focuses on the class that consists of the plasma membrane-bound leucine-rich repeat proteins known as receptor-like proteins (RLPs). The first isolated

  17. Fnr is involved in oxygen control of Herbaspirillum seropedicae N-truncated NifA protein activity in Escherichia coli.

    Science.gov (United States)

    Monteiro, Rose A; de Souza, Emanuel M; Yates, M Geoffrey; Pedrosa, Fabio O; Chubatsu, Leda S

    2003-03-01

    Herbaspirillum seropedicae is an endophytic diazotroph belonging to the beta-subclass of the class Proteobacteria, which colonizes many members of the Gramineae. The activity of the NifA protein, a transcriptional activator of nif genes in H. seropedicae, is controlled by ammonium ions through its N-terminal domain and by oxygen through mechanisms that are not well understood. Here we report that the NifA protein of H. seropedicae is inactive and more susceptible to degradation in an fnr Escherichia coli background. Both effects correlate with oxygen exposure and iron deprivation. Our results suggest that the oxygen sensitivity and iron requirement for H. seropedicae NifA activity involve the Fnr protein.

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

    Science.gov (United States)

    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.

  19. Occurrence and properties of Petunia peroxidase a

    NARCIS (Netherlands)

    Hendriks, T.

    1989-01-01

    Peroxidases are probably the most extensively studied enzymes in higher plants. Various isoenzymes occur as soluble proteins in the apoplast and in the vacuole, or are bound to membranes and cell walls. Their occurrence is often organ-specific and developmentally controlled, and there is

  20. The GIP gamma-tubulin complex-associated proteins are involved in nuclear architecture in Arabidopsis thaliana.

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    Morgane eBatzenschlager

    2013-11-01

    Full Text Available During interphase, the microtubular cytoskeleton of cycling plant cells is organized in both cortical and perinuclear arrays. Perinuclear microtubules (MTs are nucleated from γ-Tubulin Complexes (γ-TuCs located at the surface of the nucleus. The molecular mechanisms of γ-TuC association to the nuclear envelope are currently unknown. The γ-TuC Protein 3 (GCP3-Interacting Protein 1 (GIP1 is the smallest γ-TuC component identified so far. AtGIP1 and its homologous protein AtGIP2 participate in the localization of active γ-TuCs at interphasic and mitotic MT nucleation sites. Arabidopsis gip1gip2 mutants are impaired in establishing a fully functional mitotic spindle and exhibit severe developmental defects.In this study, gip1gip2 knock down mutants were further characterized at the cellular level. In addition to defects in both the localization of γ-TuC core proteins and MT fibre robustness, gip1gip2 mutants exhibited a severe alteration of the nuclear shape associated with an abnormal distribution of the nuclear pore complexes. Simultaneously, they showed a misorganization of the inner nuclear membrane protein AtSUN1. Furthermore, AtGIP1 was identified as an interacting partner of AtTSA1 which was detected, like the AtGIP proteins, at the nuclear envelope.These results provide the first evidence for the involvement of a γ-TuC component in both nuclear shaping and nuclear envelope organization. Functional hypotheses are discussed in order to propose a model for a GIP-dependent nucleo-cytoplasmic continuum.

  1. Apoplastic Venom Allergen-like Proteins of Cyst Nematodes Modulate the Activation of Basal Plant Innate Immunity by Cell Surface Receptors

    NARCIS (Netherlands)

    Lozano Torres, J.L.; Wilbers, R.H.P.; Warmerdam, S.; Finkers-Tomczak, A.M.; Diaz Granados Muñoz, A.; Schaik, van C.C.; Helder, J.; Bakker, J.; Goverse, A.; Schots, A.; Smant, G.

    2014-01-01

    Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of

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

    Science.gov (United States)

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

    2015-06-12

    The mitochondrial contact site and cristae organizing system (MICOS) is a recently discovered protein complex that is crucial for establishing and maintaining the proper inner membrane architecture and contacts with the outer membrane of mitochondria. The ways in which the MICOS complex is assembled and its integrity is regulated remain elusive. Here, we report a direct link between Cox17, a protein involved in the assembly of cytochrome c oxidase, and the MICOS complex. Cox17 interacts with Mic60, thereby modulating MICOS complex integrity. This interaction does not involve Sco1, a partner of Cox17 in transferring copper ions to cytochrome c oxidase. However, the Cox17-MICOS interaction is regulated by copper ions. We propose that Cox17 is a newly identified factor involved in maintaining the architecture of the MICOS complex. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Mining the Human Complexome Database Identifies RBM14 as an XPO1-Associated Protein Involved in HIV-1 Rev Function

    OpenAIRE

    Budhiraja, Sona; Liu, Hongbing; Couturier, Jacob; Malovannaya, Anna; Qin, Jun; Lewis, Dorothy E.; Rice, Andrew P.

    2015-01-01

    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 ...

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

    Science.gov (United States)

    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.

  5. A polycomb group protein, PHF1, is involved in the response to DNA double-strand breaks in human cell

    OpenAIRE

    Hong, Zehui; Jiang, Jie; Lan, Li; Nakajima, Satoshi; Kanno, Shin-ichiro; Koseki, Haruhiko; Yasui, Akira

    2008-01-01

    DNA double-strand breaks (DSBs) represent the most toxic DNA damage arisen from endogenous and exogenous genotoxic stresses and are known to be repaired by either homologous recombination or nonhomologous end-joining processes. Although many proteins have been identified to participate in either of the processes, the whole processes still remain elusive. Polycomb group (PcG) proteins are epigenetic chromatin modifiers involved in gene silencing, cancer development and the maintenance of embry...

  6. Schizosaccharomyces pombe Homologs of Human DJ-1 Are Stationary Phase-Associated Proteins That Are Involved in Autophagy and Oxidative Stress Resistance.

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    Yang Su

    Full Text Available The Parkinson's disease protein DJ-1 is involved in various cellular functions including detoxification of dicarbonyl compounds, autophagy and oxidative stress response. DJ-1 homologs are widely found in both prokaryotes and eukaryotes, constituting a superfamily of proteins that appear to be involved in stress response. Schizosaccharomyces pombe contains six DJ-1 homologs, designated Hsp3101-Hsp3105 and Sdj1 (previously named SpDJ-1. Here we show that deletion of any one of these six genes somehow affects autophagy during prolonged stationary phase. Furthermore, deletions of each of these DJ-1 homologs result in reduced stationary phase survival. Deletion of sdj1 also increases the sensitivity of stationary-phase cells to oxidative stress induced by hydrogen peroxide (H2O2 whereas overexpression of sdj1 has the opposite effect. Consistent with their role in stationary phase, expression of hsp3101, hsp3102, hsp3105 and sdj1, and to a lesser extent hsp3103 and hsp3104, is increased in stationary phase. The induction of hsp3101, hsp3102, hsp3105 and sdj1 involves the Sty1-regulated transcription factor Atf1 but not the transcription factor Pap1. Our results firmly establish that S. pombe homologs of DJ-1 are stationary-phase associated proteins and are likely involved in autophagy and antioxidant defense in stationary phase of S. pombe cells.

  7. Identification and characterization of proteins involved in nuclear organization using Drosophila GFP protein trap lines.

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    Margaret Rohrbaugh

    Full Text Available Strains from a collection of Drosophila GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern.We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named CG11138. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of Drosophila insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the Decondensation factor 31 (Df31 gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that Drosophila Nucleoplasmin (dNlp is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl, a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology.These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these proteins.

  8. Proteins involved in biophoton emission and flooding-stress responses in soybean under light and dark conditions.

    Science.gov (United States)

    Kamal, Abu Hena Mostafa; Komatsu, Setsuko

    2016-02-01

    To know the molecular systems basically flooding conditions in soybean, biophoton emission measurements and proteomic analyses were carried out for flooding-stressed roots under light and dark conditions. Photon emission was analyzed using a photon counter. Gel-free quantitative proteomics were performed to identify significant changes proteins using the nano LC-MS along with SIEVE software. Biophoton emissions were significantly increased in both light and dark conditions after flooding stress, but gradually decreased with continued flooding exposure compared to the control plants. Among the 120 significantly identified proteins in the roots of soybean plants, 73 and 19 proteins were decreased and increased in the light condition, respectively, and 4 and 24 proteins were increased and decreased, respectively, in the dark condition. The proteins were mainly functionally grouped into cell organization, protein degradation/synthesis, and glycolysis. The highly abundant lactate/malate dehydrogenase proteins were decreased in flooding-stressed roots exposed to light, whereas the lysine ketoglutarate reductase/saccharopine dehydrogenase bifunctional enzyme was increased in both light and dark conditions. Notably, however, specific enzyme assays revealed that the activities of these enzymes and biophoton emission were sharply increased after 3 days of flooding stress. This finding suggests that the source of biophoton emission in roots might involve the chemical excitation of electron or proton through enzymatic or non-enzymatic oxidation and reduction reactions. Moreover, the lysine ketoglutarate reductase/saccharopine dehydrogenase bifunctional enzyme may play important roles in responses in flooding stress of soybean under the light condition and as a contributing factor to biophoton emission.

  9. Identification of ER Proteins Involved in the Functional Organisation of the Early Secretory Pathway in Drosophila Cells by a Targeted RNAi Screen

    Science.gov (United States)

    Kondylis, Vangelis; Tang, Yang; Fuchs, Florian; Boutros, Michael; Rabouille, Catherine

    2011-01-01

    Background In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation. Results To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for “more and smaller Golgi”) upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER. Conclusions This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation. PMID:21383842

  10. Glycation inhibitors extend yeast chronological lifespan by reducing advanced glycation end products and by back regulation of proteins involved in mitochondrial respiration.

    Science.gov (United States)

    Kazi, Rubina S; Banarjee, Reema M; Deshmukh, Arati B; Patil, Gouri V; Jagadeeshaprasad, Mashanipalya G; Kulkarni, Mahesh J

    2017-03-06

    Advanced Glycation End products (AGEs) are implicated in aging process. Thus, reducing AGEs by using glycation inhibitors may help in attenuating the aging process. In this study using Saccharomyces cerevisiae yeast system, we show that Aminoguanidine (AMG), a well-known glycation inhibitor, decreases the AGE modification of proteins in non-calorie restriction (NR) (2% glucose) and extends chronological lifespan (CLS) similar to that of calorie restriction (CR) condition (0.5% glucose). Proteomic analysis revealed that AMG back regulates the expression of differentially expressed proteins especially those involved in mitochondrial respiration in NR condition, suggesting that it switches metabolism from fermentation to respiration, mimicking CR. AMG induced back regulation of differentially expressed proteins could be possibly due to its chemical effect or indirectly by glycation inhibition. To delineate this, Metformin (MET), a structural analog of AMG and a mild glycation inhibitor and Hydralazine (HYD), another potent glycation inhibitor but not structural analog of AMG were used. HYD was more effective than MET in mimicking AMG suggesting that glycation inhibition was responsible for restoration of differentially expressed proteins. Thus glycation inhibitors particularly AMG, HYD and MET extend yeast CLS by reducing AGEs, modulating the expression of proteins involved in mitochondrial respiration and possibly by scavenging glucose. This study reports the role of glycation in aging process. In the non-caloric restriction condition, carbohydrates such as glucose promote protein glycation and reduce CLS. While, the inhibitors of glycation such as AMG, HYD, MET mimic the caloric restriction condition by back regulating deregulated proteins involved in mitochondrial respiration which could facilitate shift of metabolism from fermentation to respiration and extend yeast CLS. These findings suggest that glycation inhibitors can be potential molecules that can be used

  11. Differential Proteomic Analysis of the Pancreas of Diabetic db/db Mice Reveals the Proteins Involved in the Development of Complications of Diabetes Mellitus

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    Victoriano Pérez-Vázquez

    2014-05-01

    Full Text Available Type 2 diabetes mellitus is characterized by hyperglycemia and insulin-resistance. Diabetes results from pancreatic inability to secrete the insulin needed to overcome this resistance. We analyzed the protein profile from the pancreas of ten-week old diabetic db/db and wild type mice through proteomics. Pancreatic proteins were separated in two-dimensional polyacrylamide gel electrophoresis (2D-PAGE and significant changes in db/db mice respect to wild type mice were observed in 27 proteins. Twenty five proteins were identified by matrix-assisted laser desorption/ionization (MALDI time-of-flight (TOF and their interactions were analyzed using search tool for the retrieval of interacting genes/proteins (STRING and database for annotation, visualization and integrated discovery (DAVID. Some of these proteins were Pancreatic α-amylase, Cytochrome b5, Lithostathine-1, Lithostathine-2, Chymotrypsinogen B, Peroxiredoxin-4, Aspartyl aminopeptidase, Endoplasmin, and others, which are involved in the metabolism of carbohydrates and proteins, as well as in oxidative stress, and inflammation. Remarkably, these are mostly endoplasmic reticulum proteins related to peptidase activity, i.e., they are involved in proteolysis, glucose catabolism and in the tumor necrosis factor-mediated signaling pathway. These results suggest mechanisms for insulin resistance, and the chronic inflammatory state observed in diabetes.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    Highlights: ► We isolated xanthorrhizol, a sesquiterpenoid compound from Curcuma xanthorrhiza. ► Xanthorrhizol induced apoptosis in HepG2 cells as observed using SEM. ► Apoptosis in xanthorrhizol-treated HepG2 cells involved Bcl-2 family proteins. ► DNA fragmentation was observed in xanthorrhizol-treated HepG2 cells. ► 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 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tee, Thiam-Tsui, E-mail: thiamtsu@yahoo.com [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Cheah, Yew-Hoong [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, Jalan Pahang, Kuala Lumpur (Malaysia); Meenakshii, Nallappan [Biology Department, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia)

    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. Comparative proteomic analysis reveals proteins putatively involved in toxin biosynthesis in the marine dinoflagellate Alexandrium catenella.

    Science.gov (United States)

    Wang, Da-Zhi; Gao, Yue; Lin, Lin; Hong, Hua-Sheng

    2013-01-22

    Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing) coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to), alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

  15. Developmental expression of a cell surface protein involved in sea urchin skeleton formation

    International Nuclear Information System (INIS)

    Farach, M.C.; Valdizan, M.; Park, H.R.; Decker, G.L.; Lennarz, W.J.

    1986-01-01

    The authors have previously used a monoclonal antibody (1223) to identify a 130 Kd cell surface protein involved in skeleton formation is sea urchin embryos. In the current study the authors have examined the expression of the 1223 antigen over the course of development of embryos of two species, Strongylocentrotus purpuratus and Lytechinus pictus. The 130 Kd protein is detected in S. purp eggs on immunoblots. Labeling with [ 3 H] leucine and immunoaffinity chromatography show that it also is synthesized shortly after fertilization. Immunofluroescence reveals that at this early stage the 1223 antigen is uniformly distributed on all of the cells. Synthesis decreases to a minimum by the time of hatching (18 h), as does the total amount of antigen present in the embryo. A second period of synthesis commences at the mesenchyme blastula stage, when the spicule-forming primary mesenchyme cells (PMCs) have appeared. During this later stage, synthesis and cell surface expression are restricted to the PMCs. In contrast to S. purp., in L. pictus the 130 Kd protein does not appear until the PMCs are formed. Hybrid embryos demonstrate a pattern of expression of the maternal species. These results suggest that early expression of 1223 antigen in S. purp. is due to utilization of maternal transcripts present in the egg. In both species later expression in PMCs appears to be the result of cell-type specific synthesis, perhaps encoded by embryonic transcripts

  16. Matrix Gla Protein is Involved in Crystal Formation in Kidney of Hyperoxaluric Rats

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    Xiuli Lu

    2013-02-01

    Full Text Available Background: Matrix Gla protein (MGP is a molecular determinant regulating vascular calcification of the extracellular matrix. However, it is still unclear how MGP may be invovled in crystal formation in the kidney of hyperoxaluric rats. Methods: Male Sprague-Dawley rats were divided into the hyperoxaluric group and control group. Hyperoxaluric rats were administrated by 0.75% ethylene glycol (EG for up to 8 weeks. Renal MGP expression was detected by the standard avidin-biotin complex (ABC method. Renal crystal deposition was observed by a polarizing microscope. Total RNA and protein from the rat kidney tissue were extracted. The levels of MGP mRNA and protein expression were analyzed by the real-time polymerase chain reaction (RT-PCR and Western blot. Results: Hyperoxaluria was induced successfully in rats. The MGP was polarly distributed, on the apical membrane of renal tubular epithelial cells, and was found in the ascending thick limbs of Henle's loop (cTAL and the distal convoluted tubule (DCT in hyperoxaluric rats, its expression however, was present in the medullary collecting duct (MCD in stone-forming rats. Crystals with multilaminated structure formed in the injurious renal tubules with lack of MGP expression.MGP mRNA expression was significantly upregulated by the crystals' stimulations. Conclusion: Our results suggested that the MGP was involved in crystals formation by the continuous expression, distributing it polarly in the renal tubular cells and binding directly to the crystals.

  17. DELLA proteins regulate expression of a subset of AM symbiosis-induced genes in Medicago truncatula.

    Science.gov (United States)

    Floss, Daniela S; Lévesque-Tremblay, Véronique; Park, Hee-Jin; Harrison, Maria J

    2016-01-01

    The majority of the vascular flowering plants form symbiotic associations with fungi from the phylum Glomeromycota through which both partners gain access to nutrients, either mineral nutrients in the case of the plant, or carbon, in the case of the fungus. (1) The association develops in the roots and requires substantial remodeling of the root cortical cells where branched fungal hyphae, called arbuscules, are housed in a new membrane-bound apoplastic compartment. (2) Nutrient exchange between the symbionts occurs over this interface and its development and maintenance is critical for symbiosis. Previously, we showed that DELLA proteins, which are well known as repressors of gibberellic acid signaling, also regulate development of AM symbiosis and are necessary to enable arbuscule development. (3) Furthermore, constitutive overexpression of a dominant DELLA protein (della1-Δ18) is sufficient to induce transcripts of several AM symbiosis-induced genes, even in the absence of the fungal symbiont. (4) Here we further extend this approach and identify AM symbiosis genes that respond transcriptionally to constitutive expression of a dominant DELLA protein and also genes that do respond to this treatment. Additionally, we demonstrate that DELLAs interact with REQUIRED FOR ARBUSCULE DEVELOPMENT 1 (RAD1) which further extends our knowledge of GRAS factor complexes that have the potential to regulate gene expression during AM symbiosis.

  18. Identification of a novel centrosomal protein CrpF46 involved in cell cycle progression and mitosis

    International Nuclear Information System (INIS)

    Wei Yi; Shen Enzhi; Zhao Na; Liu Qian; Fan Jinling; Marc, Jan; Wang Yongchao; Sun Le; Liang Qianjin

    2008-01-01

    A novel centrosome-related protein Crp F46 was detected using a serum F46 from a patient suffering from progressive systemic sclerosis. We identified the protein by immunoprecipitation and Western blotting followed by tandem mass spectrometry sequencing. The protein Crp F46 has an apparent molecular mass of ∼ 60 kDa, is highly homologous to a 527 amino acid sequence of the C-terminal portion of the protein Golgin-245, and appears to be a splice variant of Golgin-245. Immunofluorescence microscopy of synchronized HeLa cells labeled with an anti-Crp F46 monoclonal antibody revealed that Crp F46 localized exclusively to the centrosome during interphase, although it dispersed throughout the cytoplasm at the onset of mitosis. Domain analysis using Crp F46 fragments in GFP-expression vectors transformed into HeLa cells revealed that centrosomal targeting is conferred by a C-terminal coiled-coil domain. Antisense Crp F46 knockdown inhibited cell growth and proliferation and the cell cycle typically stalled at S phase. The knockdown also resulted in the formation of poly-centrosomal and multinucleate cells, which finally became apoptotic. These results suggest that Crp F46 is a novel centrosome-related protein that associates with the centrosome in a cell cycle-dependent manner and is involved in the progression of the cell cycle and M phase mechanism

  19. Induction of protein body formation in plant leaves by elastin-like polypeptide fusions

    Directory of Open Access Journals (Sweden)

    Joensuu Jussi J

    2009-08-01

    Full Text Available Abstract Background Elastin-like polypeptides are synthetic biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the simple non-chromatographic purification of recombinant proteins. In addition, elastin-like polypeptide fusions have been shown to enhance the accumulation of a range of different recombinant proteins in plants, thus addressing the major limitation of plant-based expression systems, which is a low production yield. This study's main objectives were to determine the general utility of elastin-like polypeptide protein fusions in various intracellular compartments and to elucidate elastin-like polypeptide's mechanism of action for increasing recombinant protein accumulation in the endoplasmic reticulum of plants. Results The effect of elastin-like polypeptide fusions on the accumulation of green fluorescent protein targeted to the cytoplasm, chloroplasts, apoplast, and endoplasmic reticulum was evaluated. The endoplasmic reticulum was the only intracellular compartment in which an elastin-like polypeptide tag was shown to significantly enhance recombinant protein accumulation. Interestingly, endoplasmic reticulum-targeted elastin-like polypeptide fusions induced the formation of a novel type of protein body, which may be responsible for elastin-like polypeptide's positive effect on recombinant protein accumulation by excluding the heterologous protein from normal physiological turnover. Although expressed in the leaves of plants, these novel protein bodies appeared similar in size and morphology to the prolamin-based protein bodies naturally found in plant seeds. The elastin-like polypeptide-induced protein bodies were highly mobile organelles, exhibiting various dynamic patterns of movement throughout the cells, which were dependent on intact actin microfilaments and a functional actomyosin motility system. Conclusion An endoplasmic reticulum-targeted elastin-like polypeptide fusion approach

  20. PsVPS1, a dynamin-related protein, is involved in cyst germination and soybean infection of Phytophthora sojae.

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    Delong Li

    Full Text Available Plant pathogens secrete effector proteins to suppress plant immunity. However, the mechanism by which oomycete pathogens deliver effector proteins during plant infection remains unknown. In this report, we characterized a Phytophthora sojae vps1 gene. This gene encodes a homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene vps1 that mediates budding of clathrin-coated vesicles from the late Golgi, which are diverted from the general secretory pathway to the vacuole. PsVPS1-silenced mutants were generated using polyethylene glycol-mediated protoplast stable transformation and were viable but had reduced extracellular protein activity. The PsVPS1-silenced mutants showed impaired hyphal growth, and the shapes of the vacuoles were highly fragmented. Silencing of PsVPS1 affected cyst germination as well as the polarized growth of germinated cysts. Silenced mutants showed impaired invasion of susceptible soybean plants regardless of wounding. These results suggest that PsVPS1 is involved in vacuole morphology and cyst development. Moreover, it is essential for the virulence of P. sojae and extracellular protein secretion.

  1. Plasma membrane H(+)-ATPase is involved in methyl jasmonate-induced root hair formation in lettuce (Lactuca sativa L.) seedlings.

    Science.gov (United States)

    Zhu, Changhua; Yang, Na; Ma, Xiaoling; Li, Guijun; Qian, Meng; Ng, Denny; Xia, Kai; Gan, Lijun

    2015-06-01

    Our results show that methyl jasmonate induces plasma membrane H (+) -ATPase activity and subsequently influences the apoplastic pH of trichoblasts to maintain a cell wall pH environment appropriate for root hair development. Root hairs, which arise from root epidermal cells, are tubular structures that increase the efficiency of water absorption and nutrient uptake. Plant hormones are critical regulators of root hair development. In this study, we investigated the regulatory role of the plasma membrane (PM) H(+)-ATPase in methyl jasmonate (MeJA)-induced root hair formation. We found that MeJA had a pronounced effect on the promotion of root hair formation in lettuce seedlings, but that this effect was blocked by the PM H(+)-ATPase inhibitor vanadate. Furthermore, MeJA treatment increased PM H(+)-ATPase activity in parallel with H(+) efflux from the root tips of lettuce seedlings and rhizosphere acidification. Our results also showed that MeJA-induced root hair formation was accompanied by hydrogen peroxide accumulation. The apoplastic acidification acted in concert with reactive oxygen species to modulate root hair formation. Our results suggest that the effect of MeJA on root hair formation is mediated by modulation of PM H(+)-ATPase activity.

  2. An Apoplastic β-Glucosidase is Essential for the Degradation of Flavonol 3-O-β-Glucoside-7-O-α-Rhamnosides in Arabidopsis.

    Science.gov (United States)

    Roepke, Jonathon; Gordon, Harley O W; Neil, Kevin J A; Gidda, Satinder; Mullen, Robert T; Freixas Coutin, José A; Bray-Stone, Delaney; Bozzo, Gale G

    2017-06-01

    Flavonol bisglycosides accumulate in plant vegetative tissues in response to abiotic stress, including simultaneous environmental perturbations (i.e. nitrogen deficiency and low temperature, NDLT), but disappear with recovery from NDLT. Previously, we determined that a recombinant Arabidopsis β-glucosidase (BGLU), BGLU15, hydrolyzes flavonol 3-O-β-glucoside-7-O-α-rhamnosides and flavonol 3-O-β-glucosides, forming flavonol 7-O-α-rhamnosides and flavonol aglycones, respectively. In this study, the transient expression of a BGLU15-Cherry fusion protein in onion epidermal cells demonstrated that BGLU15 was localized to the apoplast. Analysis of BGLU15 T-DNA insertional inactivation lines (bglu15-1 and bglu15-2) revealed negligible levels of BGLU15 transcripts, whereas its paralogs BGLU12 and BGLU16 were expressed in wild-type and bglu15 plants. The recombinant BGLU16 did not hydrolyze quercetin 3-O-β-glucoside-7-O-α-rhamnoside or rhamnosylated flavonols, but was active with the synthetic substrate, p-nitrophenyl-β-d-glucoside. In addition, shoots of both bglu15 mutants contained negligible flavonol 3-O-β-glucoside-7-O-α-rhamnoside hydrolase activity, whereas this activity increased by 223% within 2 d of NDLT recovery in wild-type plants. The levels of flavonol 3-O-β-glucoside-7-O-α-rhamnosides and quercetin 3-O-β-glucoside were high and relatively unchanged in shoots of bglu15 mutants during recovery from NDLT, whereas rapid losses were apparent in wild-type shoots. Moreover, losses of two flavonol 3-O-β-neohesperidoside-7-O-α-rhamnosides and kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside were evident during recovery from NDLT, regardless of whether BGLU15 was present. A spike in a kaempferol 7-O-α-rhamnoside occurred with stress recovery, regardless of germplasm, suggesting a contribution from hydrolysis of kaempferol 3-O-β-neohesperidoside-7-O-α-rhamnosides and/or kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside by hitherto unknown mechanisms. Thus

  3. Regulation of myofibrillar accumulation in chick muscle cultures - Evidence for the involvement of calcium and lysosomes in non-uniform turnover of contractile proteins

    Science.gov (United States)

    Silver, Geri; Etlinger, Joseph D.

    1985-01-01

    The effects of calcium on the synthesis and the degradation of individual myofibrillar proteins were investigated using primary chick-leg skeletal muscle cultures labeled with S-35-methionine (for protein accumulation experiments) or Ca(2+)-45 (for calcium efflux experiments). It was found that the turnover of individual contractile proteins is regulated nonuniformly by a calcium-dependent mechanism involving lysosomes. The results also indicate that contractile proteins are released from the myofibril before their breakdown to amino acids.

  4. Isoforms of acyl carrier protein involved in seed-specific fatty acid synthesis.

    Science.gov (United States)

    Suh, M C; Schultz, D J; Ohlrogge, J B

    1999-03-01

    Seeds of coriandrum sativum (coriander) and Thunbergia alata (black-eyed Susan vine) produce unusual monoenoic fatty acids which constitute over 80% of the total fatty acids of the seed oil. The initial step in the formation of these fatty acids is the desaturation of palmitoyl-ACP (acyl carrier protein) at the delta(4) or delta(6) positions to produce delta(4)-hexadecenoic acid (16:1(delta(4)) or delta(6)-hexadecenoic acid (16:1(delta(6)), respectively. The involvement of specific forms of ACP in the production of these novel monoenoic fatty acids was studied. ACPs were partially purified from endosperm of coriander and T. alata and used to generate 3H- and 14C-labelled palmitoyl-ACP substrates. In competition assays with labelled palmitoyl-ACP prepared from spinach (Spinacia oleracea), delta(4)-acyl-ACP desaturase activity was two- to threefold higher with coriander ACP than with spinach ACP. Similarly, the T. alata delta(6) desaturase favoured T. alata ACP over spinach ACP. A cDNA clone, Cs-ACP-1, encoding ACP was isolated from a coriander endosperm cDNA library. Cs-ACP-1 mRNA was predominantly expressed in endosperm rather than leaves. The Cs-ACP-1 mature protein was expressed in E. coli and comigrated on SDS-PAGE with the most abundant ACP expressed in endosperm tissues. In in vitro delta(4)-palmitoyl-ACP desaturase assays, the Cs-ACP-1 expressed from E. coli was four- and 10-fold more active than spinach ACP or E. coli ACP, respectively, in the synthesis of delta(4)-hexadecenoic acid from palmitoyl-ACP. In contrast, delta(9)-stearoyl-ACP desaturase activity from coriander endosperm did not discriminate strongly between different ACP species. These results indicate that individual ACP isoforms are specifically involved in the biosynthesis of unusual seed fatty acids and further suggest that expression of multiple ACP isoforms may participate in determining the products of fatty acid biosynthesis.

  5. Identification of Proteins Using iTRAQ and Virus-Induced Gene Silencing Reveals Three Bread Wheat Proteins Involved in the Response to Combined Osmotic-Cold Stress.

    Science.gov (United States)

    Zhang, Ning; Zhang, Lingran; Shi, Chaonan; Zhao, Lei; Cui, Dangqun; Chen, Feng

    2018-05-25

    Crops are often subjected to a combination of stresses in the field. To date, studies on the physiological and molecular responses of common wheat to a combination of osmotic and cold stresses, however, remain unknown. In this study, wheat seedlings exposed to osmotic-cold stress for 24 h showed inhibited growth, as well as increased lipid peroxidation, relative electrolyte leakage, and soluble sugar contents. iTRAQ-based quantitative proteome method was employed to determine the proteomic profiles of the roots and leaves of wheat seedlings exposed to osmotic-cold stress conditions. A total of 250 and 258 proteins with significantly altered abundance in the roots and leaves were identified, respectively, and the majority of these proteins displayed differential abundance, thereby revealing organ-specific differences in adaptation to osmotic-cold stress. Yeast two hybrid assay examined five pairs of stress/defense-related protein-protein interactions in the predicted protein interaction network. Furthermore, quantitative real-time PCR analysis indicated that abiotic stresses increased the expression of three candidate protein genes, i.e., TaGRP2, CDCP, and Wcor410c in wheat leaves. Virus-induced gene silencing indicated that three genes TaGRP2, CDCP, and Wcor410c were involved in modulating osmotic-cold stress in common wheat. Our study provides useful information for the elucidation of molecular and genetics bases of osmotic-cold combined stress in bread wheat.

  6. Identification of differentially accumulated proteins involved in regulating independent and combined osmosis and cadmium stress response in Brachypodium seedling roots.

    Science.gov (United States)

    Chen, Ziyan; Zhu, Dong; Wu, Jisu; Cheng, Zhiwei; Yan, Xing; Deng, Xiong; Yan, Yueming

    2018-05-17

    In this study, we aimed to identify differentially accumulated proteins (DAPs) involved in PEG mock osmotic stress, cadmium (Cd 2+ ) stress, and their combined stress responses in Brachypodium distachyon seedling roots. The results showed that combined PEG and Cd 2+ stresses had more significant effects on Brachypodium seedling root growth, physiological traits, and ultrastructures when compared with each individual stress. Totally, 106 DAPs were identified that are responsive to individual and combined stresses in roots. These DAPs were mainly involved in energy metabolism, detoxification and stress defense and protein metabolism. Principal component analysis revealed that DAPs from Cd 2+ and combined stress treatments were grouped closer than those from osmotic stress treatment, indicating that Cd 2+ and combined stresses had more severe influences on the root proteome than osmotic stress alone. Protein-protein interaction analyses highlighted a 14-3-3 centered sub-network that synergistically responded to osmotic and Cd 2+ stresses and their combined stresses. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 14 key DAP genes revealed that most genes showed consistency between transcriptional and translational expression patterns. A putative pathway of proteome metabolic changes in Brachypodium seedling roots under different stresses was proposed, which revealed a complicated synergetic responsive network of plant roots to adverse environments.

  7. Physical Interaction between Replication Protein A (RPA) and MRN: Involvement of RPA2 Phosphorylation and the N-terminus of RPA1

    OpenAIRE

    Oakley, Greg; Tillison, Kristin; Opiyo, Stephen; Glanzer, Jason; Horn, Jeffrey M.; Patrick, Steve M.

    2009-01-01

    Replication protein A (RPA) is a heterotrimeric protein consisting of RPA1, RPA2 and RPA3 subunits that binds to ssDNA with high affinity. The response to replication stress requires the recruitment of RPA and the MRE11/RAD50/NBS1 (MRN) complex. RPA bound to ssDNA stabilizes stalled replication forks by recruiting checkpoint proteins involved in fork stabilization. MRN can bind DNA structures encountered at stalled or collapsed replication forks, such as ssDNA-dsDNA junctions or breaks and pr...

  8. Functional analysis of thermostable proteins involved in carbohydrate metabolism

    NARCIS (Netherlands)

    Akerboom, A.P.

    2007-01-01

    Thermostable proteins can resist temperature stress whilst keeping their integrity and functionality. In many cases, thermostable proteins originate from hyperthermophilic microorganisms that thrive in extreme environments. These systems are generally located close to geothermal (volcanic) activity,

  9. Evidence for the involvement of a 66 kDa membrane protein in the synthesis of sterolglucoside in ''Saccharomyces cerevisiae''

    International Nuclear Information System (INIS)

    Lenart, U.; Palamarczyk, G.

    1995-01-01

    The membrane-bound sterolglucoside synthase from the yeast ''Saccharomyces cerevisiae'' has been solubilized by nonionic detergent, Nonidet P-40, Triton X-100, and partially purified by DEAE-cellulose column chromatography and ammonium sulfate fractionation. SDS/PAGE of the purified fraction revealed the presence of two protein bands of molecular mass 66 kDa and 54 kDa. In an attempt to identify further the polypeptide chain of sterolglucoside synthase, the partially purified enzyme was treated with [di- 125 I]-5-[3-(p-azidosalicylamide)]allyl-UDPglucose, a photoactive analogue of UDPglucose, which is a substrate for this enzyme. Upon photolysis the 125 I-labelled probe was shown to link covalently to the 66 kDa protein. The photoinsertion was competed out by the presence of unlabeled UDPglucose thus suggesting that this protein contains substrate binding site for UDPglucose. Since photoinsertion of the probe to protein of 66 kDa correlated with the molecular mass of the protein visualized upon enzyme purification we postulate that the 66 kDa protein is involved in sterolglucoside synthesis in yeast. (author). 10 refs, 5 figs, 1 tab

  10. The SGS3 protein involved in PTGS finds a family

    Directory of Open Access Journals (Sweden)

    Bateman Alex

    2002-08-01

    Full Text Available Abstract Background Post transcriptional gene silencing (PTGS is a recently discovered phenomenon that is an area of intense research interest. Components of the PTGS machinery are being discovered by genetic and bioinformatics approaches, but the picture is not yet complete. Results The gene for the PTGS impaired Arabidopsis mutant sgs3 was recently cloned and was not found to have similarity to any other known protein. By a detailed analysis of the sequence of SGS3 we have defined three new protein domains: the XH domain, the XS domain and the zf-XS domain, that are shared with a large family of uncharacterised plant proteins. This work implicates these plant proteins in PTGS. Conclusion The enigmatic SGS3 protein has been found to contain two predicted domains in common with a family of plant proteins. The other members of this family have been predicted to be transcription factors, however this function seems unlikely based on this analysis. A bioinformatics approach has implicated a new family of plant proteins related to SGS3 as potential candidates for PTGS related functions.

  11. A PerR-like protein involved in response to oxidative stress in the extreme bacterium Deinococcus radiodurans

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chengzhi; Wang, Liangyan; Li, Tao; Lin, Lin; Dai, Shang; Tian, Bing, E-mail: tianbing@zju.edu.cn; Hua, Yuejin, E-mail: yjhua@zju.edu.cn

    2014-07-18

    Highlights: • We report a novel PerR-like protein of Fur family in D. radiodurans that is not annotated in the current database. • drperR responses to H{sub 2}O{sub 2} and functions as a negative regulator of katE and dps. • We provided implications on how to utilize sequenced genome data and the importance of genome data mining. • This study adds knowledge to complicated regulatory network that responds to ROS stress in D. radiodurans. - Abstract: Response and defense systems against reactive oxygen species (ROS) contribute to the remarkable resistance of Deinococcus radiodurans to oxidative stress induced by oxidants or radiation. However, mechanisms involved in ROS response and defense systems of D. radiodurans are not well understood. Fur family proteins are important in ROS response. Only a single Fur homolog is predicted by sequence similarity in the current D. radiodurans genome database. Our bioinformatics analysis demonstrated an additional guanine nucleotide in the genome of D. radiodurans that is not in the database, leading to the discovery of another Fur homolog DrPerR. Gene disruption mutant of DrPerR showed enhanced resistance to hydrogen peroxide (H{sub 2}O{sub 2}) and increased catalase activity in cell extracts. Real-time PCR results indicated that DrPerR functions as a repressor of the catalase gene katE. Meanwhile, derepression of dps (DNA-binding proteins from starved cells) gene under H{sub 2}O{sub 2} stress by DrPerR point to its regulatory role in metal ions hemostasis. Thus, DrPerR might function as a Fur homolog protein which is involved in ROS response and defense. These results help clarify the complicated regulatory network that responds to ROS stress in D. radiodurans.

  12. A PerR-like protein involved in response to oxidative stress in the extreme bacterium Deinococcus radiodurans

    International Nuclear Information System (INIS)

    Liu, Chengzhi; Wang, Liangyan; Li, Tao; Lin, Lin; Dai, Shang; Tian, Bing; Hua, Yuejin

    2014-01-01

    Highlights: • We report a novel PerR-like protein of Fur family in D. radiodurans that is not annotated in the current database. • drperR responses to H 2 O 2 and functions as a negative regulator of katE and dps. • We provided implications on how to utilize sequenced genome data and the importance of genome data mining. • This study adds knowledge to complicated regulatory network that responds to ROS stress in D. radiodurans. - Abstract: Response and defense systems against reactive oxygen species (ROS) contribute to the remarkable resistance of Deinococcus radiodurans to oxidative stress induced by oxidants or radiation. However, mechanisms involved in ROS response and defense systems of D. radiodurans are not well understood. Fur family proteins are important in ROS response. Only a single Fur homolog is predicted by sequence similarity in the current D. radiodurans genome database. Our bioinformatics analysis demonstrated an additional guanine nucleotide in the genome of D. radiodurans that is not in the database, leading to the discovery of another Fur homolog DrPerR. Gene disruption mutant of DrPerR showed enhanced resistance to hydrogen peroxide (H 2 O 2 ) and increased catalase activity in cell extracts. Real-time PCR results indicated that DrPerR functions as a repressor of the catalase gene katE. Meanwhile, derepression of dps (DNA-binding proteins from starved cells) gene under H 2 O 2 stress by DrPerR point to its regulatory role in metal ions hemostasis. Thus, DrPerR might function as a Fur homolog protein which is involved in ROS response and defense. These results help clarify the complicated regulatory network that responds to ROS stress in D. radiodurans

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

    Science.gov (United States)

    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 phigh Fe

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

    Science.gov (United States)

    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. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Attenuated expression of the tight junction proteins is involved in clopidogrel-induced gastric injury through p38 MAPK activation

    International Nuclear Information System (INIS)

    Wu, Hai-Lu; Gao, Xin; Jiang, Zong-Dan; Duan, Zhao-Tao; Wang, Shu-Kui; He, Bang-Shun; Zhang, Zhen-Yu; Xie, Hong-Guang

    2013-01-01

    Highlights: ► Clopidogrel suppressed GES-1 cell viability in a concentration- and time-dependent manner. ► Clopidogrel significantly increased dextran permeability, reduced occludin and ZO-1 expression, and induced cell apoptosis. ► Clopidogrel activated p38 MAPK signaling pathway. ► Activation of p38 activity was involved in clopidogrel-induced increase in gastric epithelial cells permeability and disruption of TJ. -- Abstract: Bleeding complications and delayed healing of gastric ulcer associated with use of clopidogrel is a common clinical concern; however, the underlying mechanisms remain to be determined. This study aimed to clarify whether clopidogrel could cause the damage of the human gastric epithelial cells and to further elucidate the mechanisms involved. After human gastric epithelial cell line GES-1 had been treated with clopidogrel (0.5–2.5 mM), the cell proliferation was examined by MTT assay, apoptosis was measured with DAPI staining and flow cytometry analysis, and the barrier function of the tight junctions (TJ) was evaluated by permeability measurement and transmission electron microscopy. Moreover, expression of the TJ proteins occludin and ZO-1 and the phosphorylation of the mitogen-activated protein kinases (MAPK) p38, ERK, and JNK were examined by western blot. In addition, three MAPK inhibitors specific to p38, ERK and JNK were used, respectively, to verify the signaling pathways responsible for regulating the expression of the TJ proteins being tested. Results showed that clopidogrel significantly increased dextran permeability, induced apoptosis, suppressed GES-1 cell viability, and reduced the expression of the TJ proteins (occludin and ZO-1), acting through p38 MAPK phosphorylation. Furthermore, these observed effects were partially abolished by SB-203580 (a p38 MAPK inhibitor), rather than by either U-0126 (an ERK inhibitor) or SP-600125 (a JNK inhibitor), suggesting that clopidogrel-induced disruption in the gastric

  16. Reconstruction of the yeast protein-protein interaction network involved in nutrient sensing and global metabolic regulation

    DEFF Research Database (Denmark)

    Nandy, Subir Kumar; Jouhten, Paula; Nielsen, Jens

    2010-01-01

    proteins. Despite the value of BioGRID for studying protein-protein interactions, there is a need for manual curation of these interactions in order to remove false positives. RESULTS: Here we describe an annotated reconstruction of the protein-protein interactions around four key nutrient......) and for all the interactions between them (edges). The annotated information is readily available utilizing the functionalities of network modelling tools such as Cytoscape and CellDesigner. CONCLUSIONS: The reported fully annotated interaction model serves as a platform for integrated systems biology studies...

  17. At the border: the plasma membrane-cell wall continuum.

    Science.gov (United States)

    Liu, Zengyu; Persson, Staffan; Sánchez-Rodríguez, Clara

    2015-03-01

    Plant cells rely on their cell walls for directed growth and environmental adaptation. Synthesis and remodelling of the cell walls are membrane-related processes. During cell growth and exposure to external stimuli, there is a constant exchange of lipids, proteins, and other cell wall components between the cytosol and the plasma membrane/apoplast. This exchange of material and the localization of cell wall proteins at certain spots in the plasma membrane seem to rely on a particular membrane composition. In addition, sensors at the plasma membrane detect changes in the cell wall architecture, and activate cytoplasmic signalling schemes and ultimately cell wall remodelling. The apoplastic polysaccharide matrix is, on the other hand, crucial for preventing proteins diffusing uncontrollably in the membrane. Therefore, the cell wall-plasma membrane link is essential for plant development and responses to external stimuli. This review focuses on the relationship between the cell wall and plasma membrane, and its importance for plant tissue organization. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  18. Redox stress proteins are involved in adaptation response of the hyperthermoacidophilic archaeon Sulfolobus solfataricus to nickel challenge

    Directory of Open Access Journals (Sweden)

    Scaloni Andrea

    2007-08-01

    Full Text Available Abstract Background Exposure to nickel (Ni and its chemical derivatives has been associated with severe health effects in human. On the contrary, poor knowledge has been acquired on target physiological processes or molecular mechanisms of this metal in model organisms, including Bacteria and Archaea. In this study, we describe an analysis focused at identifying proteins involved in the recovery of the archaeon Sulfolobus solfataricus strain MT4 from Ni-induced stress. Results To this purpose, Sulfolobus solfataricus was grown in the presence of the highest nickel sulphate concentration still allowing cells to survive; crude extracts from treated and untreated cells were compared at the proteome level by using a bi-dimensional chromatography approach. We identified several proteins specifically repressed or induced as result of Ni treatment. Observed up-regulated proteins were largely endowed with the ability to trigger recovery from oxidative and osmotic stress in other biological systems. It is noteworthy that most of the proteins induced following Ni treatment perform similar functions and a few have eukaryal homologue counterparts. Conclusion These findings suggest a series of preferential gene expression pathways activated in adaptation response to metal challenge.

  19. Polycystin-1 C terminus cleavage and its relation with polycystin-2, two proteins involved in polycystic kidney disease

    Directory of Open Access Journals (Sweden)

    Claudia A. Bertuccio

    2013-04-01

    Full Text Available Autosomal dominant polycystic kidney disease (ADPKD, a most common genetic cause of chronic renal failure, is characterized by the progressive development and enlargement of cysts in kidneys and other organs. The cystogenic process is highly complex and involves a high proliferative rate, increased apoptosis, altered protein sorting, changed secretory characteristics, and disorganization of the extracellular matrix. ADPKD is caused by mutations in the genes encoding polycystin-1 (PC-1 or polycystin-2 (PC-2. PC-1 undergoes multiple cleavages that intervene in several signaling pathways involved in cellular proliferation and differentiation mechanisms. One of these cleavages releases the cytoplasmic C-terminal tail of PC-1. In addition, the C-terminal cytoplasmic tails of PC-1 and PC-2 interact in vitro and in vivo. The purpose of this review is to summarize recent literature that suggests that PC-1 and PC-2 may function through a common signaling pathway necessary for normal tubulogenesis. We hope that a better understanding of PC-1 and PC-2 protein function will lead to progress in diagnosis and treatment for ADPKD.

  20. Wheat F-Box Protein Gene TaFBA1 Is Involved in Plant Tolerance to Heat Stress

    Directory of Open Access Journals (Sweden)

    Qinxue Li

    2018-04-01

    Full Text Available Adverse environmental conditions, including high temperature, often affect the growth and production of crops worldwide. F-box protein, a core component of the Skp1-Cullin-F-box (SCF E3 ligase complex, plays an important role in abiotic stress responses. A previously cloned gene from wheat, TaFBA1, encodes a homologous F-box protein. A Yeast two-Hybrid (Y2H assay showed that TaFBA1 interacted with other SCF proteins. We found that the expression of TaFBA1 could be induced by heat stress (45°C. Overexpression of TaFBA1 enhanced heat stress tolerance in transgenic tobacco, because growth inhibition was reduced and photosynthesis increased as compared with those in the wild type (WT plants. Furthermore, the accumulation of H2O2, O2-, and carbonyl protein decreased and cell damage was alleviated in transgenic plants under heat stress, which resulted in less oxidative damage. However, the transgenic plants contained more enzymatic antioxidants after heat stress, which might be related to the regulation of some antioxidant gene expressions. The qRT-PCR analysis showed that the overexpression of TaFBA1 upregulated the expression of genes involved in reactive oxygen species (ROS scavenging, proline biosynthesis, and abiotic stress responses. We identified the interaction of TaFBA1 with Triticum aestivum stress responsive protein 1 (TaASRP1 by Y2H assay and bimolecular fluorescence complementation (BiFC assay. The results suggested that TaFBA1 may improve enzymatic antioxidant levels and regulate gene expression by interacting with other proteins, such as TaASRP1, which leads to the enhanced heat stress tolerance seen in the transgenic plants.

  1. Energy Stress Regulates Hippo-YAP Signaling Involving AMPK-Mediated Regulation of Angiomotin-like 1 Protein

    Directory of Open Access Journals (Sweden)

    Michael DeRan

    2014-10-01

    Full Text Available Hippo signaling is a tumor-suppressor pathway involved in organ size control and tumorigenesis through the inhibition of YAP and TAZ. Here, we show that energy stress induces YAP cytoplasmic retention and S127 phosphorylation and inhibits YAP transcriptional activity and YAP-dependent transformation. These effects require the central metabolic sensor AMP-activated protein kinase (AMPK and the upstream Hippo pathway components Lats1/Lats2 and angiomotin-like 1 (AMOTL1. Furthermore, we show that AMPK directly phosphorylates S793 of AMOTL1. AMPK activation stabilizes and increases AMOTL1 steady-state protein levels, contributing to YAP inhibition. The phosphorylation-deficient S793Ala mutant of AMOTL1 showed a shorter half-life and conferred resistance to energy-stress-induced YAP inhibition. Our findings link energy sensing to the Hippo-YAP pathway and suggest that YAP may integrate spatial (contact inhibition, mechanical, and metabolic signals to control cellular proliferation and survival.

  2. SLXL1, a novel acrosomal protein, interacts with DKKL1 and is involved in fertilization in mice.

    Directory of Open Access Journals (Sweden)

    Xin-jie Zhuang

    Full Text Available BACKGROUND: Spermatogenesis is a complex cellular developmental process which involves diverse families of genes. The Xlr (X-linked, lymphocyte regulated family includes multiple members, only a few of which have reported functions in meiosis, post-meiotic maturation, and fertilization of germ cells. Slx-like1 (Slxl1 is a member of the Xlr family, whose expression and function in spermatogenesis need to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: The mRNA and protein expression and localization of Slxl1 were investigated by RT-PCR, Western blotting and immunohistochemistry in different tissues and at different stages of spermatogenesis. The interacting partner of SLXL1 was examined by co-immunoprecipitation and co-localization. Assessment of the role of SLXL1 in capacitation, acrosome reaction, zona pellucida binding/penetration, and fertilization was carried out in vitro using blocking antisera. The results showed that Slxl1 mRNA and protein were specifically expressed in the testis. SLXL1 was exclusively located in the acrosome of post-meiotic germ cells and interacts with DKKL1 (Dickkopf-like1, which is an acrosome-associated protein and plays an important role in fertilization. The rates of zona pellucida binding/penetration and fertilization were significantly reduced by the anti-SLXL1 polyclonal antiserum. CONCLUSIONS/SIGNIFICANCE: SLXL1 is the first identified member of the XLR family that is associated with acrosome and is involved in zona pellucid binding/penetration and subsequent fertilization. These results, together with previous studies, suggest that Xlr family members participate in diverse processes from meiosis to fertilization during spermatogenesis.

  3. Different cAMP sources are critically involved in G protein-coupled receptor CRHR1 signaling.

    Science.gov (United States)

    Inda, Carolina; Dos Santos Claro, Paula A; Bonfiglio, Juan J; Senin, Sergio A; Maccarrone, Giuseppina; Turck, Christoph W; Silberstein, Susana

    2016-07-18

    Corticotropin-releasing hormone receptor 1 (CRHR1) activates G protein-dependent and internalization-dependent signaling mechanisms. Here, we report that the cyclic AMP (cAMP) response of CRHR1 in physiologically relevant scenarios engages separate cAMP sources, involving the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). cAMP produced by tmACs and sAC is required for the acute phase of extracellular signal regulated kinase 1/2 activation triggered by CRH-stimulated CRHR1, but only sAC activity is essential for the sustained internalization-dependent phase. Thus, different cAMP sources are involved in different signaling mechanisms. Examination of the cAMP response revealed that CRH-activated CRHR1 generates cAMP after endocytosis. Characterizing CRHR1 signaling uncovered a specific link between CRH-activated CRHR1, sAC, and endosome-based signaling. We provide evidence of sAC being involved in an endocytosis-dependent cAMP response, strengthening the emerging model of GPCR signaling in which the cAMP response does not occur exclusively at the plasma membrane and introducing the notion of sAC as an alternative source of cAMP. © 2016 Inda et al.

  4. Ricinus communis cyclophilin: functional characterisation of a sieve tube protein involved in protein folding.

    Science.gov (United States)

    Gottschalk, Maren; Dolgener, Elmar; Xoconostle-Cázares, Beatriz; Lucas, William J; Komor, Ewald; Schobert, Christian

    2008-09-01

    The phloem translocation stream of the angiosperms contains a special population of proteins and RNA molecules which appear to be produced in the companion cells prior to being transported into the sieve tube system through the interconnecting plasmodesmata. During this process, these non-cell-autonomous proteins are thought to undergo partial unfolding. Recent mass spectroscopy studies identified peptidyl-prolyl cis-trans isomerase (PPIases) as potential molecular chaperones functioning in the phloem translocation stream (Giavalisco et al. 2006). In the present study, we describe the cloning and characterisation of a castor bean phloem cyclophilin, RcCYP1 that has high peptidyl-prolyl cis-trans isomerase activity. Equivalent enzymatic activity was detected with phloem sap or purified recombinant (His)(6)-tagged RcCYP1. Mass spectrometry analysis of proteolytic peptides, derived from a 22 kDa band in HPLC-fractionated phloem sap, immunolocalisation studies and Western analysis of proteins extracted from castor bean tissues/organs indicated that RcCYP1 is an abundant protein in the companion cell-sieve element complex. Microinjection experiments established that purified recombinant (His)(6)-RcCYP1 can interact with plasmodesmata to both induce an increase in size exclusion limit and mediate its own cell-to-cell trafficking. Collectively, these findings support the hypothesis that RcCYP1 plays a role in the refolding of non-cell-autonomous proteins after their entry into the phloem translocation stream.

  5. KH-type splicing regulatory protein is involved in esophageal squamous cell carcinoma progression.

    Science.gov (United States)

    Fujita, Yuji; Masuda, Kiyoshi; Hamada, Junichi; Shoda, Katsutoshi; Naruto, Takuya; Hamada, Satoshi; Miyakami, Yuko; Kohmoto, Tomohiro; Watanabe, Miki; Takahashi, Rizu; Tange, Shoichiro; Saito, Masako; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Tangoku, Akira; Otsuji, Eigo; Imoto, Issei

    2017-11-24

    KH-type splicing regulatory protein (KHSRP) is a multifunctional RNA-binding protein, which is involved in several post-transcriptional aspects of RNA metabolism, including microRNA (miRNA) biogenesis. It affects distinct cell functions in different tissues and can have an impact on various pathological conditions. In the present study, we investigated the oncogenic functions of KHSRP and their underlying mechanisms in the pathogenesis of esophageal squamous cell carcinoma (ESCC). KHSRP expression levels were elevated in ESCC tumors when compared with those in non-tumorous tissues by immunohistochemistry, and cytoplasmic KHSRP overexpression was found to be an independent prognosticator for worse overall survival in a cohort of 104 patients with ESCC. KHSRP knockdown inhibited growth, migration, and invasion of ESCC cells. KHSRP knockdown also inhibited the maturation of cancer-associated miRNAs, such as miR-21, miR-130b, and miR-301, and induced the expression of their target mRNAs, such as BMP6, PDCD4, and TIMP3, resulting in the inhibition of epithelial-to-mesenchymal transition. Our findings uncover a novel oncogenic function of KHSRP in esophageal tumorigenesis and implicate its use as a marker for prognostic evaluation and as a putative therapeutic target in ESCC.

  6. Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl-like molecules binding.

    Science.gov (United States)

    Isvoran, Adriana; Craciun, Dana; Martiny, Virginie; Sperandio, Olivier; Miteva, Maria A

    2013-06-14

    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. 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. 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. 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.

  7. Identification and characterization of a stage specific membrane protein involved in flagellar attachment in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Katherine Woods

    Full Text Available Flagellar attachment is a visibly striking morphological feature of African trypanosomes but little is known about the requirements for attachment at a molecular level. This study characterizes a previously undescribed membrane protein, FLA3, which plays an essential role in flagellar attachment in Trypanosoma brucei. FLA3 is heavily N-glycosylated, locates to the flagellar attachment zone and appears to be a bloodstream stage specific protein. Ablation of the FLA3 mRNA rapidly led to flagellar detachment and a concomitant failure of cytokinesis in the long slender bloodstream form but had no effect on the procyclic form. Flagellar detachment was obvious shortly after induction of the dsRNA and the newly synthesized flagellum was often completely detached after it emerged from the flagellar pocket. Within 12 h most cells possessed detached flagella alongside the existing attached flagellum. These results suggest that proteins involved in attachment are not shared between the new and old attachment zones. In other respects the detached flagella appear normal, they beat rapidly although directional motion was lost, and they possess an apparently normal axoneme and paraflagellar rod structure. The flagellar attachment zone appeared to be disrupted when FLA3 was depleted. Thus, while flagellar attachment is a constitutive feature of the life cycle of trypanosomes, attachment requires stage specific elements at the protein level.

  8. LocZ Is a New Cell Division Protein Involved in Proper Septum Placement in Streptococcus pneumoniae

    Science.gov (United States)

    Holečková, Nela; Molle, Virginie; Buriánková, Karolína; Benada, Oldřich; Kofroňová, Olga; Ulrych, Aleš; Branny, Pavel

    2014-01-01

    ABSTRACT How bacteria control proper septum placement at midcell, to guarantee the generation of identical daughter cells, is still largely unknown. Although different systems involved in the selection of the division site have been described in selected species, these do not appear to be widely conserved. Here, we report that LocZ (Spr0334), a newly identified cell division protein, is involved in proper septum placement in Streptococcus pneumoniae. We show that locZ is not essential but that its deletion results in cell division defects and shape deformation, causing cells to divide asymmetrically and generate unequally sized, occasionally anucleated, daughter cells. LocZ has a unique localization profile. It arrives early at midcell, before FtsZ and FtsA, and leaves the septum early, apparently moving along with the equatorial rings that mark the future division sites. Consistently, cells lacking LocZ also show misplacement of the Z-ring, suggesting that it could act as a positive regulator to determine septum placement. LocZ was identified as a substrate of the Ser/Thr protein kinase StkP, which regulates cell division in S. pneumoniae. Interestingly, homologues of LocZ are found only in streptococci, lactococci, and enterococci, indicating that this close phylogenetically related group of bacteria evolved a specific solution to spatially regulate cell division. PMID:25550321

  9. Cell wall-bound invertase limits sucrose export and is involved in symptom development and inhibition of photosynthesis during compatible interaction between tomato and Xanthomonas campestris pv vesicatoria.

    Science.gov (United States)

    Kocal, Nurcan; Sonnewald, Uwe; Sonnewald, Sophia

    2008-11-01

    Cell wall-bound invertase (cw-Inv) plays an important role in carbohydrate partitioning and regulation of sink-source interaction. There is increasing evidence that pathogens interfere with sink-source interaction, and induction of cw-Inv activity has frequently been shown in response to pathogen infection. To investigate the role of cw-Inv, transgenic tomato (Solanum lycopersicum) plants silenced for the major leaf cw-Inv isoforms were generated and analyzed during normal growth and during the compatible interaction with Xanthomonas campestris pv vesicatoria. Under normal growth conditions, activities of sucrolytic enzymes as well as photosynthesis and respiration were unaltered in the transgenic plants compared with wild-type plants. However, starch levels of source leaves were strongly reduced, which was most likely caused by an enhanced sucrose exudation rate. Following X. campestris pv vesicatoria infection, cw-Inv-silenced plants showed an increased sucrose to hexose ratio in the apoplast of leaves. Symptom development, inhibition of photosynthesis, and expression of photosynthetic genes were clearly delayed in transgenic plants compared with wild-type plants. In addition, induction of senescence-associated and pathogenesis-related genes observed in infected wild-type plants was abolished in cw-Inv-silenced tomato lines. These changes were not associated with decreased bacterial growth. In conclusion, cw-Inv restricts carbon export from source leaves and regulates the sucrose to hexose ratio in the apoplast. Furthermore, an increased apoplastic hexose to sucrose ratio can be linked to inhibition of photosynthesis and induction of pathogenesis-related gene expression but does not significantly influence bacterial growth. Indirectly, bacteria may benefit from low invertase activity, since the longevity of host cells is raised and basal defense might be dampened.

  10. Composite Transport Model and Water and Solute Transport across Plant Roots: An Update.

    Science.gov (United States)

    Kim, Yangmin X; Ranathunge, Kosala; Lee, Seulbi; Lee, Yejin; Lee, Deogbae; Sung, Jwakyung

    2018-01-01

    The present review examines recent experimental findings in root transport phenomena in terms of the composite transport model (CTM). It has been a well-accepted conceptual model to explain the complex water and solute flows across the root that has been related to the composite anatomical structure. There are three parallel pathways involved in the transport of water and solutes in roots - apoplast, symplast, and transcellular paths. The role of aquaporins (AQPs), which facilitate water flows through the transcellular path, and root apoplast is examined in terms of the CTM. The contribution of the plasma membrane bound AQPs for the overall water transport in the whole plant level was varying depending on the plant species, age of roots with varying developmental stages of apoplastic barriers, and driving forces (hydrostatic vs. osmotic). Many studies have demonstrated that the apoplastic barriers, such as Casparian bands in the primary anticlinal walls and suberin lamellae in the secondary cell walls, in the endo- and exodermis are not perfect barriers and unable to completely block the transport of water and some solute transport into the stele. Recent research on water and solute transport of roots with and without exodermis triggered the importance of the extension of conventional CTM adding resistances that arrange in series (epidermis, exodermis, mid-cortex, endodermis, and pericycle). The extension of the model may answer current questions about the applicability of CTM for composite water and solute transport of roots that contain complex anatomical structures with heterogeneous cell layers.

  11. Yeast two-hybrid screens imply involvement of Fanconi anemia proteins in transcription regulation, cell signaling, oxidative metabolism, and cellular transport.

    Science.gov (United States)

    Reuter, Tanja Y; Medhurst, Annette L; Waisfisz, Quinten; Zhi, Yu; Herterich, Sabine; Hoehn, Holger; Gross, Hans J; Joenje, Hans; Hoatlin, Maureen E; Mathew, Christopher G; Huber, Pia A J

    2003-10-01

    Mutations in one of at least eight different genes cause bone marrow failure, chromosome instability, and predisposition to cancer associated with the rare genetic syndrome Fanconi anemia (FA). The cloning of seven genes has provided the tools to study the molecular pathway disrupted in Fanconi anemia patients. The structure of the genes and their gene products provided few clues to their functional role. We report here the use of 3 FA proteins, FANCA, FANCC, and FANCG, as "baits" in the hunt for interactors to obtain clues for FA protein functions. Using five different human cDNA libraries we screened 36.5x10(6) clones with the technique of the yeast two-hybrid system. We identified 69 proteins which have not previously been linked to the FA pathway as direct interactors of FANCA, FANCC, or FANCG. Most of these proteins are associated with four functional classes including transcription regulation (21 proteins), signaling (13 proteins), oxidative metabolism (10 proteins), and intracellular transport (11 proteins). Interaction with 6 proteins, DAXX, Ran, IkappaBgamma, USP14, and the previously reported SNX5 and FAZF, was additionally confirmed by coimmunoprecipitation and/or colocalization studies. Taken together, our data strongly support the hypothesis that FA proteins are functionally involved in several complex cellular pathways including transcription regulation, cell signaling, oxidative metabolism, and cellular transport.

  12. Isolation of proteins involved in the replication of adenoviral DNA in vitro

    International Nuclear Information System (INIS)

    Lichy, J.H.; Nagata, K.; Friefeld, B.R.; Enomoto, T.; Field, J.; Guggenheimer, R.A.; Ikeda, J.E.; Horwitz, M.S.; Hurwitz, J.

    1983-01-01

    The simple mechanism of replication of adenoviral DNA has made adenovirus an especially useful model system for studies of eukaryotic replication mechanisms. The availability of this in vitro system that replicates exogenously added Ad DNA-pro has made it possible to characterize the factors involved in replication. The results presented in this paper summarize our further fractionation of the in vitro system. First, the properties of two factors purified from the uninfected nuclear extract are described. Second, the separation of the pTP/Ad Pol complex into subunits and the properties of the isolated subunits are presented. The 140K protein is shown to possess the Ad DNA polymerase activity. The results suggest that the only DNA polymerase required for adenoviral DNA replication in vitro is the 140K Ad DNA polymerase and that this enzyme is probably a viral gene product. 50 references, 10 figures, 3 tables

  13. Postnatal growth velocity modulates alterations of proteins involved in metabolism and neuronal plasticity in neonatal hypothalamus in rats born with intrauterine growth restriction.

    Science.gov (United States)

    Alexandre-Gouabau, Marie-Cécile F; Bailly, Emilie; Moyon, Thomas L; Grit, Isabelle C; Coupé, Bérengère; Le Drean, Gwenola; Rogniaux, Hélène J; Parnet, Patricia

    2012-02-01

    Intrauterine growth restriction (IUGR) due to maternal protein restriction is associated in rats with an alteration in hypothalamic centers involved in feeding behaviour. In order to gain insight into the mechanism of perinatal maternal undernutrition in the brain, we used proteomics approach to identify hypothalamic proteins that are altered in their expression following protein restriction in utero. We used an animal model in which restriction of the protein intake of pregnant rats (8% vs. 20%) produces IUGR pups which were randomized to a nursing regimen leading to either rapid or slow catch-up growth. We identified several proteins which allowed, by multivariate analysis, a very good discrimination of the three groups according to their perinatal nutrition. These proteins were related to energy-sensing pathways (Eno 1, E(2)PDH, Acot 1 and Fabp5), redox status (Bcs 1L, PrdX3 and 14-3-3 protein) or amino acid pathway (Acy1) as well as neurodevelopment (DRPs, MAP2, Snca). In addition, the differential expressions of several key proteins suggested possible shunts towards ketone-body metabolism and lipid oxidation, providing the energy and carbon skeletons necessary to lipogenesis. Our results show that maternal protein deprivation during pregnancy only (IUGR with rapid catch-up growth) or pregnancy and lactation (IUGR with slow postnatal growth) modulates numerous metabolic pathways resulting in alterations of hypothalamic energy supply. As several of these pathways are involved in signalling, it remains to be determined whether hypothalamic proteome adaptation of IUGR rats in response to different postnatal growth rates could also interfere with cerebral plasticity or neuronal maturation. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

    Directory of Open Access Journals (Sweden)

    Petra Procházková

    Full Text Available Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs of the 5'- or 3'-untranslated regions (UTR of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP. The earthworm IRE site in 5'-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant.

  15. Involvement of the Iron Regulatory Protein from Eisenia andrei Earthworms in the Regulation of Cellular Iron Homeostasis

    Science.gov (United States)

    Procházková, Petra; Škanta, František; Roubalová, Radka; Šilerová, Marcela; Dvořák, Jiří; Bilej, Martin

    2014-01-01

    Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5′- or 3′-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5′-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant. PMID:25279857

  16. Proteins involved in attack and defence in Zygomycete-aphid interactions

    DEFF Research Database (Denmark)

    Grell, Morten Nedergaard; Jensen, Annette Bruun; Lange, Lene

    2009-01-01

    and defense related proteins of the host. Now, selected proteins are being produced in an expression host for further studies. The conservation of the fungal secreted proteins in the species dominating the collected material, namely Pandora neoaphidis, Entomophthora planchoniana and Conidiobolus obscurus......, and additional related fungi are under investigation. We anticipate that our work will shed light on this highly specialized group of fungi that has attracted substantial attention as potential bio-control agents....

  17. APC/C-mediated degradation of dsRNA-binding protein 4 (DRB4 involved in RNA silencing.

    Directory of Open Access Journals (Sweden)

    Katia Marrocco

    Full Text Available Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome is a master ubiquitin protein ligase (E3 that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized.In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA. This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2 of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed.Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular-protein concentrations and homeostasis of DRB4.

  18. Involvement of Trichoderma harzianum Epl-1 Protein in the Regulation of Botrytis Virulence- and Tomato Defense-Related Genes.

    Science.gov (United States)

    Gomes, Eriston V; Ulhoa, Cirano J; Cardoza, Rosa E; Silva, Roberto N; Gutiérrez, Santiago

    2017-01-01

    Several Trichoderma spp. are well known for their ability to: (i) act as important biocontrol agents against phytopathogenic fungi; (ii) function as biofertilizers; (iii) increase the tolerance of plants to biotic and abiotic stresses; and (iv) induce plant defense responses via the production and secretion of elicitor molecules. In this study, we analyzed the gene-regulation effects of Trichoderma harzianum Epl-1 protein during the interactions of mutant Δ epl-1 or wild-type T. harzianum strains with: (a) the phytopathogen Botrytis cinerea and (b) with tomato plants, on short (24 h hydroponic cultures) and long periods (4-weeks old plants) after Trichoderma inoculation. Our results indicate that T. harzianum Epl-1 protein affects the in vitro expression of B. cinerea virulence genes, especially those involved in the botrydial biosynthesis ( BcBOT genes), during the mycoparasitism interaction. The tomato defense-related genes were also affected, indicating that Epl-1 is involved in the elicitation of the salicylic acid pathway. Moreover, Epl-1 also regulates the priming effect in host tomato plants and contributes to enhance the interaction with the host tomato plant during the early stage of root colonization.

  19. Involvement of Trichoderma harzianum Epl-1 Protein in the Regulation of Botrytis Virulence- and Tomato Defense-Related Genes

    Directory of Open Access Journals (Sweden)

    Eriston V. Gomes

    2017-05-01

    Full Text Available Several Trichoderma spp. are well known for their ability to: (i act as important biocontrol agents against phytopathogenic fungi; (ii function as biofertilizers; (iii increase the tolerance of plants to biotic and abiotic stresses; and (iv induce plant defense responses via the production and secretion of elicitor molecules. In this study, we analyzed the gene-regulation effects of Trichoderma harzianum Epl-1 protein during the interactions of mutant Δepl-1 or wild-type T. harzianum strains with: (a the phytopathogen Botrytis cinerea and (b with tomato plants, on short (24 h hydroponic cultures and long periods (4-weeks old plants after Trichoderma inoculation. Our results indicate that T. harzianum Epl-1 protein affects the in vitro expression of B. cinerea virulence genes, especially those involved in the botrydial biosynthesis (BcBOT genes, during the mycoparasitism interaction. The tomato defense-related genes were also affected, indicating that Epl-1 is involved in the elicitation of the salicylic acid pathway. Moreover, Epl-1 also regulates the priming effect in host tomato plants and contributes to enhance the interaction with the host tomato plant during the early stage of root colonization.

  20. AMP-activated protein kinase is involved in the activation of the Fanconi anemia/BRCA pathway in response to DNA interstrand crosslinks.

    Science.gov (United States)

    Chun, Min Jeong; Kim, Sunshin; Hwang, Soo Kyung; Kim, Bong Sub; Kim, Hyoun Geun; Choi, Hae In; Kim, Jong Heon; Goh, Sung Ho; Lee, Chang-Hun

    2016-08-16

    Fanconi anemia complementation group (FANC) proteins constitute the Fanconi Anemia (FA)/BRCA pathway that is activated in response to DNA interstrand crosslinks (ICLs). We previously performed yeast two-hybrid screening to identify novel FANC-interacting proteins and discovered that the alpha subunit of AMP-activated protein kinase (AMPKα1) was a candidate binding partner of the FANCG protein, which is a component of the FA nuclear core complex. We confirmed the interaction between AMPKα and both FANCG using co-immunoprecipitation experiments. Additionally, we showed that AMPKα interacted with FANCA, another component of the FA nuclear core complex. AMPKα knockdown in U2OS cells decreased FANCD2 monoubiquitination and nuclear foci formation upon mitomycin C-induced ICLs. Furthermore, AMPKα knockdown enhanced cellular sensitivity to MMC. MMC treatment resulted in an increase in AMPKα phosphorylation/activation, indicating AMPK is involved in the cellular response to ICLs. FANCA was phosphorylated by AMPK at S347 and phosphorylation increased with MMC treatment. MMC-induced FANCD2 monoubiquitination and nuclear foci formation were compromised in a U2OS cell line that stably overexpressed the S347A mutant form of FANCA compared to wild-type FANCA-overexpressing cells, indicating a requirement for FANCA phosphorylation at S347 for proper activation of the FA/BRCA pathway. Our data suggest AMPK is involved in the activation of the FA/BRCA pathway.

  1. Physico-Pathologic Mechanisms Involved in Neurodegeneration: Misfolded Protein-Plasma Membrane Interactions.

    Science.gov (United States)

    Shrivastava, Amulya Nidhi; Aperia, Anita; Melki, Ronald; Triller, Antoine

    2017-07-05

    Several neurodegenerative disorders, such as Alzheimer's and Parkinson's disease, are characterized by prominent loss of synapses and neurons associated with the presence of abnormally structured or misfolded protein assemblies. Cell-to-cell transfer of misfolded proteins has been proposed for the intra-cerebral propagation of these diseases. When released, misfolded proteins diffuse in the 3D extracellular space before binding to the plasma membrane of neighboring cells, where they diffuse on a 2D plane. This reduction in diffusion dimension and the cell surface molecular crowding promote deleterious interactions with native membrane proteins, favoring clustering and further aggregation of misfolded protein assemblies. These processes open up new avenues for therapeutics development targeting the initial interactions of deleterious proteins with the plasma membrane or the subsequent pathological signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Involvement of protein kinase C in the mechanism of action of Escherichia coli heat-stable enterotoxin (STa) in a human colonic carcinoma cell line, COLO-205

    International Nuclear Information System (INIS)

    Gupta, Dyuti Datta; Saha, Subhrajit; Chakrabarti, Manoj K.

    2005-01-01

    The present study was undertaken to determine the involvement of calcium-protein kinase C pathway in the mechanism of action of Escherichia coli heat stable enterotoxin (STa) apart from STa-induced activation of guanylate cyclase in human colonic carcinoma cell line COLO-205, which was used as a model cultured cell line to study the mechanism of action of E. coli STa. In response to E. coli STa, protein kinase C (PKC) activity was increased in a time-dependent manner with its physical translocation from cytosol to membrane. Inhibition of the PKC activity in membrane fraction and inhibition of its physical translocation in response to IP 3 -mediated calcium release inhibitor dantrolene suggested the involvement of intracellular store depletion in the regulation of PKC activity. Among different PKC isoforms, predominant involvement of calcium-dependent protein kinase C (PKCα) was specified using isotype-specific pseudosubstrate, which showed pronounce enzyme activity. Inhibition of enzyme activity by PKCα-specific inhibitor Goe6976 and immunoblott study employing isotype-specific antibody further demonstrated the involvement of calcium-dependent isoform of PKC in the mechanism of action of E. coli STa. Moreover, inhibition of guanylate cyclase activity by PKCα-specific inhibitor Goe6976 suggested the involvement of PKCα in the regulation of guanylate cyclase activity

  3. Primary structure and subcellular localization of two fimbrial subunit-like proteins involved in the biosynthesis of K99 fibrillae.

    Science.gov (United States)

    Roosendaal, E; Jacobs, A A; Rathman, P; Sondermeyer, C; Stegehuis, F; Oudega, B; de Graaf, F K

    1987-09-01

    Analysis of the nucleotide sequence of the distal part of the fan gene cluster encoding the proteins involved in the biosynthesis of the fibrillar adhesin, K99, revealed the presence of two structural genes, fanG and fanH. The amino acid sequence of the gene products (FanG and FanH) showed significant homology to the amino acid sequence of the fibrillar subunit protein (FanC). Introduction of a site-specific frameshift mutation in fanG or fanH resulted in a simultaneous decrease in fibrillae production and adhesive capacity. Analysis of subcellular fractions showed that, in contrast to the K99 fibrillar subunit (FanC), both the FanH and the FanG protein were loosely associated with the outer membrane, possibly on the periplasmic side, but were not components of the fimbriae themselves.

  4. NDV-induced apoptosis in absence of Bax; evidence of involvement of apoptotic proteins upstream of mitochondria

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    Molouki Aidin

    2012-08-01

    Full Text Available Abstract Background Recently it was shown that following infection of HeLa cells with Newcastle disease virus (NDV, the matrix (M protein binds to Bax and subsequently the intrinsic pathway of apoptosis is activated. Moreover, there was very little alteration on mRNA and protein levels of Bax and Bcl-2 after infection with NDV. Finding In order to further investigate the role of members of the Bcl-2 family, Bax-knockout and wild-type HCT116 cells were infected with NDV strain AF2240. Although both cells underwent apoptosis through the activation of the intrinsic pathway and the release of cytochrome c from mitochondria, the percentage of dead Bax-knockout cells was significantly lower than wt cells (more than 10% at 48 h post-infection. In a parallel experiment, the effect of NDV on HT29 cells, that are originally Bcl-2-free, was studied. Apoptosis in HT29 cells was associated with Bax redistribution from cytoplasm to mitochondria, similar to that of HeLa and wt HCT116 cells. Conclusion Although the presence of Bax during NDV-induced apoptosis contributes to a faster cell death, it was concluded that other apoptotic protein(s upstream of mitochondria are also involved since cancer cells die whether in the presence or absence of Bax. Therefore, the classic Bax/Bcl-2 ratio may not be a major determinant in NDV-induced apoptosis.

  5. Anaplasma phagocytophilum MSP4 and HSP70 Proteins Are Involved in Interactions with Host Cells during Pathogen Infection

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    Marinela Contreras

    2017-07-01

    Full Text Available Anaplasma phagocytophilum transmembrane and surface proteins play a role during infection and multiplication in host neutrophils and tick vector cells. Recently, A. phagocytophilum Major surface protein 4 (MSP4 and Heat shock protein 70 (HSP70 were shown to be localized on the bacterial membrane, with a possible role during pathogen infection in ticks. In this study, we hypothesized that A. phagocytophilum MSP4 and HSP70 have similar functions in tick-pathogen and host-pathogen interactions. To address this hypothesis, herein we characterized the role of these bacterial proteins in interaction and infection of vertebrate host cells. The results showed that A. phagocytophilum MSP4 and HSP70 are involved in host-pathogen interactions, with a role for HSP70 during pathogen infection. The analysis of the potential protective capacity of MSP4 and MSP4-HSP70 antigens in immunized sheep showed that MSP4-HSP70 was only partially protective against pathogen infection. This limited protection may be associated with several factors, including the recognition of non-protective epitopes by IgG in immunized lambs. Nevertheless, these antigens may be combined with other candidate protective antigens for the development of vaccines for the control of human and animal granulocytic anaplasmosis. Focusing on the characterization of host protective immune mechanisms and protein-protein interactions at the host-pathogen interface may lead to the discovery and design of new effective protective antigens.

  6. Elevated atmospheric CO2 decreases the ammonia compensation point of barley plants

    DEFF Research Database (Denmark)

    Wang, Liang; Pedas, Pai; Eriksson, Ulf Dennis

    2013-01-01

    mu mol mol(-1)) or elevated (800 mu mol mol(-1)) CO2 concentration with NO3- or NH4NO3 as the nitrogen source. The concentrations of NH4+ and H+ in the leaf apoplastic solution were measured along with different foliar N pools and enzymes involved in N metabolism. Elevated CO2 caused a threefold...... decrease in the NH4+ concentration in the apoplastic solution and slightly acidified it. This resulted in a decline of the chi(NH3) from 2.25 and 2.95 nmol mol(-1) under ambient CO2 to 0.37 and 0.89 nmol mol(-1) at elevated CO2 in the NO3- and NH4NO3 treatments, respectively. The decrease in chi(NH3...

  7. Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4 exhibits growth inhibitory effects in prostate cancer cells

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    Shayan eSarkar

    2015-10-01

    Full Text Available The gene Par-4 (Prostate Apoptosis Response 4 was originally identified in prostate cancer cells undergoing apoptosis and its product Par-4 showed cancer specific pro-apoptotic activity. Particularly, the SAC domain of Par-4 (SAC-Par-4 selectively kills cancer cells leaving normal cells unaffected. The therapeutic significance of bioactive SAC-Par-4 is enormous in cancer biology; however, its large scale production is still a matter of concern. Here we report the production of SAC-Par-4-GFP fusion protein coupled to translational enhancer sequence (5′ AMV and apoplast signal peptide (aTP in transgenic Nicotiana tabacum cv. Samsun NN plants under the control of a unique recombinant promoter M24. Transgene integration was confirmed by genomic DNA PCR, Southern and Northern blotting, Real-time PCR and Nuclear run-on assays. Results of Western blot analysis and ELISA confirmed expression of recombinant SAC-Par-4-GFP protein and it was as high as 0.15% of total soluble protein. In addition, we found that targeting of plant recombinant SAC-Par-4-GFP to the apoplast and endoplasmic reticulum (ER was essential for the stability of plant recombinant protein in comparison to the bacterial derived SAC-Par-4. Deglycosylation analysis demonstrated that ER-targeted SAC-Par-4-GFP-SEKDEL undergoes O-linked glycosylation unlike apoplast-targeted SAC-Par-4-GFP. Furthermore, various in vitro studies like mammalian cells proliferation assay (MTT, apoptosis induction assays, and NF-κB suppression suggested the cytotoxic and apoptotic properties of plant-derived SAC-Par-4-GFP against multiple prostate cancer cell lines. Additionally, pre-treatment of MAT-LyLu prostate cancer cells with purified SAC-Par-4-GFP significantly delayed the onset of tumor in a syngeneic rat prostate cancer model. Taken altogether, we proclaim that plant made SAC-Par-4 may become a useful alternate therapy for effectively alleviating cancer in the new era.

  8. Wounding coordinately induces cell wall protein, cell cycle and pectin methyl esterase genes involved in tuber closing layer and wound periderm development.

    Science.gov (United States)

    Neubauer, Jonathan D; Lulai, Edward C; Thompson, Asunta L; Suttle, Jeffrey C; Bolton, Melvin D

    2012-04-15

    Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1-5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5-14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine-and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more

  9. A cytoskeleton-associated protein, TMAP/CKAP2, is involved in the proliferation of human foreskin fibroblasts

    International Nuclear Information System (INIS)

    Jeon, Sang-Min; Choi, Bongkun; Hong, Kyung Uk; Kim, Eunhee; Seong, Yeon-Sun; Bae, Chang-Dae; Park, Joobae

    2006-01-01

    Previously, we reported the cloning of a cytoskeleton-associated protein, TMAP/CKAP2, which was up-regulated in primary human gastric cancers. Although TMAP/CKAP2 has been found to be expressed in most cancer cell lines examined, the function of CKAP2 is not known. In this study, we found that TMAP/CKAP2 was not expressed in G0/G1 arrested HFFs, but that it was expressed in actively dividing cells. After initiating the cell cycle, TMAP/CKAP2 levels remained low throughout most of the G1 phase, but gradually increased between late G1 and G2/M. Knockdown of TMAP/CKAP2 reduced pRB phosphorylation and increased p27 expression, and consequently reduced HFF proliferation, whereas constitutive TMAP/CKAP2 expression increased pRB phosphorylation and enhanced proliferation. Our results show that this novel cytoskeleton-associated protein is expressed cell cycle dependently and that it is involved in cell proliferation

  10. A cytoskeleton-associated protein, TMAP/CKAP2, is involved in the proliferation of human foreskin fibroblasts.

    Science.gov (United States)

    Jeon, Sang-Min; Choi, Bongkun; Hong, Kyung Uk; Kim, Eunhee; Seong, Yeon-Sun; Bae, Chang-Dae; Park, Joobae

    2006-09-15

    Previously, we reported the cloning of a cytoskeleton-associated protein, TMAP/CKAP2, which was up-regulated in primary human gastric cancers. Although TMAP/CKAP2 has been found to be expressed in most cancer cell lines examined, the function of CKAP2 is not known. In this study, we found that TMAP/CKAP2 was not expressed in G0/G1 arrested HFFs, but that it was expressed in actively dividing cells. After initiating the cell cycle, TMAP/CKAP2 levels remained low throughout most of the G1 phase, but gradually increased between late G1 and G2/M. Knockdown of TMAP/CKAP2 reduced pRB phosphorylation and increased p27 expression, and consequently reduced HFF proliferation, whereas constitutive TMAP/CKAP2 expression increased pRB phosphorylation and enhanced proliferation. Our results show that this novel cytoskeleton-associated protein is expressed cell cycle dependently and that it is involved in cell proliferation.

  11. Pseudomonas aeruginosa invasion and cytotoxicity are independent events, both of which involve protein tyrosine kinase activity.

    Science.gov (United States)

    Evans, D J; Frank, D W; Finck-Barbançon, V; Wu, C; Fleiszig, S M

    1998-04-01

    Pseudomonas aeruginosa clinical isolates exhibit invasive or cytotoxic phenotypes. Cytotoxic strains acquire some of the characteristics of invasive strains when a regulatory gene, exsA, that controls the expression of several extracellular proteins, is inactivated. exsA mutants are not cytotoxic and can be detected within epithelial cells by gentamicin survival assays. The purpose of this study was to determine whether epithelial cell invasion precedes and/or is essential for cytotoxicity. This was tested by measuring invasion (gentamicin survival) and cytotoxicity (trypan blue staining) of PA103 mutants deficient in specific exsA-regulated proteins and by testing the effect of drugs that inhibit invasion for their effect on cytotoxicity. A transposon mutant in the exsA-regulated extracellular factor exoU was neither cytotoxic nor invasive. Furthermore, several of the drugs that inhibited invasion did not prevent cytotoxicity. These results show that invasion and cytotoxicity are mutually exclusive events, inversely regulated by an exsA-encoded invasion inhibitor(s). Both involve host cell protein tyrosine kinase (PTK) activity, but they differ in that invasion requires Src family tyrosine kinases and calcium-calmodulin activity. PTK inhibitor drugs such as genistein may have therapeutic potential through their ability to block both invasive and cytotoxicity pathways via an action on the host cell.

  12. Involvement of C4 protein of beet severe curly top virus (family Geminiviridae in virus movement.

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    Kunling Teng

    Full Text Available BACKGROUND: Beet severe curly top virus (BSCTV is a leafhopper transmitted geminivirus with a monopartite genome. C4 proteins encoded by geminivirus play an important role in virus/plant interaction. METHODS AND FINDINGS: To understand the function of C4 encoded by BSCTV, two BSCTV mutants were constructed by introducing termination codons in ORF C4 without affecting the amino acids encoded by overlapping ORF Rep. BSCTV mutants containing disrupted ORF C4 retained the ability to replicate in Arabidopsis protoplasts and in the agro-inoculated leaf discs of N. benthamiana, suggesting C4 is not required for virus DNA replication. However, both mutants did not accumulate viral DNA in newly emerged leaves of inoculated N. benthamiana and Arabidopsis, and the inoculated plants were asymptomatic. We also showed that C4 expression in plant could help C4 deficient BSCTV mutants to move systemically. C4 was localized in the cytosol and the nucleus in both Arabidopsis protoplasts and N. benthamiana leaves and the protein appeared to bind viral DNA and ds/ssDNA nonspecifically, displaying novel DNA binding properties. CONCLUSIONS: Our results suggest that C4 protein in BSCTV is involved in symptom production and may facilitate virus movement instead of virus replication.

  13. A cerato-platanin protein SsCP1 targets plant PR1 and contributes to virulence of Sclerotinia sclerotiorum

    DEFF Research Database (Denmark)

    Yang, Guogen; Tang, Liguang; Gong, Yingdi

    2018-01-01

    in the apoplast to facilitate infection by S. sclerotiorum. Overexpressing PR1 enhanced resistance to the wild-type strain, but not to the Sscp1 knockout strain of S. sclerotiorum. Sscp1-expressing transgenic plants showed increased concentrations of salicylic acid (SA) and higher levels of resistance to several...

  14. Identification and characterisation of a hyper-variable apoplastic effector gene family of the potato cyst nematodes.

    Science.gov (United States)

    Eves-van den Akker, Sebastian; Lilley, Catherine J; Jones, John T; Urwin, Peter E

    2014-09-01

    Sedentary endoparasitic nematodes are obligate biotrophs that modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Using assumptions about the characteristics of genes involved in plant-nematode biotrophic interactions to inform the identification strategy, we provide a description and characterisation of a novel group of hyper-variable extracellular effectors termed HYP, from the potato cyst nematode Globodera pallida. HYP effectors comprise a large gene family, with a modular structure, and have unparalleled diversity between individuals of the same population: no two nematodes tested had the same genetic complement of HYP effectors. Individuals vary in the number, size, and type of effector subfamilies. HYP effectors are expressed throughout the biotrophic stages in large secretory cells associated with the amphids of parasitic stage nematodes as confirmed by in situ hybridisation. The encoded proteins are secreted into the host roots where they are detectable by immunochemistry in the apoplasm, between the anterior end of the nematode and the feeding site. We have identified HYP effectors in three genera of plant parasitic nematodes capable of infecting a broad range of mono- and dicotyledon crop species. In planta RNAi targeted to all members of the effector family causes a reduction in successful parasitism.

  15. Involvement of both protein kinase C and G proteins in superoxide production after IgE triggering in guinea pig eosinophils

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    Toshiya Aizawa

    1997-01-01

    Full Text Available To study the function and mechanism of eosinophils via the low affinity IgE receptor (FceRII, we examined the production of 02 metabolites by measuring the luminol-dependent chemiluminescence (LDCL response and the generation of cysteinyl leukotrienes. Eosinophils obtained from guinea pig peritoneal fluid sensitized with horse serum were purified. Luminol-dependent chemiluminescence was induced by stimulation with monoclonal anti-CD23 antibody, but not by mouse serum (controls. The mean (±SEM value of LDCL was 20.6±1.3X103 c.p.m. This reaction consisted of an initial rapid phase and a propagation phase and ended within lOmin. Guinea pig eosinophils were histochemically stained with monoclonal anti-CD23 antibody. The major product generated in the LDCL response was superoxide, as determined by the measurement of superoxide by cytochrome c reduction and the complete inhibitory effect of superoxide dismutase on the LDCL response. Pretreatment with either pertussis toxin or cholera toxin inhibited the LDCL reaction. Depletion of bivalent ions by EDTA inhibited this response and the protein kinase C inhibitor D-sphingosin inhibited both 1-oleoyl-2-acetyl-glycerol-induced and FcϵRII-mediated LDCL. These findings suggest that the NADPH-protein kinase C pathway may be involved in the FceRII-mediated LDCL response in guinea pig eosinophils.

  16. Proteome profiling of neuroblastoma-derived exosomes reveal the expression of proteins potentially involved in tumor progression.

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    Danilo Marimpietri

    Full Text Available Neuroblastoma (NB is the most common extracranial solid tumor in childhood, with grim prognosis in a half of patients. Exosomes are nanometer-sized membrane vesicles derived from the multivesicular bodies (MVBs of the endocytic pathway and released by normal and neoplastic cells. Tumor-derived exosomes have been shown in different model systems to carry molecules that promote cancer growth and dissemination. In this respect, we have here performed the first characterization and proteomic analysis of exosomes isolated from human NB cell lines by filtration and ultracentrifugation. Electron microscopy demonstrated that NB-derived exosomes exhibited the characteristic cup-shaped morphology. Dynamic light scattering studies showed a bell-shaped curve and a polydispersity factor consistent with those of exosomes. Zeta potential values suggested a good nanoparticle stability. We performed proteomic analysis of NB-derived exosomes by two dimension liquid chromatography separation and mass spectrometry analyses using the multidimensional protein identification technology strategy. We found that the large majority of the proteins identified in NB derived exosomes are present in Exocarta database including tetraspanins, fibronectin, heat shock proteins, MVB proteins, cytoskeleton-related proteins, prominin-1 (CD133, basigin (CD147 and B7-H3 (CD276. Expression of the CD9, CD63 and CD81 tetraspanins, fibronectin, CD133, CD147 and CD276 was validated by flow cytometry. Noteworthy, flow cytometric analysis showed that NB-derived exosomes expressed the GD2 disialoganglioside, the most specific marker of NB. In conclusion, this study shows that NB-derived exosomes express a discrete set of molecules involved in defense response, cell differentiation, cell proliferation and regulation of other important biological process. Thus, NB-derived exosomes may play an important role in the modulation of tumor microenvironment and represent potential tumor biomarkers.

  17. Prediction of Chlamydia pneumoniae protein localization in host mitochondria and cytoplasm and possible involvements in lung cancer etiology: a computational approach

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    Aws Alshamsan

    2017-12-01

    Full Text Available Collecting evidence suggests that the intercellular infection of Chlamydia pneumoniae in lungs contributes to the etiology of lung cancer. Many proteins of Chlamydia pneumoniae outmanoeuvre the various system of the host. The infection may regulate various factors, which can influence the growth of lung cancer in affected persons. In this in-silico study, we predict potential targeting of Chlamydia pneumoniae proteins in mitochondrial and cytoplasmic comportments of host cell and their possible involvement in growth and development of lung cancer. Various cellular activities are controlled in mitochondria and cytoplasm, where the localization of Chlamydia pneumoniae proteins may alter the normal functioning of host cells. The rationale of this study is to find out and explain the connection between Chlamydia pneumoniae infection and lung cancer. A sum of 183 and 513 proteins were predicted to target in mitochondria and cytoplasm of host cell out of total 1112 proteins of Chlamydia pneumoniae. In particular, many targeted proteins may interfere with normal growth behaviour of host cells, thereby altering the decision of program cell death. Present article provides a potential connection of Chlamydia pneumoniae protein targeting and proposed that various targeted proteins may play crucial role in lung cancer etiology through diverse mechanisms.

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.

  19. Generation of mariner-based transposon insertion mutant library of Bacillus sphaericus 2297 and investigation of genes involved in sporulation and mosquito-larvicidal crystal protein synthesis.

    Science.gov (United States)

    Wu, Yiming; Hu, Xiaomin; Ge, Yong; Zheng, Dasheng; Yuan, Zhiming

    2012-05-01

    Bacillus sphaericus has been used with great success in mosquito control programs worldwide. Under conditions of nutrient limitation, it undergoes sporulation via a series of well defined morphological stages. However, only a small number of genes involved in sporulation have been identified. To identify genes associated with sporulation, and to understand the relationship between sporulation and crystal protein synthesis, a random mariner-based transposon insertion mutant library of B. sphaericus strain 2297 was constructed and seven sporulation-defective mutants were selected. Sequencing of the DNA flanking of the transposon insertion identified several genes involved in sporulation. The morphologies of mutants were determined by electron microscopy and synthesis of crystal proteins was analyzed by SDS-PAGE and Western blot. Four mutants blocked at early stages of sporulation failed to produce crystal proteins and had lower larvicidal activity. However, the other three mutants were blocked at later stages and were able to form crystal proteins, and the larvicidal activity was similar to wild type. These results indicated that crystal protein synthesis in B. sphaericus is dependent on sporulation initiation. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  20. Ice recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic Antarctic hair grass Deschampsia antarctica E. Desv.

    Science.gov (United States)

    John, Ulrik P; Polotnianka, Renatam M; Sivakumaran, Kailayapillai A; Chew, Orinda; Mackin, Leanne; Kuiper, Micheal J; Talbot, Jonathan P; Nugent, Gregory D; Mautord, Julie; Schrauf, Gustavo E; Spangenberg, German C

    2009-04-01

    Antarctic hair grass (Deschampsia antarctica E. Desv.), the only grass indigenous to Antarctica, has well-developed freezing tolerance, strongly induced by cold acclimation. Here, we show that in response to low temperatures, D. antarctica expresses potent recrystallization inhibition (RI) activity that, inhibits the growth of small ice crystals into potentially damaging large ones, is proteinaceous and localized to the apoplasm. A gene family from D. antarctica encoding putative homologs of an ice recrystallization inhibition protein (IRIP) has been isolated and characterized. IRIPs are apoplastically targeted proteins with two potential ice-binding motifs: 1-9 leucine-rich repeats (LRRs) and c. 16 'IRIP' repeats. IRIP genes appear to be confined to the grass subfamily Pooideae and their products, exhibit sequence similarity to phytosulphokine receptors and are predicted to adopt conformations with two ice-binding surfaces. D. antarctica IRIP (DaIRIP) transcript levels are greatly enhanced in leaf tissue following cold acclimation. Transgenic Arabidopsis thaliana expressing a DaIRIP has novel RI activity, and purified DaIRIP, when added back to extracts of leaves from non-acclimated D. antarctica, can reconstitute the activity found in acclimated plants. We propose that IRIP-mediated RI activity may contribute to the cryotolerance of D. antarctica, and thus to its unique ability to have colonized Antarctica.

  1. Composite Transport Model and Water and Solute Transport across Plant Roots: An Update

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    Yangmin X. Kim

    2018-02-01

    Full Text Available The present review examines recent experimental findings in root transport phenomena in terms of the composite transport model (CTM. It has been a well-accepted conceptual model to explain the complex water and solute flows across the root that has been related to the composite anatomical structure. There are three parallel pathways involved in the transport of water and solutes in roots – apoplast, symplast, and transcellular paths. The role of aquaporins (AQPs, which facilitate water flows through the transcellular path, and root apoplast is examined in terms of the CTM. The contribution of the plasma membrane bound AQPs for the overall water transport in the whole plant level was varying depending on the plant species, age of roots with varying developmental stages of apoplastic barriers, and driving forces (hydrostatic vs. osmotic. Many studies have demonstrated that the apoplastic barriers, such as Casparian bands in the primary anticlinal walls and suberin lamellae in the secondary cell walls, in the endo- and exodermis are not perfect barriers and unable to completely block the transport of water and some solute transport into the stele. Recent research on water and solute transport of roots with and without exodermis triggered the importance of the extension of conventional CTM adding resistances that arrange in series (epidermis, exodermis, mid-cortex, endodermis, and pericycle. The extension of the model may answer current questions about the applicability of CTM for composite water and solute transport of roots that contain complex anatomical structures with heterogeneous cell layers.

  2. Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII.

    Science.gov (United States)

    Inturi, Raviteja; Mun, Kwangchol; Singethan, Katrin; Schreiner, Sabrina; Punga, Tanel

    2018-02-01

    Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and

  3. Nuclear substructure reorganization during late stageerythropoiesis is selective and does not involve caspase cleavage ofmajor nuclear substructural proteins

    Energy Technology Data Exchange (ETDEWEB)

    Krauss, Sharon Wald; Lo, Annie J.; Short, Sarah A.; Koury, MarkJ.; Mohandas, Narla; Chasis, Joel Anne

    2005-04-06

    Enucleation, a rare feature of mammalian differentiation, occurs in three cell types: erythroblasts, lens epithelium and keratinocytes. Previous investigations suggest that caspase activation functions in lens epithelial and keratinocyte enucleation, as well as in early erythropoiesis encompassing BFU-E differentiation to proerythroblast. To determine whether caspase activation contributes to later erythropoiesis and whether nuclear substructures other than chromatin reorganize, we analyzed distributions of nuclear subcompartment proteins and assayed for caspase-induced cleavage of subcompartmental target proteins in mouse erythroblasts. We found that patterns of lamin B in the filamentous network interacting with both the nuclear envelope and DNA, nuclear matrix protein NuMA, and splicing factors Sm and SC35 persisted during nuclear condensation, consistent with effective transcription of genes expressed late in differentiation. Thus nuclear reorganization prior to enucleation is selective, allowing maintenance of critical transcriptional processes independent of extensive chromosomal reorganization. Consistent with these data, we found no evidence for caspase-induced cleavage of major nuclear subcompartment proteins during late erythropoiesis, in contrast to what has been observed in early erythropoiesis and in lens epithelial and keratinocyte differentiation. These findings imply that nuclear condensation and extrusion during terminal erythroid differentiation involve novel mechanisms that do not entail major activation of apoptotic machinery.

  4. Two Sets of Piwi Proteins Are Involved in Distinct sRNA Pathways Leading to Elimination of Germline-Specific DNA

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    Dominique I. Furrer

    2017-07-01

    Full Text Available Piwi proteins and piRNAs protect eukaryotic germlines against the spread of transposons. During development in the ciliate Paramecium, two Piwi-dependent sRNA classes are involved in the elimination of transposons and transposon-derived DNA: scan RNAs (scnRNAs, associated with Ptiwi01 and Ptiwi09, and iesRNAs, whose binding partners we now identify as Ptiwi10 and Ptiwi11. scnRNAs derive from the maternal genome and initiate DNA elimination during development, whereas iesRNAs continue DNA targeting until the removal process is complete. Here, we show that scnRNAs and iesRNAs are processed by distinct Dicer-like proteins and bind Piwi proteins in a mutually exclusive manner, suggesting separate biogenesis pathways. We also demonstrate that the PTIWI10 gene is transcribed from the developing nucleus and that its transcription depends on prior DNA excision, suggesting a mechanism of gene expression control triggered by the removal of short DNA segments interrupting the gene.

  5. The Cerato-Platanin protein Epl-1 from Trichoderma harzianum is involved in mycoparasitism, plant resistance induction and self cell wall protection.

    Science.gov (United States)

    Gomes, Eriston Vieira; Costa, Mariana do Nascimento; de Paula, Renato Graciano; de Azevedo, Rafael Ricci; da Silva, Francilene Lopes; Noronha, Eliane F; Ulhoa, Cirano José; Monteiro, Valdirene Neves; Cardoza, Rosa Elena; Gutiérrez, Santiago; Silva, Roberto Nascimento

    2015-12-09

    Trichoderma harzianum species are well known as biocontrol agents against important fungal phytopathogens. Mycoparasitism is one of the strategies used by this fungus in the biocontrol process. In this work, we analyzed the effect of Epl-1 protein, previously described as plant resistance elicitor, in expression modulation of T. harzianum genes involved in mycoparasitism process against phytopathogenic fungi; self cell wall protection and recognition; host hyphae coiling and triggering expression of defense-related genes in beans plants. The results indicated that the absence of Epl-1 protein affects the expression of all mycoparasitism genes analyzed in direct confrontation assays against phytopathogen Sclerotinia sclerotiorum as well as T. harzianum itself; the host mycoparasitic coiling process and expression modulation of plant defense genes showing different pattern compared with wild type strain. These data indicated the involvement T. harzianum Epl-1 in self and host interaction and also recognition of T. harzianum as a symbiotic fungus by the bean plants.

  6. High Prevalence of Neutrophil Cytoplasmic Autoantibodies in Infants with Food Protein-Induced Proctitis/Proctocolitis: Autoimmunity Involvement?

    Directory of Open Access Journals (Sweden)

    Alena Sekerkova

    2015-01-01

    Full Text Available Background. Food protein-induced proctitis/proctocolitis (FPIP is the most common noninfectious colitis in children in the first year of life. Along with the overall clinical symptoms, diarrhoea and rectal bleeding are the main manifestations of the disease. There is no routine noninvasive test that would be specific for this type of colitis. The aim of our study was to find a noninvasive laboratory test or tests that may be helpful in differential diagnosis of food protein-induced proctitis/proctocolitis. Methods. ANA, ANCA, ASCA, a-EMA, a-tTg, specific IgE, total IgE, IgG, IgA, IgM, and concentration of serum calprotectin were measured in a group of 25 patients with colitis and 18 children with other diagnoses. Results. Atypical-pANCA antibodies of IgG isotype were detected in the sera of 24 patients by the method of indirect immunofluorescence, and 5 patients showed also the positivity of IgA isotype. In control samples these autoantibodies were not detected. Other autoantibodies were not demonstrated in either patient or control group. Conclusions. Of the parameters tested in noninfectious colitis, atypical-pANCA on ethanol-fixed granulocytes appears to be a suitable serological marker of food protein-induced proctitis/proctocolitis and suggests a possible involvement of an autoimmune mechanisms in the pathogenesis of this disease.

  7. Yeast Interacting Proteins Database: YGR013W, YKL012W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available tion U1 snRNP protein involved in splicing, interacts with the branchpoint-binding protein during the formation of the second commitm... PRP40 U1 snRNP protein involved in splicing, interacts with the branchpoint-binding protein during the form...ation of the second commitment complex Rows with this prey as prey (1) Rows with

  8. The RNA-binding protein xCIRP2 is involved in apoptotic tail regression during metamorphosis in Xenopus laevis tadpoles.

    Science.gov (United States)

    Eto, Ko; Iwama, Tomoyuki; Tajima, Tatsuya; Abe, Shin-ichi

    2012-10-01

    Frog metamorphosis induced by thyroid hormone (TH) involves not only cell proliferation and differentiation in reconstituted organs such as limbs, but also apoptotic cell death in degenerated organs such as tails. However, the molecular mechanisms directing the TH-dependent cell fate determination remain unclear. We have previously identified from newts an RNA-binding protein (nRBP) acting as the regulator governing survival and death in germ cells during spermatogenesis. To investigate the molecular events leading the tail resorption during metamorphosis, we analyzed the expression, the functional role in apoptosis, and the regulation of xCIRP2, a frog homolog of nRBP, in tails of Xenopus laevis tadpoles. At the prometamorphic stage, xCIRP2 protein is expressed in fibroblast, epidermal, nerve, and muscular cells and localized in their cytoplasm. When spontaneous metamorphosis progressed, the level of xCIRP2 mRNA remained unchanged but the amount of the protein decreased. In organ cultures of tails at the prometamorphic stage, xCIRP2 protein decreased before their lengths shortened during TH-dependent metamorphosis. The inhibition of calpain or proteasome attenuated the TH-induced decrease of xCIRP2 protein in tails, impairing their regression. These results suggest that xCIRP2 protein is downregulated through calpain- and proteasome-mediated proteolysis in response to TH at the onset of metamorphosis, inducing apoptosis in tails and thereby degenerating them. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Nitrile-specifier Proteins Involved in Glucosinolate Hydrolysis in Arabidopsis thaliana*S⃞

    Science.gov (United States)

    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. Elevated level of human RPA interacting protein α (hRIPα) in cervical tumor cells is involved in cell proliferation through regulating RPA transport.

    Science.gov (United States)

    Namkoong, Sim; Lee, Eun-Ju; Jang, Ik-Soon; Park, Junsoo

    2012-10-19

    Replication protein A (RPA) is a eukaryotic single-stranded DNA binding protein that is essential for DNA replication, repair, and recombination, and human RPA interacting protein α (hRIPα) is the nuclear transporter of RPA. Here, we report the regulatory role of hRIPα protein in cell proliferation. Western blot analysis revealed that the level of hRIPα was frequently elevated in cervical tumors tissues and hRIPα knockdown by siRNA inhibited cellular proliferation through deregulation of the cell cycle. In addition, overexpression of hRIPα resulted in increased clonogenicity. These results indicate that hRIPα is involved in cell proliferation through regulation of RPA transport. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  11. An apple B-box protein, MdCOL11, is involved in UV-B- and temperature-induced anthocyanin biosynthesis.

    Science.gov (United States)

    Bai, Songling; Saito, Takanori; Honda, Chikako; Hatsuyama, Yoshimichi; Ito, Akiko; Moriguchi, Takaya

    2014-11-01

    Our studies showed that an apple B-box protein, MdCOL11, the homolog of AtBBX22, is involved in UV-B- and temperature-induced anthocyanin biosynthesis in apple peel. Anthocyanin is responsible for the red pigmentation in apple peel and a R2R3 MYB gene, MdMYBA/1/10, a homolog of MdMYBA, controls its accumulation. Arabidopsis PAP1 is under the control of a series of upstream factors involved in light signal transduction and photomorphogenesis, such as ELONGATED HYPOCOTYL 5 (HY5) and B-box family (BBX) proteins. In this study, we identified and characterized the homolog of Arabidopsis BBX22 in apple, designated as MdCOL11. Overexpression of MdCOL11 in Arabidopsis enhanced the accumulation of anthocyanin. In apples, MdCOL11 was differentially expressed in all tissues, with the highest expression in petals and the lowest expression in the xylem. Transcripts of MdCOL11 noticeably accumulated at the ripening stage, concomitant with increases in the expressions of anthocyanin biosynthesis-related genes. In an in vitro treatment experiment, MdCOL11 was upregulated in an ultra-violet (UV)-B- and temperature-dependent manner, together with the inductions of anthocyanin biosynthesis-related genes and anthocyanin accumulation in apple peel. Furthermore, a dual-luciferase assay indicated that (1) MdCOL11 regulated the expression of MdMYBA and (2) MdCOL11 was a target of MdHY5. Taken together, our results suggest that MdCOL11 is involved in MdHY5-mediated signal transduction and regulates anthocyanin accumulation in apple peel, which sheds new light on anthocyanin accumulation in apples.

  12. Identification and characterisation of a hyper-variable apoplastic effector gene family of the potato cyst nematodes.

    Directory of Open Access Journals (Sweden)

    Sebastian Eves-van den Akker

    2014-09-01

    Full Text Available Sedentary endoparasitic nematodes are obligate biotrophs that modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Using assumptions about the characteristics of genes involved in plant-nematode biotrophic interactions to inform the identification strategy, we provide a description and characterisation of a novel group of hyper-variable extracellular effectors termed HYP, from the potato cyst nematode Globodera pallida. HYP effectors comprise a large gene family, with a modular structure, and have unparalleled diversity between individuals of the same population: no two nematodes tested had the same genetic complement of HYP effectors. Individuals vary in the number, size, and type of effector subfamilies. HYP effectors are expressed throughout the biotrophic stages in large secretory cells associated with the amphids of parasitic stage nematodes as confirmed by in situ hybridisation. The encoded proteins are secreted into the host roots where they are detectable by immunochemistry in the apoplasm, between the anterior end of the nematode and the feeding site. We have identified HYP effectors in three genera of plant parasitic nematodes capable of infecting a broad range of mono- and dicotyledon crop species. In planta RNAi targeted to all members of the effector family causes a reduction in successful parasitism.

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

    Science.gov (United States)

    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

  14. No evidence for involvement of plasma proteins or blood-borne cells in amyloid plaque formation in scrapie-affected mice. An immunohistoperoxidase study

    NARCIS (Netherlands)

    Eikelenboom, P.; Scott, J. R.; McBride, P. A.; Rozemuller, J. M.; Bruce, M. E.; Fraser, H.

    1987-01-01

    The present study was designed to investigate blood-brain permeability and the possible involvement of plasma proteins and blood-borne cells in amyloid plaque formation in scrapie-affected mice. No abnormal extravasation of intravenously injected horseradish peroxidase (HRP) was found and with

  15. Integral UBL domain proteins: a family of proteasome interacting proteins

    DEFF Research Database (Denmark)

    Hartmann-Petersen, Rasmus; Gordon, Colin

    2004-01-01

    The family of ubiquitin-like (UBL) domain proteins (UDPs) comprises a conserved group of proteins involved in a multitude of different cellular activities. However, recent studies on UBL-domain proteins indicate that these proteins appear to share a common property in their ability to interact...

  16. Identification of proteins involved in the functioning of Riftia pachyptila symbiosis by Subtractive Suppression Hybridization.

    Science.gov (United States)

    Sanchez, Sophie; Hourdez, Stéphane; Lallier, François H

    2007-09-24

    Since its discovery around deep sea hydrothermal vents of the Galapagos Rift about 30 years ago, the chemoautotrophic symbiosis between the vestimentiferan tubeworm Riftia pachyptila and its symbiotic sulfide-oxidizing gamma-proteobacteria has been extensively studied. However, studies on the tubeworm host were essentially targeted, biochemical approaches. We decided to use a global molecular approach to identify new proteins involved in metabolite exchanges and assimilation by the host. We used a Subtractive Suppression Hybridization approach (SSH) in an unusual way, by comparing pairs of tissues from a single individual. We chose to identify the sequences preferentially expressed in the branchial plume tissue (the only organ in contact with the sea water) and in the trophosome (the organ housing the symbiotic bacteria) using the body wall as a reference tissue because it is supposedly not involved in metabolite exchanges in this species. We produced four cDNA libraries: i) body wall-subtracted branchial plume library (BR-BW), ii) and its reverse library, branchial plume-subtracted body wall library (BW-BR), iii) body wall-subtracted trophosome library (TR-BW), iv) and its reverse library, trophosome-subtracted body wall library (BW-TR). For each library, we sequenced about 200 clones resulting in 45 different sequences on average in each library (58 and 59 cDNAs for BR-BW and TR-BW libraries respectively). Overall, half of the contigs matched records found in the databases with good E-values. After quantitative PCR analysis, it resulted that 16S, Major Vault Protein, carbonic anhydrase (RpCAbr), cathepsin and chitinase precursor transcripts were highly represented in the branchial plume tissue compared to the trophosome and the body wall tissues, whereas carbonic anhydrase (RpCAtr), myohemerythrin, a putative T-Cell receptor and one non identified transcript were highly specific of the trophosome tissue. Quantitative PCR analyses were congruent with our libraries

  17. Identification of proteins involved in the functioning of Riftia pachyptila symbiosis by Subtractive Suppression Hybridization

    Directory of Open Access Journals (Sweden)

    Lallier François H

    2007-09-01

    Full Text Available Abstract Background Since its discovery around deep sea hydrothermal vents of the Galapagos Rift about 30 years ago, the chemoautotrophic symbiosis between the vestimentiferan tubeworm Riftia pachyptila and its symbiotic sulfide-oxidizing γ-proteobacteria has been extensively studied. However, studies on the tubeworm host were essentially targeted, biochemical approaches. We decided to use a global molecular approach to identify new proteins involved in metabolite exchanges and assimilation by the host. We used a Subtractive Suppression Hybridization approach (SSH in an unusual way, by comparing pairs of tissues from a single individual. We chose to identify the sequences preferentially expressed in the branchial plume tissue (the only organ in contact with the sea water and in the trophosome (the organ housing the symbiotic bacteria using the body wall as a reference tissue because it is supposedly not involved in metabolite exchanges in this species. Results We produced four cDNA libraries: i body wall-subtracted branchial plume library (BR-BW, ii and its reverse library, branchial plume-subtracted body wall library (BW-BR, iii body wall-subtracted trophosome library (TR-BW, iv and its reverse library, trophosome-subtracted body wall library (BW-TR. For each library, we sequenced about 200 clones resulting in 45 different sequences on average in each library (58 and 59 cDNAs for BR-BW and TR-BW libraries respectively. Overall, half of the contigs matched records found in the databases with good E-values. After quantitative PCR analysis, it resulted that 16S, Major Vault Protein, carbonic anhydrase (RpCAbr, cathepsin and chitinase precursor transcripts were highly represented in the branchial plume tissue compared to the trophosome and the body wall tissues, whereas carbonic anhydrase (RpCAtr, myohemerythrin, a putative T-Cell receptor and one non identified transcript were highly specific of the trophosome tissue. Conclusion Quantitative PCR

  18. ALDH16A1 is a novel non-catalytic enzyme that may be involved in the etiology of gout via protein-protein interactions with HPRT1.

    Science.gov (United States)

    Vasiliou, Vasilis; Sandoval, Monica; Backos, Donald S; Jackson, Brian C; Chen, Ying; Reigan, Philip; Lanaspa, Miguel A; Johnson, Richard J; Koppaka, Vindhya; Thompson, David C

    2013-02-25

    Gout, a common form of inflammatory arthritis, is strongly associated with elevated uric acid concentrations in the blood (hyperuricemia). A recent study in Icelanders identified a rare missense single nucleotide polymorphism (SNP) in the ALDH16A1 gene, ALDH16A1*2, to be associated with gout and serum uric acid levels. ALDH16A1 is a novel and rather unique member of the ALDH superfamily in relation to its gene and protein structures. ALDH16 genes are present in fish, amphibians, protista, bacteria but absent from archaea, fungi and plants. In most mammalian species, two ALDH16A1 spliced variants (ALDH16A1, long form and ALDH16A1_v2, short form) have been identified and both are expressed in HepG-2, HK-2 and HK-293 human cell lines. The ALDH16 proteins contain two ALDH domains (as opposed to one in the other members of the superfamily), four transmembrane and one coiled-coil domains. The active site of ALDH16 proteins from bacterial, frog and lower animals contain the catalytically important cysteine residue (Cys-302); this residue is absent from the mammalian and fish orthologs. Molecular modeling predicts that both the short and long forms of human ALDH16A1 protein would lack catalytic activity but may interact with the hypoxanthine-guanine phosphoribosyltransferase (HPRT1) protein, a key enzyme involved in uric acid metabolism and gout. Interestingly, such protein-protein interactions with HPRT1 are predicted to be impaired for the long or short forms of ALDH16A1*2. These results lead to the intriguing possibility that association between ALDH16A1 and HPRT1 may be required for optimal HPRT activity with disruption of this interaction possibly contributing to the hyperuricemia seen in ALDH16A1*2 carriers. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. Alignment of non-covalent interactions at protein-protein interfaces.

    Directory of Open Access Journals (Sweden)

    Hongbo Zhu

    Full Text Available BACKGROUND: The study and comparison of protein-protein interfaces is essential for the understanding of the mechanisms of interaction between proteins. While there are many methods for comparing protein structures and protein binding sites, so far no methods have been reported for comparing the geometry of non-covalent interactions occurring at protein-protein interfaces. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a method for aligning non-covalent interactions between different protein-protein interfaces. The method aligns the vector representations of van der Waals interactions and hydrogen bonds based on their geometry. The method has been applied to a dataset which comprises a variety of protein-protein interfaces. The alignments are consistent to a large extent with the results obtained using two other complementary approaches. In addition, we apply the method to three examples of protein mimicry. The method successfully aligns respective interfaces and allows for recognizing conserved interface regions. CONCLUSIONS/SIGNIFICANCE: The Galinter method has been validated in the comparison of interfaces in which homologous subunits are involved, including cases of mimicry. The method is also applicable to comparing interfaces involving non-peptidic compounds. Galinter assists users in identifying local interface regions with similar patterns of non-covalent interactions. This is particularly relevant to the investigation of the molecular basis of interaction mimicry.

  20. Synthesis of Lipidated Proteins.

    Science.gov (United States)

    Mejuch, Tom; Waldmann, Herbert

    2016-08-17

    Protein lipidation is one of the major post-translational modifications (PTM) of proteins. The attachment of the lipid moiety frequently determines the localization and the function of the lipoproteins. Lipidated proteins participate in many essential biological processes in eukaryotic cells, including vesicular trafficking, signal transduction, and regulation of the immune response. Malfunction of these cellular processes usually leads to various diseases such as cancer. Understanding the mechanism of cellular signaling and identifying the protein-protein and protein-lipid interactions in which the lipoproteins are involved is a crucial task. To achieve these goals, fully functional lipidated proteins are required. However, access to lipoproteins by means of standard expression is often rather limited. Therefore, semisynthetic methods, involving the synthesis of lipidated peptides and their subsequent chemoselective ligation to yield full-length lipoproteins, were developed. In this Review we summarize the commonly used methods for lipoprotein synthesis and the development of the corresponding chemoselective ligation techniques. Several key studies involving full-length semisynthetic lipidated Ras, Rheb, and LC3 proteins are presented.

  1. Evolutionary history of callose synthases in terrestrial plants with emphasis on proteins involved in male gametophyte development.

    Directory of Open Access Journals (Sweden)

    Lenka Záveská Drábková

    Full Text Available Callose is a plant-specific polysaccharide (β-1,3-glucan playing an important role in angiosperms in many developmental processes and responses to biotic and abiotic stresses. Callose is synthesised at the plasma membrane of plant cells by callose synthase (CalS and, among others, represents the main polysaccharide in the callose wall surrounding the tetrads of developing microspores and in the growing pollen tube wall. CalS proteins involvement in spore development is a plesiomorphic feature of terrestrial plants, but very little is known about their evolutionary origin and relationships amongst the members of this protein family. We performed thorough comparative analyses of callose synthase family proteins from major plant lineages to determine their evolutionary history across the plant kingdom. A total of 1211 candidate CalS sequences were identified and compared amongst diverse taxonomic groups of plants, from bryophytes to angiosperms. Phylogenetic analyses identified six main clades of CalS proteins and suggested duplications during the evolution of specialised functions. Twelve family members had previously been identified in Arabidopsis thaliana. We focused on five CalS subfamilies directly linked to pollen function and found that proteins expressed in pollen evolved twice. CalS9/10 and CalS11/12 formed well-defined clades, whereas pollen-specific CalS5 was found within subfamilies that mostly did not express in mature pollen vegetative cell, although were found in sperm cells. Expression of five out of seven mature pollen-expressed CalS genes was affected by mutations in bzip transcription factors. Only three subfamilies, CalS5, CalS10, and CalS11, however, formed monophyletic, mostly conserved clades. The pairs CalS9/CalS10, CalS11/CalS12 and CalS3 may have diverged after angiosperms diversified from lycophytes and bryophytes. Our analysis of fully sequenced plant proteins identified new evolutionary lineages of callose synthase

  2. In vivo inhibition of cysteine proteases provides evidence for the involvement of 'senescence-associated vacuoles' in chloroplast protein degradation during dark-induced senescence of tobacco leaves.

    Science.gov (United States)

    Carrión, Cristian A; Costa, María Lorenza; Martínez, Dana E; Mohr, Christina; Humbeck, Klaus; Guiamet, Juan J

    2013-11-01

    Breakdown of leaf proteins, particularly chloroplast proteins, is a massive process in senescing leaves. In spite of its importance in internal N recycling, the mechanism(s) and the enzymes involved are largely unknown. Senescence-associated vacuoles (SAVs) are small, acidic vacuoles with high cysteine peptidase activity. Chloroplast-targeted proteins re-localize to SAVs during senescence, suggesting that SAVs might be involved in chloroplast protein degradation. SAVs were undetectable in mature, non-senescent tobacco leaves. Their abundance, visualized either with the acidotropic marker Lysotracker Red or by green fluorescent protein (GFP) fluorescence in a line expressing the senescence-associated cysteine protease SAG12 fused to GFP, increased during senescence induction in darkness, and peaked after 2-4 d, when chloroplast dismantling was most intense. Increased abundance of SAVs correlated with higher levels of SAG12 mRNA. Activity labelling with a biotinylated derivative of the cysteine protease inhibitor E-64 was used to detect active cysteine proteases. The two apparently most abundant cysteine proteases of senescing leaves, of 40kDa and 33kDa were detected in isolated SAVs. Rubisco degradation in isolated SAVs was completely blocked by E-64. Treatment of leaf disks with E-64 in vivo substantially reduced degradation of Rubisco and leaf proteins. Overall, these results indicate that SAVs contain most of the cysteine protease activity of senescing cells, and that SAV cysteine proteases are at least partly responsible for the degradation of stromal proteins of the chloroplast.

  3. Increased Uptake of Chelated Copper Ions by Lolium perenne Attributed to Amplified Membrane and Endodermal Damage

    Directory of Open Access Journals (Sweden)

    Anthea Johnson

    2015-10-01

    Full Text Available The contributions of mechanisms by which chelators influence metal translocation to plant shoot tissues are analyzed using a combination of numerical modelling and physical experiments. The model distinguishes between apoplastic and symplastic pathways of water and solute movement. It also includes the barrier effects of the endodermis and plasma membrane. Simulations are used to assess transport pathways for free and chelated metals, identifying mechanisms involved in chelate-enhanced phytoextraction. Hypothesized transport mechanisms and parameters specific to amendment treatments are estimated, with simulated results compared to experimental data. Parameter values for each amendment treatment are estimated based on literature and experimental values, and used for model calibration and simulation of amendment influences on solute transport pathways and mechanisms. Modeling indicates that chelation alters the pathways for Cu transport. For free ions, Cu transport to leaf tissue can be described using purely apoplastic or transcellular pathways. For strong chelators (ethylenediaminetetraacetic acid (EDTA and diethylenetriaminepentaacetic acid (DTPA, transport by the purely apoplastic pathway is insufficient to represent measured Cu transport to leaf tissue. Consistent with experimental observations, increased membrane permeability is required for simulating translocation in EDTA and DTPA treatments. Increasing the membrane permeability is key to enhancing phytoextraction efficiency.

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

    Science.gov (United States)

    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. Minor Coat and Heat Shock Proteins Are Involved in the Binding of Citrus Tristeza Virus to the Foregut of Its Aphid Vector, Toxoptera citricida.

    Science.gov (United States)

    Killiny, N; Harper, S J; Alfaress, S; El Mohtar, C; Dawson, W O

    2016-11-01

    Vector transmission is a critical stage in the viral life cycle, yet for most plant viruses how they interact with their vector is unknown or is explained by analogy with previously described relatives. Here we examined the mechanism underlying the transmission of citrus tristeza virus (CTV) by its aphid vector, Toxoptera citricida, with the objective of identifying what virus-encoded proteins it uses to interact with the vector. Using fluorescently labeled virions, we demonstrated that CTV binds specifically to the lining of the cibarium of the aphid. Through in vitro competitive binding assays between fluorescent virions and free viral proteins, we determined that the minor coat protein is involved in vector interaction. We also found that the presence of two heat shock-like proteins, p61 and p65, reduces virion binding in vitro Additionally, treating the dissected mouthparts with proteases did not affect the binding of CTV virions. In contrast, chitinase treatment reduced CTV binding to the foregut. Finally, competition with glucose, N-acetyl-β-d-glucosamine, chitobiose, and chitotriose reduced the binding. These findings together suggest that CTV binds to the sugar moieties of the cuticular surface of the aphid cibarium, and the binding involves the concerted activity of three virus-encoded proteins. Limited information is known about the specific interactions between citrus tristeza virus and its aphid vectors. These interactions are important for the process of successful transmission. In this study, we localized the CTV retention site as the cibarium of the aphid foregut. Moreover, we demonstrated that the nature of these interactions is protein-carbohydrate binding. The viral proteins, including the minor coat protein and two heat shock proteins, bind to sugar moieties on the surface of the foregut. These findings will help in understanding the transmission mechanism of CTV by the aphid vector and may help in developing control strategies which interfere

  6. Involvement of TRPV3 and TRPM8 ion channel proteins in induction of mammalian cold-inducible proteins.

    Science.gov (United States)

    Fujita, Takanori; Liu, Yu; Higashitsuji, Hiroaki; Itoh, Katsuhiko; Shibasaki, Koji; Fujita, Jun; Nishiyama, Hiroyuki

    2018-01-01

    Cold-inducible RNA-binding protein (CIRP), RNA-binding motif protein 3 (RBM3) and serine and arginine rich splicing factor 5 (SRSF5) are RNA-binding proteins that are transcriptionally upregulated in response to moderately low temperatures and a variety of cellular stresses in mammalian cells. Induction of these cold-inducible proteins (CIPs) is dependent on transient receptor potential (TRP) V4 channel protein, but seems independent of its ion channel activity. We herein report that in addition to TRPV4, TRPV3 and TRPM8 are necessary for the induction of CIPs. We established cell lines from the lung of TRPV4-knockout (KO) mouse, and observed induction of CIPs in them by western blot analysis. A TRPV4 antagonist RN1734 suppressed the induction in wild-type mouse cells, but not in TRPV4-KO cells. A TRPV3 channel blocker S408271 and a TRPM8 channel blocker AMTB as well as siRNAs against TRPV3 and TRPM8 suppressed the CIP induction in mouse TRPV4-KO cells and human U-2 OS cells. A TRPV3 channel agonist 2-APB induced CIP expression, but camphor did not. Neither did a TRPM8 channel agonist WS-12. These results suggest that TRPV4, TRPV3 and TRPM8 proteins, but not their ion channel activities are necessary for the induction of CIPs at 32 °C. Identification of proteins that differentially interact with these TRP channels at 37 °C and 32 °C would help elucidate the underlying mechanisms of CIP induction by hypothermia. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Involvement of Mζ-Like Protein Kinase in the Mechanisms of Conditioned Food Aversion Memory Reconsolidation in the Helix lucorum.

    Science.gov (United States)

    Solntseva, S V; Kozyrev, S A; Nikitin, V P

    2015-06-01

    We studied the involvement of Mζ-like protein kinase (PKMζ) into mechanisms of conditioned food aversion memory reconsolidation in Helix lucorum. Injections PKMζ inhibitor ZIP in a dose of 5 mg/kg on day 2 or 10 after learning led to memory impairment and amnesia development. Injections of the inhibitor in doses of 1.5 or 2.5 mg/kg had no effect. Repeated training on day 11 after induction of amnesia resulted in the formation of memory on the same type of food aversion similar to first training. The number of combinations of conditional (food) and reinforcing (electrical shock) stimuli was similar during initial and repeated training. We hypothesize that the inhibition of Mζ-like protein kinase erases the memory trace and a new memory is formed during repeated training.

  8. Changes over lactation in breast milk serum proteins involved in the maturation of immune and digestive system of the infant.

    Science.gov (United States)

    Zhang, Lina; de Waard, Marita; Verheijen, Hester; Boeren, Sjef; Hageman, Jos A; van Hooijdonk, Toon; Vervoort, Jacques; van Goudoever, Johannes B; Hettinga, Kasper

    2016-09-16

    To objective of this study was to better understand the biological functions of breast milk proteins in relation to the growth and development of infants over the first six months of life. Breast milk samples from four individual women collected at seven time points in the first six months after delivery were analyzed by filter aided sample preparation and dimethyl labeling combined with liquid chromatography tandem mass spectrometry. A total of 247 and 200 milk serum proteins were identified and quantified, respectively. The milk serum proteome showed a high similarity (80% overlap) on the qualitative level between women and over lactation. The quantitative changes in milk serum proteins were mainly caused by three groups of proteins, enzymes, and transport and immunity proteins. Of these 21 significantly changed proteins, 30% were transport proteins, such as serum albumin and fatty acid binding protein, which are both involved in transporting nutrients to the infant. The decrease of the enzyme bile salt-activated lipase as well as the immunity proteins immunoglobulins and lactoferrin coincide with the gradual maturation of the digestive and immune system of infants. The human milk serum proteome didn't differ qualitatively but it did quantitatively, both between mothers and as lactation advanced. The changes of the breast milk serum proteome over lactation corresponded with the development of the digestive and immune system of infants. Breast milk proteins provide nutrition, but also contribute to healthy development of infants. Despite the previously reported large number of identified breast milk proteins and their changes over lactation, less is known on the changes of these proteins in individual mothers. This study is the first to determine the qualitative and quantitative changes of milk proteome over lactation between individual mothers. The results indicate that the differences in the milk proteome between individual mothers are more related to the

  9. A cathepsin F-like peptidase involved in barley grain protein mobilization, HvPap-1, is modulated by its own propeptide and by cystatins

    Science.gov (United States)

    Diaz, Isabel

    2012-01-01

    Among the C1A cysteine proteases, the plant cathepsin F-like group has been poorly studied. This paper describes the molecular and functional characterization of the HvPap-1 cathepsin F-like protein from barley. This peptidase is N-glycosylated and has to be processed to become active by its own propeptide being an important modulator of the peptidase activity. The expression pattern of its mRNA and protein suggest that it is involved in different proteolytic processes in the barley plant. HvPap-1 peptidase has been purified in Escherichia coli and the recombinant protein is able to degrade different substrates, including barley grain proteins (hordeins, albumins, and globulins) stored in the barley endosperm. It has been localized in protein bodies and vesicles of the embryo and it is induced in aleurones by gibberellin treatment. These three features support the implication of HvPap-1 in storage protein mobilization during grain germination. In addition, a complex regulation exerted by the barley cystatins, which are cysteine protease inhibitors, and by its own propeptide, is also described PMID:22791822

  10. The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant-pathogen interactions

    Directory of Open Access Journals (Sweden)

    Mansoor eKarimi Jashni

    2015-08-01

    Full Text Available Upon host penetration, fungal pathogens secrete a plethora of effectors to promote disease, including proteases that degrade plant antimicrobial proteins, and protease inhibitors (PIs that inhibit plant proteases with antimicrobial activity. Conversely, plants secrete proteases and PIs to protect themselves against pathogens or to mediate recognition of pathogen proteases and PIs, which leads to induction of defense responses. Many examples of proteases and PIs mediating effector-triggered immunity in host plants have been reported in the literature, but little is known about their role in compromising basal defense responses induced by microbe-associated molecular patterns. Recently, several reports appeared in literature on secreted fungal proteases that modify or degrade pathogenesis-related proteins, including plant chitinases or PIs that compromise their activities. This prompted us to review the recent advances on proteases and PIs involved in fungal virulence and plant defense. Proteases and PIs from plants and their fungal pathogens play an important role in the arms race between plants and pathogens, which has resulted in co-evolutionary diversification and adaptation shaping pathogen lifestyles.

  11. Proteomic analysis of stipe explants reveals differentially expressed proteins involved in early direct somatic embryogenesis of the tree fern Cyathea delgadii Sternb.

    Science.gov (United States)

    Domżalska, Lucyna; Kędracka-Krok, Sylwia; Jankowska, Urszula; Grzyb, Małgorzata; Sobczak, Mirosław; Rybczyński, Jan J; Mikuła, Anna

    2017-05-01

    Using cyto-morphological analysis of somatic embryogenesis (SE) in the tree fern Cyathea delgadii as a guide, we performed a comparative proteomic analysis in stipe explants undergoing direct SE. Plant material was cultured on hormone-free medium supplemented with 2% sucrose. Phenol extracted proteins were separated using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed for protein identification. A total number of 114 differentially regulated proteins was identified during early SE, i.e. when the first cell divisions started and several-cell pro-embryos were formed. Proteins were assigned to seven functional categories: carbohydrate metabolism, protein metabolism, cell organization, defense and stress responses, amino acid metabolism, purine metabolism, and fatty acid metabolism. Carbohydrate and protein metabolism were found to be the most sensitive SE functions with the greatest number of alterations in the intensity of spots in gel. Differences, especially in non-enzymatic and structural protein abundance, are indicative for cell organization, including cytoskeleton rearrangement and changes in cell wall components. The highest induced changes concern those enzymes related to fatty acid metabolism. Global analysis of the proteome reveals several proteins that can represent markers for the first 16days of SE induction and expression in fern. The findings of this research improve the understanding of molecular processes involved in direct SE in C. delgadii. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: Involvement of a membrane receptor and a cytosolic protein

    International Nuclear Information System (INIS)

    Price, E.M.; Ratnam, M.; Rodeman, K.M.; Freisheim, J.H.

    1988-01-01

    A radioiodinated photoaffinity analogue of methotrexate, N α -(4-amino-4-deoxy-10-methyl-pteroyl)-N ε -(4-azidosalicylyl)-L-lysine (APA-ASA-Lys), was recently used to identify the plasma membrane derived binding protein involved in the transport of this folate antagonist into murine L1210 cells. The labeled protein has an apparent molecular weight of 46K-48K when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no such labeling occurs in a methotrexate transport-defective cell line (L1210/R81). Labeling of the total cytosolic protein from disrupted cells, followed by electrophoresis and autoradiography, showed, among other proteins, a 21K band, corresponding to dihydrofolate reductase (DHFR), in both the parent and R81 cells and a 38K band only in the parent cells. However, when whole cells were UV irradiated at various times at 37 degree C following addition of radiolabeled APA-ASA-Lys, the 38K protein and DHFR were the only cytosolic proteins labeled in the parent cells, while the intact R81 cells showed no labeled cytosolic protein, since the photoprobe is not transported. Further, when the parent cells were treated with a pulse of radiolabeled photoprobe, followed by UV irradiation at different times at 37 degree C, the probe appeared sequentially on the 48K membrane protein and both the 38K cytosolic protein and dihydrofolate reductase. A 48K protein could be detected in both parent L1210 cells and the R81 cells on Western blots using antisera to a membrane folate binding protein from human placenta. These results suggest a vectorial transport of APA-ASA-Lys or methotrexate and reduced folate coenzymes into murine L1210 cells mediated by a 48K integral membrane protein and a 38K cytosolic or peripheral membrane protein. The 38K protein may help in the trafficking of reduced folate coenzymes, shuttling them to various cytosolic targets

  13. p38 mitogen-activated protein kinase is involved in arginase-II-mediated eNOS-uncoupling in obesity.

    Science.gov (United States)

    Yu, Yi; Rajapakse, Angana G; Montani, Jean-Pierre; Yang, Zhihong; Ming, Xiu-Fen

    2014-07-18

    Endothelial nitric oxide synthase (eNOS)-uncoupling links obesity-associated insulin resistance and type-II diabetes to the increased incidence of cardiovascular disease. Studies have indicated that increased arginase is involved in eNOS-uncoupling through competing with the substrate L-arginine. Given that arginase-II (Arg-II) exerts some of its biological functions through crosstalk with signal transduction pathways, and that p38 mitogen-activated protein kinase (p38mapk) is involved in eNOS-uncoupling, we investigated here whether p38mapk is involved in Arg-II-mediated eNOS-uncoupling in a high fat diet (HFD)-induced obesity mouse model. Obesity was induced in wild type (WT) and Arg-II-deficient (Arg-II(-/-)) mice on C57BL/6 J background by high-fat diet (HFD, 55% fat) for 14 weeks starting from age of 7 weeks. The entire aortas were isolated and subjected to 1) immunoblotting analysis of the protein level of eNOS, Arg-II and p38mapk activation; 2) arginase activity assay; 3) endothelium-dependent and independent vasomotor responses; 4) en face staining of superoxide anion and NO production with Dihydroethidium and 4,5-Diaminofluorescein Diacetate, respectively, to assess eNOS-uncoupling. To evaluate the role of p38mapk, isolated aortas were treated with p38mapk inhibitor SB203580 (10 μmol/L, 1 h) prior to the analysis. In addition, the role of p38mapk in Arg-II-induced eNOS-uncoupling was investigated in cultured human endothelial cells overexpressing Arg-II in the absence or presence of shRNA against p38mapk. HFD enhanced Arg-II expression/activity and p38mapk activity, which was associated with eNOS-uncoupling as revealed by decreased NO and enhanced L-NAME-inhibitable superoxide in aortas of WT obese mice. In accordance, WT obese mice revealed decreased endothelium-dependent relaxations to acetylcholine despite of higher eNOS protein level, whereas Arg-II(-/-) obese mice were protected from HFD-induced eNOS-uncoupling and endothelial dysfunction, which

  14. The knottin-like Blufensin family regulates genes involved in nuclear import and the secretory pathway in barley-powdery mildew interactions

    Science.gov (United States)

    Xu, Weihui; Meng, Yan; Surana, Priyanka; Fuerst, Greg; Nettleton, Dan; Wise, Roger P.

    2015-01-01

    Plants have evolved complex regulatory mechanisms to control a multi-layered defense response to microbial attack. Both temporal and spatial gene expression are tightly regulated in response to pathogen ingress, modulating both positive and negative control of defense. BLUFENSINs, small knottin-like peptides in barley, wheat, and rice, are highly induced by attack from fungal pathogens, in particular, the obligate biotrophic fungus, Blumeria graminis f. sp. hordei (Bgh), causal agent of barley powdery mildew. Previous research indicated that Blufensin1 (Bln1) functions as a negative regulator of basal defense mechanisms. In the current report, we show that BLN1 and BLN2 can both be secreted to the apoplast and Barley stripe mosaic virus (BSMV)-mediated overexpression of Bln2 increases susceptibility of barley to Bgh. Bimolecular fluorescence complementation (BiFC) assays signify that BLN1 and BLN2 can interact with each other, and with calmodulin. We then used BSMV-induced gene silencing to knock down Bln1, followed by Barley1 GeneChip transcriptome analysis, to identify additional host genes influenced by Bln1. Analysis of differential expression revealed a gene set enriched for those encoding proteins annotated to nuclear import and the secretory pathway, particularly Importin α1-b and Sec61 γ subunits. Further functional analysis of these two affected genes showed that when silenced, they also reduced susceptibility to Bgh. Taken together, we postulate that Bln1 is co-opted by Bgh to facilitate transport of disease-related host proteins or effectors, influencing the establishment of Bgh compatibility on its barley host. PMID:26089830

  15. Yeast Interacting Proteins Database: YNL216W, YLR453C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YNL216W RAP1 DNA-binding protein involved in either activation or repression of transcription, depending...NA-binding protein involved in either activation or repression of transcription, depending on binding site c

  16. Proteins involved in difference of sorbitol fermentation rates of the toxigenic and nontoxigenic Vibrio cholerae El Tor strains revealed by comparative proteome analysis

    Science.gov (United States)

    2009-01-01

    Background The nontoxigenic V. cholerae El Tor strains ferment sorbitol faster than the toxigenic strains, hence fast-fermenting and slow-fermenting strains are defined by sorbitol fermentation test. This test has been used for more than 40 years in cholera surveillance and strain analysis in China. Understanding of the mechanisms of sorbitol metabolism of the toxigenic and nontoxigenic strains may help to explore the genome and metabolism divergence in these strains. Here we used comparative proteomic analysis to find the proteins which may be involved in such metabolic difference. Results We found the production of formate and lactic acid in the sorbitol fermentation medium of the nontoxigenic strain was earlier than of the toxigenic strain. We compared the protein expression profiles of the toxigenic strain N16961 and nontoxigenic strain JS32 cultured in sorbitol fermentation medium, by using fructose fermentation medium as the control. Seventy-three differential protein spots were found and further identified by MALDI-MS. The difference of product of fructose-specific IIA/FPR component gene and mannitol-1-P dehydrogenase, may be involved in the difference of sorbitol transportation and dehydrogenation in the sorbitol fast- and slow-fermenting strains. The difference of the relative transcription levels of pyruvate formate-lyase to pyruvate dehydrogenase between the toxigenic and nontoxigenic strains may be also responsible for the time and ability difference of formate production between these strains. Conclusion Multiple factors involved in different metabolism steps may affect the sorbitol fermentation in the toxigenic and nontoxigenic strains of V. cholerae El Tor. PMID:19589152

  17. A CheR/CheB fusion protein is involved in cyst cell development and chemotaxis in Azospirillum brasilense Sp7.

    Science.gov (United States)

    Wu, Lixian; Cui, Yanhua; Hong, Yuanyuan; Chen, Sanfeng

    2011-12-20

    We here report the sequence and functional analysis of cstB of Azospirillum brasilense Sp7. The predicted cstB contains C-terminal two PAS domains and N-terminal part which has similarity with CheB-CheR fusion protein. cstB mutants had reduced swarming ability compared to that of A. brasilense wild-type strain, implying that cstB was involved in chemotaxis in A. brasilense. A microscopic analysis revealed that cstB mutants developed mature cyst cells more quickly than wild type, indicating that cstB is involved in cyst formation. cstB mutants were affected in colony morphology and the production of exopolysaccharides (EPS) which are essential for A. brasilense cells to differentiate into cyst-like forms. These observations suggested that cstB was a multi-effector involved in cyst development and chemotaxis in A. brasilense. Copyright © 2010 Elsevier GmbH. All rights reserved.

  18. Many rivers to cross: the journey of zinc from soil to seed

    Directory of Open Access Journals (Sweden)

    Lene Irene Olsen

    2014-02-01

    Full Text Available An important goal of micronutrient biofortification is to enhance the amount of bioavailable zinc in the edible seed of cereals and more specifically in the endosperm. The picture is starting to emerge for how zinc is translocated from the soil through the mother plant to the developing seed. On this journey, zinc is transported from symplast to symplast via multiple apoplastic spaces. During each step, zinc is imported into a symplast before it is exported again. Cellular import and export of zinc requires passage through biological membranes, which makes membrane-bound transporters of zinc especially interesting as potential transport bottlenecks. Inside the cell, zinc can be imported into or exported out of organelles by other transporters. The function of several membrane proteins involved in the transport of zinc across the tonoplast, chloroplast or plasma membranes are currently known. These include members of the ZIP (ZRT-IRT-like Protein, and MTP (Metal Tolerance Protein and Heavy Metal ATPase (HMA families. An important player in the transport process is the ligand nicotianamine that binds zinc to increase its solubility in living cells and in this way buffers the intracellular zinc concentration.

  19. Enhanced heterologous expression of biologically active human granulocyte colony stimulating factor in transgenic tobacco BY-2 cells by localization to endoplasmic reticulum.

    Science.gov (United States)

    Nair, Nisha R; Chidambareswaren, M; Manjula, S

    2014-09-01

    Tobacco Bright Yellow-2 (BY-2) cells, one of the best characterized cell lines is an attractive expression system for heterologous protein expression. However, the expression of foreign proteins is currently hampered by their low yield, which is partially the result of proteolytic degradation. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine. Recombinant hG-CSF is successfully being used for the treatment of chemotherapy-induced neutropenia in cancer patients. Here, we describe a simple strategy for producing biologically active hG-CSF in tobacco BY-2 cells, localized in the apoplast of BY-2 cells, as well as targeted to the endoplasmic reticulum (ER). ER targeting significantly enhanced recombinant production which scaled to 17.89 mg/l from 4.19 mg/l when expressed in the apoplasts. Southern blotting confirmed the stable integration of hG-CSF in the BY-2 nuclear genome, and the expression of hG-CSF was analysed by Western blotting. Total soluble protein containing hG-CSF isolated from positive calli showed proliferative potential when tested on HL-60 cell lines by MTT assay. We also report the potential of a Fluorescence-activated cell sorting approach for an efficient sorting of the hG-CSF-expressing cell lines, which will enable the generation of homogenous high-producing cell lines.

  20. The Involvement of Thaumatin-Like Proteins in Plant Food Cross-Reactivity: A Multicenter Study Using a Specific Protein Microarray

    Science.gov (United States)

    Palacín, Arantxa; Rivas, Luis A.; Gómez-Casado, Cristina; Aguirre, Jacobo; Tordesillas, Leticia; Bartra, Joan; Blanco, Carlos; Carrillo, Teresa; Cuesta-Herranz, Javier; Bonny, José A. Cumplido; Flores, Enrique; García-Alvarez-Eire, Mar G.; García-Nuñez, Ignacio; Fernández, Francisco J.; Gamboa, Pedro; Muñoz, Rosa; Sánchez-Monge, Rosa; Torres, Maria; Losada, Susana Varela; Villalba, Mayte; Vega, Francisco; Parro, Victor; Blanca, Miguel; Salcedo, Gabriel; Díaz-Perales, Araceli

    2012-01-01

    Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited >50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy. PMID:22970164

  1. The involvement of thaumatin-like proteins in plant food cross-reactivity: a multicenter study using a specific protein microarray.

    Directory of Open Access Journals (Sweden)

    Arantxa Palacín

    Full Text Available Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG and another against pollens but tolerant to food-plant allergens (PAG, were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%, chestnut TLP (24% and plane pollen TLP (22% proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited >50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy.

  2. A tandem sequence motif acts as a distance-dependent enhancer in a set of genes involved in translation by binding the proteins NonO and SFPQ

    Directory of Open Access Journals (Sweden)

    Roepcke Stefan

    2011-12-01

    Full Text Available Abstract Background Bioinformatic analyses of expression control sequences in promoters of co-expressed or functionally related genes enable the discovery of common regulatory sequence motifs that might be involved in co-ordinated gene expression. By studying promoter sequences of the human ribosomal protein genes we recently identified a novel highly specific Localized Tandem Sequence Motif (LTSM. In this work we sought to identify additional genes and LTSM-binding proteins to elucidate potential regulatory mechanisms. Results Genome-wide analyses allowed finding a considerable number of additional LTSM-positive genes, the products of which are involved in translation, among them, translation initiation and elongation factors, and 5S rRNA. Electromobility shift assays then showed specific signals demonstrating the binding of protein complexes to LTSM in ribosomal protein gene promoters. Pull-down assays with LTSM-containing oligonucleotides and subsequent mass spectrometric analysis identified the related multifunctional nucleotide binding proteins NonO and SFPQ in the binding complex. Functional characterization then revealed that LTSM enhances the transcriptional activity of the promoters in dependency of the distance from the transcription start site. Conclusions Our data demonstrate the power of bioinformatic analyses for the identification of biologically relevant sequence motifs. LTSM and the here found LTSM-binding proteins NonO and SFPQ were discovered through a synergistic combination of bioinformatic and biochemical methods and are regulators of the expression of a set of genes of the translational apparatus in a distance-dependent manner.

  3. Interactions involved in pH protection of the alphavirus fusion protein

    Energy Technology Data Exchange (ETDEWEB)

    Fields, Whitney; Kielian, Margaret, E-mail: margaret.kielian@einstein.yu.edu

    2015-12-15

    The alphavirus membrane protein E1 mediates low pH-triggered fusion of the viral and endosome membranes during virus entry. During virus biogenesis E1 associates as a heterodimer with the transmembrane protein p62. Late in the secretory pathway, cellular furin cleaves p62 to the mature E2 protein and a peripheral protein E3. E3 remains bound to E2 at low pH, stabilizing the heterodimer and thus protecting E1 from the acidic pH of the secretory pathway. Release of E3 at neutral pH then primes the virus for fusion during entry. Here we used site-directed mutagenesis and revertant analysis to define residues important for the interactions at the E3–E2 interface. Our data identified a key residue, E2 W235, which was required for E1 pH protection and alphavirus production. Our data also suggest additional residues on E3 and E2 that affect their interacting surfaces and thus influence the pH protection of E1 during alphavirus exit.

  4. Quantification of the main digestive processes in ruminants: the equations involved in the renewed energy and protein feed evaluation systems.

    Science.gov (United States)

    Sauvant, D; Nozière, P

    2016-05-01

    The evolution of feeding systems for ruminants towards evaluation of diets in terms of multiple responses requires the updating of the calculation of nutrient supply to the animals to make it more accurate on aggregated units (feed unit, or UF, for energy and protein digestible in the intestine, or PDI, for metabolizable protein) and to allow prediction of absorbed nutrients. The present update of the French system is based on the building and interpretation through meta-analysis of large databases on digestion and nutrition of ruminants. Equations involved in the calculation of UF and PDI have been updated, allowing: (1) prediction of the out flow rate of particles and liquid depending on the level of intake and the proportion of concentrate, and the use of this in the calculation of ruminal digestion of protein and starch from in situ data; (2) the system to take into account the effects of the main factors of digestive interactions (level of intake, proportion of concentrate, rumen protein balance) on organic matter digestibility, energy losses in methane and in urine; (3) more accurate calculation of the energy available in the rumen and the efficiency of its use for the microbial protein synthesis. In this renewed model UF and PDI values of feedstuffs vary depending on diet composition, and intake level. Consequently, standard feed table values can be considered as being only indicative. It is thus possible to predict the nutrient supply on a wider range of diets more accurately and in particular to better integrate energy×protein interactions occurring in the gut.

  5. Arabidopsis IQM4, a Novel Calmodulin-Binding Protein, Is Involved With Seed Dormancy and Germination in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Yu Ping Zhou

    2018-06-01

    Full Text Available Seed dormancy and germination are regulated by complex mechanisms controlled by diverse hormones and environmental cues. Abscisic acid (ABA promotes seed dormancy and inhibits seed germination and post-germination growth. Calmodulin (CaM signals are involved with the inhibition of ABA during seed germination and seedling growth. In this study, we showed that Arabidopsis thaliana IQM4 could bind with calmodulin 5 (CaM5 both in vitro and in vivo, and that the interaction was the Ca2+-independent type. The IQM4 protein was localized in the chloroplast and the IQM4 gene was expressed in most tissues, especially the embryo and germinated seedlings. The T-DNA insertion mutants of IQM4 exhibited the reduced primary seed dormancy and lower ABA levels compared with wild type seeds. Moreover, IQM4 plays key roles in modulating the responses to ABA, salt, and osmotic stress during seed germination and post-germination growth. T-DNA insertion mutants exhibited ABA-insensitive and salt-hypersensitive phenotypes during seed germination and post-germination growth, whereas IQM4-overexpressing lines had ABA- and osmotic-hypersensitive, and salt-insensitive phenotypes. Gene expression analyses showed that mutation of IQM4 inhibited the expression of ABA biosynthetic genes NCED6 and NCED9, and seed maturation regulators LEC1, LEC2, ABI3, and ABI5 during the silique development, as well as promoted the expression of WRKY40 and inhibited that of ABI5 in ABA-regulated seed germination. These observations suggest that IQM4 is a novel Ca2+-independent CaM-binding protein, which is positively involved with seed dormancy and germination in Arabidopsis.

  6. Identification of a conserved protein involved in anaerobic unsaturated fatty acid synthesis in Neiserria gonorrhoeae: implications for facultative and obligate anaerobes that lack FabA

    Science.gov (United States)

    Isabella, Vincent M.; Clark, Virginia L.

    2011-01-01

    SUMMARY Transcriptome analysis of the facultative anaerobe, Neisseria gonorrhoeae, revealed that many genes of unknown function were induced under anaerobic conditions. Mutation of one such gene, NGO1024, encoding a protein belonging to the 2-nitropropane dioxygenase-like superfamiliy of proteins, was found to result in an inability of gonococci to grow anaerobically. Anaerobic growth of an NG1024 mutant was restored upon supplementation with unsaturated fatty acids (UFA), but not with the saturated fatty acid palmitate. Gonococcal fatty acid profiles confirmed that NGO1024 was involved in UFA synthesis anaerobically, but not aerobically, demonstrating that gonococci contain two distinct pathways for the production of UFAs, with a yet unidentified aerobic mechanism, and an anaerobic mechanism involving NGO1024. Expression of genes involved in classical anaerobic UFA synthesis, fabA, fabM, and fabB, was toxic in gonococci and unable to complement a NGO1024 mutation, suggesting that the chemistry involved in gonococcal anaerobic UFA synthesis is distinct from that of the classical pathway. NGO1024 homologs, which we suggest naming UfaA, form a distinct lineage within the 2-nitropropane dioxygenase-like superfamily, and are found in many facultative and obligate anaerobes that produce UFAs but lack fabA, suggesting that UfaA is part of a widespread pathway involved in UFA synthesis. PMID:21895795

  7. Adaptation of model proteins from cold to hot environments involves continuous and small adjustments of average parameters related to amino acid composition.

    Science.gov (United States)

    De Vendittis, Emmanuele; Castellano, Immacolata; Cotugno, Roberta; Ruocco, Maria Rosaria; Raimo, Gennaro; Masullo, Mariorosario

    2008-01-07

    The growth temperature adaptation of six model proteins has been studied in 42 microorganisms belonging to eubacterial and archaeal kingdoms, covering optimum growth temperatures from 7 to 103 degrees C. The selected proteins include three elongation factors involved in translation, the enzymes glyceraldehyde-3-phosphate dehydrogenase and superoxide dismutase, the cell division protein FtsZ. The common strategy of protein adaptation from cold to hot environments implies the occurrence of small changes in the amino acid composition, without altering the overall structure of the macromolecule. These continuous adjustments were investigated through parameters related to the amino acid composition of each protein. The average value per residue of mass, volume and accessible surface area allowed an evaluation of the usage of bulky residues, whereas the average hydrophobicity reflected that of hydrophobic residues. The specific proportion of bulky and hydrophobic residues in each protein almost linearly increased with the temperature of the host microorganism. This finding agrees with the structural and functional properties exhibited by proteins in differently adapted sources, thus explaining the great compactness or the high flexibility exhibited by (hyper)thermophilic or psychrophilic proteins, respectively. Indeed, heat-adapted proteins incline toward the usage of heavier-size and more hydrophobic residues with respect to mesophiles, whereas the cold-adapted macromolecules show the opposite behavior with a certain preference for smaller-size and less hydrophobic residues. An investigation on the different increase of bulky residues along with the growth temperature observed in the six model proteins suggests the relevance of the possible different role and/or structure organization played by protein domains. The significance of the linear correlations between growth temperature and parameters related to the amino acid composition improved when the analysis was

  8. Cell wall accumulation of fluorescent proteins derived from a trans-Golgi cisternal membrane marker and paramural bodies in interdigitated Arabidopsis leaf epidermal cells.

    Science.gov (United States)

    Akita, Kae; Kobayashi, Megumi; Sato, Mayuko; Kutsuna, Natsumaro; Ueda, Takashi; Toyooka, Kiminori; Nagata, Noriko; Hasezawa, Seiichiro; Higaki, Takumi

    2017-01-01

    In most dicotyledonous plants, leaf epidermal pavement cells develop jigsaw puzzle-like shapes during cell expansion. The rapid growth and complicated cell shape of pavement cells is suggested to be achieved by targeted exocytosis that is coordinated with cytoskeletal rearrangement to provide plasma membrane and/or cell wall materials for lobe development during their morphogenesis. Therefore, visualization of membrane trafficking in leaf pavement cells should contribute an understanding of the mechanism of plant cell morphogenesis. To reveal membrane trafficking in pavement cells, we observed monomeric red fluorescent protein-tagged rat sialyl transferases, which are markers of trans-Golgi cisternal membranes, in the leaf epidermis of Arabidopsis thaliana. Quantitative fluorescence imaging techniques and immunoelectron microscopic observations revealed that accumulation of the red fluorescent protein occurred mostly in the curved regions of pavement cell borders and guard cell ends during leaf expansion. Transmission electron microscopy observations revealed that apoplastic vesicular membrane structures called paramural bodies were more frequent beneath the curved cell wall regions of interdigitated pavement cells and guard cell ends in young leaf epidermis. In addition, pharmacological studies showed that perturbations in membrane trafficking resulted in simple cell shapes. These results suggested possible heterogeneity of the curved regions of plasma membranes, implying a relationship with pavement cell morphogenesis.

  9. Identification of proteins likely to be involved in morphogenesis, cell division, and signal transduction in Planctomycetes by comparative genomics.

    Science.gov (United States)

    Jogler, Christian; Waldmann, Jost; Huang, Xiaoluo; Jogler, Mareike; Glöckner, Frank Oliver; Mascher, Thorsten; Kolter, Roberto

    2012-12-01

    Members of the Planctomycetes clade share many unusual features for bacteria. Their cytoplasm contains membrane-bound compartments, they lack peptidoglycan and FtsZ, they divide by polar budding, and they are capable of endocytosis. Planctomycete genomes have remained enigmatic, generally being quite large (up to 9 Mb), and on average, 55% of their predicted proteins are of unknown function. Importantly, proteins related to the unusual traits of Planctomycetes remain largely unknown. Thus, we embarked on bioinformatic analyses of these genomes in an effort to predict proteins that are likely to be involved in compartmentalization, cell division, and signal transduction. We used three complementary strategies. First, we defined the Planctomycetes core genome and subtracted genes of well-studied model organisms. Second, we analyzed the gene content and synteny of morphogenesis and cell division genes and combined both methods using a "guilt-by-association" approach. Third, we identified signal transduction systems as well as sigma factors. These analyses provide a manageable list of candidate genes for future genetic studies and provide evidence for complex signaling in the Planctomycetes akin to that observed for bacteria with complex life-styles, such as Myxococcus xanthus.

  10. Involvement of protein kinase C-δ activation in betulininduced ...

    African Journals Online (AJOL)

    Purpose: To investigate the clinical benefits and underlying mechanisms of action of betulin in the treatment of cancer using a neuroblastoma (NB) cell model. Method: Cell viability ... of tumor recurrence. Keywords: Betulin, Neuroblastoma, Apoptosis, protein kinase C-δ, Adjuvant chemotherapy, Tumor recurrence, Caspase ...

  11. Lower Squalene Epoxidase and Higher Scavenger Receptor Class B Type 1 Protein Levels Are Involved in Reduced Serum Cholesterol Levels in Stroke-Prone Spontaneously Hypertensive Rats.

    Science.gov (United States)

    Michihara, Akihiro; Mido, Mayuko; Matsuoka, Hiroshi; Mizutani, Yurika

    2015-01-01

    A lower serum cholesterol level was recently shown to be one of the causes of stroke in an epidemiological study. Spontaneously hypertensive rats stroke-prone (SHRSP) have lower serum cholesterol levels than normotensive Wistar-Kyoto rats (WKY). To elucidate the mechanisms responsible for the lower serum cholesterol levels in SHRSP, we determined whether the amounts of cholesterol biosynthetic enzymes or the receptor and transporter involved in cholesterol uptake and efflux in the liver were altered in SHRSP. When the mRNA levels of seven cholesterol biosynthetic enzymes were measured using real-time polymerase chain reaction (PCR), farnesyl pyrophosphate synthase and squalene epoxidase (SQE) levels in the liver of SHRSP were significantly lower than those in WKY. SQE protein levels were significantly reduced in tissues other than the brain of SHRSP. No significant differences were observed in low-density lipoprotein (LDL) receptor (uptake of serum LDL-cholesterol) or ATP-binding cassette transporter A1 (efflux of cholesterol from the liver/formation of high-density lipoprotein (HDL)) protein levels in the liver and testis between SHRSP and WKY, whereas scavenger receptor class B type 1 (SRB1: uptake of serum HDL-cholesterol) protein levels were higher in the livers of SHRSP. These results indicated that the lower protein levels of SQE and higher protein levels of SRB1 in the liver were involved in the reduced serum cholesterol levels in SHRSP.

  12. Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid.

    Directory of Open Access Journals (Sweden)

    Hyong Woo Choi

    2016-03-01

    Full Text Available Damage-associated molecular pattern molecules (DAMPs signal the presence of tissue damage to induce immune responses in plants and animals. Here, we report that High Mobility Group Box 3 (HMGB3 is a novel plant DAMP. Extracellular HMGB3, through receptor-like kinases BAK1 and BKK1, induced hallmark innate immune responses, including i MAPK activation, ii defense-related gene expression, iii callose deposition, and iv enhanced resistance to Botrytis cinerea. Infection by necrotrophic B. cinerea released HMGB3 into the extracellular space (apoplast. Silencing HMGBs enhanced susceptibility to B. cinerea, while HMGB3 injection into apoplast restored resistance. Like its human counterpart, HMGB3 binds salicylic acid (SA, which results in inhibition of its DAMP activity. An SA-binding site mutant of HMGB3 retained its DAMP activity, which was no longer inhibited by SA, consistent with its reduced SA-binding activity. These results provide cross-kingdom evidence that HMGB proteins function as DAMPs and that SA is their conserved inhibitor.

  13. Tim50a, a nuclear isoform of the mitochondrial Tim50, interacts with proteins involved in snRNP biogenesis

    Directory of Open Access Journals (Sweden)

    Robinson Melvin L

    2005-07-01

    Full Text Available Abstract Background The Cajal body (CB is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs, which are vital for pre-mRNA splicing. Newly imported Sm-class snRNPs traffic through CBs, where the snRNA component of the snRNP is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. It is not known how nascent snRNPs localize to the CB and are released from this structure after modification. The marker protein for CBs, coilin, may play a role in snRNP biogenesis given that it can interact with snRNPs and SMN, the protein mutated in Spinal Muscular Atrophy. Loss of coilin function in mice leads to significant viability and fertility problems and altered CB formation. Results In this report, we identify a minor isoform of the mitochondrial Tim50, Tim50a, as a coilin interacting protein. The Tim50a transcript can be detected in some cancer cell lines and normal brain tissue. The Tim50a protein differs only from Tim50 in that it contains an additional 103 aa N-terminal to the translation start of Tim50. Importantly, a putative nuclear localization signal is found within these 103 residues. In contrast to Tim50, which localizes to the cytoplasm and mitochondria, Tim50a is strictly nuclear and is enriched in speckles with snRNPs. In addition to coilin, Tim50a interacts with snRNPs and SMN. Competition binding experiments demonstrate that coilin competes with Sm proteins of snRNPs and SMN for binding sites on Tim50a. Conclusion Tim50a may play a role in snRNP biogenesis given its cellular localization and protein interaction characteristics. We hypothesize that Tim50a takes part in the release of snRNPs and SMN from the CB.

  14. Estradiol-mediated internalisation of the non-activated estrogen receptor from the goat uterine plasma membrane: identification of the proteins involved.

    Science.gov (United States)

    Sreeja, S; Thampan, Raghava Varman

    2004-04-01

    An indirect approach has been made to study the molecular details associated with the estradiol-induced internalisation of the non-activated estrogen receptor (naER) from the goat uterine plasma membrane. The internalisation of naER appears to be an energy dependent process. Exposure of the plasma membrane to estradiol results in the activation of a Mg2+ dependent ATPase associated with the membrane fraction. Presence of quercetin in the medium prevented the activation of the Mg2+ ATPase as well as the dissociation of naER from the plasma membrane. Using isolated plasma membrane preparations it has been possible to identify the proteins which interact with naER during various stages of its internalisation. The main proteins identified are: (1) a 58 kDa protein, p58, which apparently recognizes the nuclear localization signals on the naER and transports it to the nucleus: (2) hsp70: (3) hsp90, the functional roles of which remain unknown at this stage; (4) a 50 kDa protein associated with the clathrin coated vesicles, presumed to be involved in recognizing the tyrosine based internalisation signals on the naER; (5) actin which mediates the plasma membrane-to-nucleus movement of the naER-p58 complex.

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

    Directory of Open Access Journals (Sweden)

    Rebecca Cook

    2015-03-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

  17. New protein involved in the replacement of cell molecules

    DEFF Research Database (Denmark)

    Poulsen, Jesper Buchhave

    2011-01-01

    In collaboration with colleagues from La Trobe University, Australia, scientists at Aarhus University have discovered and defined a novel enzyme involved in the replacement and renewal of cell molecules. The enzyme exerts its function within the so-called mitochondria - small “enclosed” compartme......In collaboration with colleagues from La Trobe University, Australia, scientists at Aarhus University have discovered and defined a novel enzyme involved in the replacement and renewal of cell molecules. The enzyme exerts its function within the so-called mitochondria - small “enclosed...

  18. PKC signaling is involved in the regulation of progranulin (acrogranin/PC-cell-derived growth factor/granulin-epithelin precursor) protein expression in human ovarian cancer cell lines.

    Science.gov (United States)

    Diaz-Cueto, Laura; Arechavaleta-Velasco, Fabian; Diaz-Arizaga, Adriana; Dominguez-Lopez, Pablo; Robles-Flores, Martha

    2012-07-01

    Overexpression of progranulin (also named acrogranin, PC-cell-derived growth factor, or granulin-epithelin precursor) is associated with ovarian cancer, specifically with cell proliferation, malignancy, chemoresistance, and shortened overall survival. The objective of the current study is to identify the signaling pathways involved in the regulation of progranulin expression in ovarian cancer cell lines. We studied the relation of protein kinase C (PKC), phosphatidylinositol 3-kinase, protein kinase A, P38, extracellular signal-regulated kinase, and Akt pathways on the modulation of progranulin expression levels in NIH-OVCAR-3 and SK-OV-3 ovarian cancer cell lines. The different pathways were examined using pharmacological inhibitors (calphostin C, LY294002, H89, SB203580, PD98059, and Akt Inhibitor), and mRNA and protein progranulin expression were analyzed by reverse transcriptase polymerase chain reaction and Western blot techniques, respectively. Inhibition of PKC signal transduction pathway by calphostin C decreased in a dose-dependent manner protein but not mRNA levels of progranulin in both ovarian cancer cell lines. LY294002 but not wortmannin, which are phosphatidylinositol 3-kinase inhibitors, also diminished the expression of progranulin in both cell lines. In addition, LY294002 treatment produced a significant reduction in cell viability. Inhibition of protein kinase A, P38, extracellular signal-regulated kinase, and Akt did not affect progranulin protein expression. These results suggest that the PKC signaling is involved in the regulation of progranulin protein expression in 2 different ovarian cancer cell lines. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the cellular proliferation and invasion in ovarian cancer produced by progranulin.

  19. Nuclear factor of activated T cell (NFAT) transcription proteins regulate genes involved in adipocyte metabolism and lipolysis

    International Nuclear Information System (INIS)

    Holowachuk, Eugene W.

    2007-01-01

    NFAT involvement in adipocyte physiological processes was examined by treatment with CsA and/or GSK3β inhibitors (Li + or TZDZ-8), which prevent or increase NFAT nuclear translocation, respectively. CsA treatment reduced basal and TNFα-induced rates of lipolysis by 50%. Adipocytes preincubated with Li + or TZDZ-8 prior to CsA and/or TNFα, exhibited enhanced basal rates of lipolysis and complete inhibition of CsA-mediated decreased rates of lipolysis. CsA treatment dramatically reduced the mRNA levels of adipocyte-specific genes (aP2, HSL, PPARγ, ACS and Adn), compared with control or TNFα-treatment, whereas Li + pretreatment blocked the inhibitory effects of CsA, and mRNA levels of aP2, HSL, PPARγ, and ACS were found at or above control levels. NFAT nuclear localization, assessed by EMSA, confirmed that CsA or Li + treatments inhibited or increased NFAT nuclear translocation, respectively. These results show that NFAT proteins in mature adipocytes participate in the transcriptional control of genes involved in adipocyte metabolism and lipolysis

  20. G-protein-coupled estrogen receptor 1 is involved in brain development during zebrafish (Danio rerio) embryogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Yanan; Liu, Xiaochun [State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275 (China); Zhu, Pei; Li, Jianzhen; Sham, Kathy W.Y. [School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong (China); Cheng, Shuk Han [Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong (China); Li, Shuisheng; Zhang, Yong [State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275 (China); Cheng, Christopher H.K., E-mail: chkcheng@cuhk.edu.hk [School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong (China); Lin, Haoran, E-mail: lsslhr@mail.sysu.edu.cn [State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275 (China); College of Ocean, Hainan University, Haikou 570228, Hainan (China)

    2013-05-24

    Highlights: •The Gper expression was detected in the developing brain of zebrafish. •Gper morpholino knockdown induced apoptosis of brain cells. •Gper morpholino knockdown reduced expression in neuron markers. •Zebrafish Gper may be involved in neuronal development. -- Abstract: G-protein-coupled estrogen receptor 1 (Gper, formerly known as GPR30) is found to be a trophic and protective factor in mediating action of estrogen in adult brain, while its role in developing brain remains to be elucidated. Here we present the expression pattern of Gper and its functions during embryogenesis in zebrafish. Both the mRNA and protein of Gper were detected throughout embryogenesis. Whole mount in situ hybridization (WISH) revealed a wide distribution of gper mRNAs in various regions of the developing brain. Gper knockdown by specific morpholinos resulted in growth retardation in embryos and morphological defects in the developing brain. In addition, induced apoptosis, decreased proliferation of the brain cells and maldevelopment of sensory and motor neurons were also found in the morphants. Our results provide novel insights into Gper functions in the developing brain, revealing that Gper can maintain the survival of the brain cells, and formation and/or differentiation of the sensory and motor neurons.

  1. G-protein-coupled estrogen receptor 1 is involved in brain development during zebrafish (Danio rerio) embryogenesis

    International Nuclear Information System (INIS)

    Shi, Yanan; Liu, Xiaochun; Zhu, Pei; Li, Jianzhen; Sham, Kathy W.Y.; Cheng, Shuk Han; Li, Shuisheng; Zhang, Yong; Cheng, Christopher H.K.; Lin, Haoran

    2013-01-01

    Highlights: •The Gper expression was detected in the developing brain of zebrafish. •Gper morpholino knockdown induced apoptosis of brain cells. •Gper morpholino knockdown reduced expression in neuron markers. •Zebrafish Gper may be involved in neuronal development. -- Abstract: G-protein-coupled estrogen receptor 1 (Gper, formerly known as GPR30) is found to be a trophic and protective factor in mediating action of estrogen in adult brain, while its role in developing brain remains to be elucidated. Here we present the expression pattern of Gper and its functions during embryogenesis in zebrafish. Both the mRNA and protein of Gper were detected throughout embryogenesis. Whole mount in situ hybridization (WISH) revealed a wide distribution of gper mRNAs in various regions of the developing brain. Gper knockdown by specific morpholinos resulted in growth retardation in embryos and morphological defects in the developing brain. In addition, induced apoptosis, decreased proliferation of the brain cells and maldevelopment of sensory and motor neurons were also found in the morphants. Our results provide novel insights into Gper functions in the developing brain, revealing that Gper can maintain the survival of the brain cells, and formation and/or differentiation of the sensory and motor neurons

  2. Genes involved in protein metabolism of the probiotic lactic acid bacterium Lactobacillus delbrueckii UFV H2b20.

    Science.gov (United States)

    Do Carmo, A P; da Silva, D F; De Oliveira, M N V; Borges, A C; De Carvalho, A F; De Moraes, C A

    2011-09-01

    A basic requirement for the prediction of the potential use of lactic acid bacteria (LAB) in the dairy industry is the identification of specific genes involved in flavour-forming pathways. The probiotic Lactobacillus delbrueckii UFV H2b20 was submitted to a genetic characterisation and phylogenetic analysis of genes involved in protein catabolism. Eight genes belonging to this system were identified, which possess a closely phylogenetic relationship to NCFM strains representative, as it was demonstrated for oppC and oppBII, encoding oligopeptide transport system components. PepC, PepN, and PepX might be essential for growth of LAB, probiotic or not, since the correspondent genes are always present, including in L. delbrueckii UFV H2b20 genome. For pepX gene, a probable link between carbohydrate catabolism and PepX expression may exists, where it is regulated by PepR1/CcpA-like, a common feature between Lactobacillus strains and also in L. delbrueckii UFV H2b20. The well conserved evolutionary history of the ilvE gene is evidence that the pathways leading to branched-chain amino acid degradation, such as isoleucine and valine, are similar among L. delbrueckii subsp. bulgaricus strains and L. delbrueckii UFV H2b20. Thus, the involvement of succinate in flavour formation can be attributed to IlvE activity. The presence of aminopeptidase G in L. delbrueckii UFV H2b20 genome, which is absent in several strains, might improve the proteolytic activity and effectiveness. The nucleotide sequence encoding PepG revealed that it is a cysteine endopeptidase, belonging to Peptidase C1 superfamily; sequence analysis showed 99% identity with L. delbrueckii subsp. bulgaricus ATCC 11842 pepG, whereas protein sequence analysis revealed 100% similarity with PepG from the same organism. The present study proposes a schematic model to explain how the proteolytic system of the probiotic L. delbrueckii UFV H2b20 works, based on the components identified so far.

  3. Brain propagation of transduced α-synuclein involves non-fibrillar protein species and is enhanced in α-synuclein null mice.

    Science.gov (United States)

    Helwig, Michael; Klinkenberg, Michael; Rusconi, Raffaella; Musgrove, Ruth E; Majbour, Nour K; El-Agnaf, Omar M A; Ulusoy, Ayse; Di Monte, Donato A

    2016-03-01

    Aggregation and neuron-to-neuron transmission are attributes of α-synuclein relevant to its pathogenetic role in human synucleinopathies such as Parkinson's disease. Intraparenchymal injections of fibrillar α-synuclein trigger widespread propagation of amyloidogenic protein species via mechanisms that require expression of endogenous α-synuclein and, possibly, its structural corruption by misfolded conformers acting as pathological seeds. Here we describe another paradigm of long-distance brain diffusion of α-synuclein that involves inter-neuronal transfer of monomeric and/or oligomeric species and is independent of recruitment of the endogenous protein. Targeted expression of human α-synuclein was induced in the mouse medulla oblongata through an injection of viral vectors into the vagus nerve. Enhanced levels of intra-neuronal α-synuclein were sufficient to initiate its caudo-rostral diffusion that likely involved at least one synaptic transfer and progressively reached specific brain regions such as the locus coeruleus, dorsal raphae and amygdala in the pons, midbrain and forebrain. Transfer of human α-synuclein was compared in two separate lines of α-synuclein-deficient mice versus their respective wild-type controls and, interestingly, lack of endogenous α-synuclein expression did not counteract diffusion but actually resulted in a more pronounced and advanced propagation of exogenous α-synuclein. Self-interaction of adjacent molecules of human α-synuclein was detected in both wild-type and mutant mice. In the former, interaction of human α-synuclein with mouse α-synuclein was also observed and might have contributed to differences in protein transmission. In wild-type and α-synuclein-deficient mice, accumulation of human α-synuclein within recipient axons in the pons, midbrain and forebrain caused morphological evidence of neuritic pathology. Tissue sections from the medulla oblongata and pons were stained with different antibodies recognizing

  4. SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea.

    Science.gov (United States)

    Kirm, Benjamin; Magdevska, Vasilka; Tome, Miha; Horvat, Marinka; Karničar, Katarina; Petek, Marko; Vidmar, Robert; Baebler, Spela; Jamnik, Polona; Fujs, Štefan; Horvat, Jaka; Fonovič, Marko; Turk, Boris; Gruden, Kristina; Petković, Hrvoje; Kosec, Gregor

    2013-12-17

    Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bld

  5. The intracellular Scots pine shoot symbiont Methylobacterium extorquens DSM13060 aggregates around the host nucleus and encodes eukaryote-like proteins.

    Science.gov (United States)

    Koskimäki, Janne J; Pirttilä, Anna Maria; Ihantola, Emmi-Leena; Halonen, Outi; Frank, A Carolin

    2015-03-24

    Endophytes are microbes that inhabit plant tissues without any apparent signs of infection, often fundamentally altering plant phenotypes. While endophytes are typically studied in plant roots, where they colonize the apoplast or dead cells, Methylobacterium extorquens strain DSM13060 is a facultatively intracellular symbiont of the meristematic cells of Scots pine (Pinus sylvestris L.) shoot tips. The bacterium promotes host growth and development without the production of known plant growth-stimulating factors. Our objective was to examine intracellular colonization by M. extorquens DSM13060 of Scots pine and sequence its genome to identify novel molecular mechanisms potentially involved in intracellular colonization and plant growth promotion. Reporter construct analysis of known growth promotion genes demonstrated that these were only weakly active inside the plant or not expressed at all. We found that bacterial cells accumulate near the nucleus in intact, living pine cells, pointing to host nuclear processes as the target of the symbiont's activity. Genome analysis identified a set of eukaryote-like functions that are common as effectors in intracellular bacterial pathogens, supporting the notion of intracellular bacterial activity. These include ankyrin repeats, transcription factors, and host-defense silencing functions and may be secreted by a recently imported type IV secretion system. Potential factors involved in host growth include three copies of phospholipase A2, an enzyme that is rare in bacteria but implicated in a range of plant cellular processes, and proteins putatively involved in gibberellin biosynthesis. Our results describe a novel endophytic niche and create a foundation for postgenomic studies of a symbiosis with potential applications in forestry and agriculture. All multicellular eukaryotes host communities of essential microbes, but most of these interactions are still poorly understood. In plants, bacterial endophytes are found inside

  6. Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium

    Directory of Open Access Journals (Sweden)

    Charlene Desbonnet

    2016-04-01

    Full Text Available The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as “cephalosporins” is reliant on the presence of class A penicillin-binding proteins (Pbps PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2 of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP. Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA. Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system.

  7. Enzymes Involved in AMPylation and deAMPylation.

    Science.gov (United States)

    Casey, Amanda K; Orth, Kim

    2018-02-14

    Posttranslational modifications are covalent changes made to proteins that typically alter the function or location of the protein. AMPylation is an emerging posttranslational modification that involves the addition of adenosine monophosphate (AMP) to a protein. Like other, more well-studied posttranslational modifications, AMPylation is predicted to regulate the activity of the modified target proteins. However, the scope of this modification both in bacteria and in eukaryotes remains to be fully determined. In this review, we provide an up to date overview of the known AMPylating enzymes, the regulation of these enzymes, and the effect of this modification on target proteins.

  8. The Haemophilus ducreyi LspA1 protein inhibits phagocytosis by using a new mechanism involving activation of C-terminal Src kinase.

    Science.gov (United States)

    Dodd, Dana A; Worth, Randall G; Rosen, Michael K; Grinstein, Sergio; van Oers, Nicolai S C; Hansen, Eric J

    2014-05-20

    Haemophilus ducreyi causes chancroid, a sexually transmitted infection. A primary means by which this pathogen causes disease involves eluding phagocytosis; however, the molecular basis for this escape mechanism has been poorly understood. Here, we report that the LspA virulence factors of H. ducreyi inhibit phagocytosis by stimulating the catalytic activity of C-terminal Src kinase (Csk), which itself inhibits Src family protein tyrosine kinases (SFKs) that promote phagocytosis. Inhibitory activity could be localized to a 37-kDa domain (designated YL2) of the 456-kDa LspA1 protein. The YL2 domain impaired ingestion of IgG-opsonized targets and decreased levels of active SFKs when expressed in mammalian cells. YL2 contains tyrosine residues in two EPIYG motifs that are phosphorylated in mammalian cells. These tyrosine residues were essential for YL2-based inhibition of phagocytosis. Csk was identified as the predominant mammalian protein interacting with YL2, and a dominant-negative Csk rescued phagocytosis in the presence of YL2. Purified Csk phosphorylated the tyrosines in the YL2 EPIYG motifs. Phosphorylated YL2 increased Csk catalytic activity, resulting in positive feedback, such that YL2 can be phosphorylated by the same kinase that it activates. Finally, we found that the Helicobacter pylori CagA protein also inhibited phagocytosis in a Csk-dependent manner, raising the possibility that this may be a general mechanism among diverse bacteria. Harnessing Csk to subvert the Fcγ receptor (FcγR)-mediated phagocytic pathway represents a new bacterial mechanism for circumventing a crucial component of the innate immune response and may potentially affect other SFK-involved cellular pathways. Phagocytosis is a critical component of the immune system that enables pathogens to be contained and cleared. A number of bacterial pathogens have developed specific strategies to either physically evade phagocytosis or block the intracellular signaling required for

  9. Evidence for the involvement of a labile protein in stimulation of adrenal steroidogenesis under conditions not inhibitory to protein synthesis

    International Nuclear Information System (INIS)

    Krueger, R.J.; Orme-Johnson, N.R.

    1988-01-01

    Evidence is presented to support the hypothesis that synthesis of a labile protein is required for stimulation of steroidogenesis in rat adrenocortical cells. Amino acids L-canavanine and L-S-aminoethylcysteine, at concentrations as high as 5 mM, each inhibited steroidogenesis to a much greater extent than they inhibited protein synthesis. S-Aminoethylcysteine caused a 50% decrease in the stimulated rate of corticosterone production under conditions where incorporation of [35S]methionine into protein was unchanged. Both amino acids block stimulation of steroid synthesis at a step subsequent to the formation of cAMP and before the synthesis of progesterone. The onset of this effect, after the addition of the amino acids, on corticosterone production is quite rapid. These results provide support, that is not dependent on inhibition of protein synthesis, for the hypothesis that a labile protein mediates stimulation of steroidogenesis. Reversal of canavanine and S-aminoethylcysteine inhibition of steroidogenesis by arginine and lysine, respectively, suggests that the inhibitors are functioning as amino acid analogs. S-Aminoethylcysteine inhibits the incorporation of [3H]lysine into protein as well as inhibits steroidogenesis; further, [3H]S-aminoethylcysteine is incorporated into protein that is nonstimulatory. These results suggest that lysine residues play an essential role in the function of the labile protein or that the labile protein contains a large number of lysine residues

  10. Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1.

    Science.gov (United States)

    Scarlatti, Francesca; Bauvy, Chantal; Ventruti, Annamaria; Sala, Giusy; Cluzeaud, Françoise; Vandewalle, Alain; Ghidoni, Riccardo; Codogno, Patrice

    2004-04-30

    The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C(2)-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B(1). Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C(2)-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C(2)-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C(2)-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.

  11. Physical interaction between replication protein A (RPA) and MRN: involvement of RPA2 phosphorylation and the N-terminus of RPA1.

    Science.gov (United States)

    Oakley, Greg G; Tillison, Kristin; Opiyo, Stephen A; Glanzer, Jason G; Horn, Jeffrey M; Patrick, Steve M

    2009-08-11

    Replication protein A (RPA) is a heterotrimeric protein consisting of RPA1, RPA2, and RPA3 subunits that binds to single-stranded DNA (ssDNA) with high affinity. The response to replication stress requires the recruitment of RPA and the MRE11-RAD50-NBS1 (MRN) complex. RPA bound to ssDNA stabilizes stalled replication forks by recruiting checkpoint proteins involved in fork stabilization. MRN can bind DNA structures encountered at stalled or collapsed replication forks, such as ssDNA-double-stranded DNA (dsDNA) junctions or breaks, and promote the restart of DNA replication. Here, we demonstrate that RPA2 phosphorylation regulates the assembly of DNA damage-induced RPA and MRN foci. Using purified proteins, we observe a direct interaction between RPA with both NBS1 and MRE11. By utilizing RPA bound to ssDNA, we demonstrate that substituting RPA with phosphorylated RPA or a phosphomimetic weakens the interaction with the MRN complex. Also, the N-terminus of RPA1 is a critical component of the RPA-MRN protein-protein interaction. Deletion of the N-terminal oligonucleotide-oligosaccharide binding fold (OB-fold) of RPA1 abrogates interactions of RPA with MRN and individual proteins of the MRN complex. Further identification of residues critical for MRN binding in the N-terminus of RPA1 shows that substitution of Arg31 and Arg41 with alanines disrupts the RPA-MRN interaction and alters cell cycle progression in response to DNA damage. Thus, the N-terminus of RPA1 and phosphorylation of RPA2 regulate RPA-MRN interactions and are important in the response to DNA damage.

  12. HIV protein sequence hotspots for crosstalk with host hub proteins.

    Directory of Open Access Journals (Sweden)

    Mahdi Sarmady

    Full Text Available HIV proteins target host hub proteins for transient binding interactions. The presence of viral proteins in the infected cell results in out-competition of host proteins in their interaction with hub proteins, drastically affecting cell physiology. Functional genomics and interactome datasets can be used to quantify the sequence hotspots on the HIV proteome mediating interactions with host hub proteins. In this study, we used the HIV and human interactome databases to identify HIV targeted host hub proteins and their host binding partners (H2. We developed a high throughput computational procedure utilizing motif discovery algorithms on sets of protein sequences, including sequences of HIV and H2 proteins. We identified as HIV sequence hotspots those linear motifs that are highly conserved on HIV sequences and at the same time have a statistically enriched presence on the sequences of H2 proteins. The HIV protein motifs discovered in this study are expressed by subsets of H2 host proteins potentially outcompeted by HIV proteins. A large subset of these motifs is involved in cleavage, nuclear localization, phosphorylation, and transcription factor binding events. Many such motifs are clustered on an HIV sequence in the form of hotspots. The sequential positions of these hotspots are consistent with the curated literature on phenotype altering residue mutations, as well as with existing binding site data. The hotspot map produced in this study is the first global portrayal of HIV motifs involved in altering the host protein network at highly connected hub nodes.

  13. Adenovirus structural protein IIIa is involved in the serotype specificity of viral DNA packaging.

    Science.gov (United States)

    Ma, Hsin-Chieh; Hearing, Patrick

    2011-08-01

    The packaging of the adenovirus (Ad) genome into a capsid displays serotype specificity. This specificity has been attributed to viral packaging proteins, the IVa2 protein and the L1-52/55K protein. We previously found that the Ad17 L1-52/55K protein was not able to complement the growth of an Ad5 L1-52/55K mutant virus, whereas two other Ad17 packaging proteins, IVa2 and L4-22K, could complement the growth of Ad5 viruses with mutations in the respective genes. In this report, we investigated why the Ad17 L1-52/55K protein was not able to complement the Ad5 L1-52/55K mutant virus. We demonstrate that the Ad17 L1-52/55K protein binds to the Ad5 IVa2 protein in vitro and the Ad5 packaging domain in vivo, activities previously associated with packaging function. The Ad17 L1-52/55K protein also associates with empty Ad5 capsids. Interestingly, we find that the Ad17 L1-52/55K protein is able to complement the growth of an Ad5 L1-52/55K mutant virus in conjunction with the Ad17 structural protein IIIa. The same result was found with the L1-52/55K and IIIa proteins of several other Ad serotypes, including Ad3 and Ad4. The Ad17 IIIa protein associates with empty Ad5 capsids. Consistent with the complementation results, we find that the IIIa protein interacts with the L1-52/55K protein in vitro and associates with the viral packaging domain in vivo. These results underscore the complex nature of virus assembly and genome encapsidation and provide a new model for how the viral genome may tether to the empty capsid during the encapsidation process.

  14. Pep1, a secreted effector protein of Ustilago maydis, is required for successful invasion of plant cells.

    Directory of Open Access Journals (Sweden)

    Gunther Doehlemann

    2009-02-01

    Full Text Available The basidiomycete Ustilago maydis causes smut disease in maize. Colonization of the host plant is initiated by direct penetration of cuticle and cell wall of maize epidermis cells. The invading hyphae are surrounded by the plant plasma membrane and proliferate within the plant tissue. We identified a novel secreted protein, termed Pep1, that is essential for penetration. Disruption mutants of pep1 are not affected in saprophytic growth and develop normal infection structures. However, Deltapep1 mutants arrest during penetration of the epidermal cell and elicit a strong plant defense response. Using Affymetrix maize arrays, we identified 116 plant genes which are differentially regulated in Deltapep1 compared to wild type infections. Most of these genes are related to plant defense. By in vivo immunolocalization, live-cell imaging and plasmolysis approaches, we detected Pep1 in the apoplastic space as well as its accumulation at sites of cell-to-cell passages. Site-directed mutagenesis identified two of the four cysteine residues in Pep1 as essential for function, suggesting that the formation of disulfide bridges is crucial for proper protein folding. The barley covered smut fungus Ustilago hordei contains an ortholog of pep1 which is needed for penetration of barley and which is able to complement the U. maydis Deltapep1 mutant. Based on these results, we conclude that Pep1 has a conserved function essential for establishing compatibility that is not restricted to the U. maydis / maize interaction.

  15. Crystallization of Spätzle, a cystine-knot protein involved in embryonic development and innate immunity in Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Hoffmann, Anita; Neumann, Piotr [Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Abteilung Physikalische Biotechnologie, Kurt-Mothes-Strasse 3, 06120 Halle (Saale) (Germany); Schierhorn, Angelika [Max-Planck-Institut für Proteinfaltung, Abteilung Massenspektrometrie, Kurt-Mothes-Strasse 3, 06120 Halle (Saale) (Germany); Stubbs, Milton T., E-mail: stubbs@biochemtech.uni-halle.de [Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Abteilung Physikalische Biotechnologie, Kurt-Mothes-Strasse 3, 06120 Halle (Saale) (Germany); Mitteldeutsches Zentrum für Struktur und Dynamik der Proteine (MZP), Martin-Luther-Universität Halle-Wittenberg (Germany)

    2008-08-01

    Crystallization of the cystine-knot protein Spätzle occurred following serendipitous limited degradation of the pro-Spätzle propeptide during the crystallization experiment. The Spätzle protein is involved in both the definition of the dorsal–ventral axis during embryonic development and in the adult innate immune response. The disulfide-linked dimeric cystine-knot protein has been expressed as a proprotein in inclusion bodies in Escherichia coli and refolded in vitro by rapid dilution. Initial orthorhombic crystals that diffracted to 7 Å resolution were obtained after three months by the sitting-drop vapour-diffusion method. Optimization of the crystallization conditions resulted in orthorhombic crystals (space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.0, b = 59.2, c = 62.5 Å) that diffracted to 2.8 Å resolution in-house. The small volume of the asymmetric unit indicated that it was not possible for the crystals to contain the complete pro-Spätzle dimer. Mass spectrometry, N-terminal sequencing and Western-blot analysis revealed that the crystals contained the C-terminal disulfide-linked cystine-knot dimer. Comparison of various crystallization experiments indicated that degradation of the N-terminal prodomain was dependent on the buffer conditions.

  16. The expression of proteins involved in digestion and detoxification are regulated in Helicoverpa armigera to cope up with chlorpyrifos insecticide.

    Science.gov (United States)

    Dawkar, Vishal V; Chikate, Yojana R; More, Tushar H; Gupta, Vidya S; Giri, Ashok P

    2016-02-01

    Helicoverpa armigera is a key pest in many vital crops, which is mainly controlled by chemical strategies. To manage this pest is becoming challenging due to its ability and evolution of resistance against insecticides. Further, its subsequent spread on nonhost plant is remarkable in recent times. Hence, decoding resistance mechanism against phytochemicals and synthetic insecticides is a major challenge. The present work describes that the digestion, defense and immunity related enzymes are associated with chlorpyrifos resistance in H. armigera. Proteomic analysis of H. armigera gut tissue upon feeding on chlorpyrifos containing diet (CH) and artificial diet (AD) using nano-liquid chromatography-mass spectrometry identified upregulated 23-proteins in CH fed larvae. Database searches combined with gene ontology analysis revealed that the identified gut proteins engrossed in digestion, proteins crucial for immunity, adaptive responses to stress, and detoxification. Biochemical and quantitative real-time polymerase chain reaction analysis of candidate proteins indicated that insects were struggling to get nutrients and energy in presence of CH, while at the same time endeavoring to metabolize chlorpyrifos. Moreover, we proposed a potential processing pathway of chlorpyrifos in H. armigera gut by examining the metabolites using gas chromatography-mass spectrometry. H. armigera exhibit a range of intriguing behavioral, morphological adaptations and resistance to insecticides by regulating expression of proteins involved in digestion and detoxification mechanisms to cope up with chlorpyrifos. In these contexts, as gut is a rich repository of biological information; profound analysis of gut tissues can give clues of detoxification and resistance mechanism in insects. © 2014 Institute of Zoology, Chinese Academy of Sciences.

  17. The tryptophan-rich sensory protein (TSPO is involved in stress-related and light-dependent processes in the cyanobacterium Fremyella diplosiphon

    Directory of Open Access Journals (Sweden)

    Andrea eBusch

    2015-12-01

    Full Text Available The tryptophan-rich sensory protein (TSPO is a membrane protein, which is a member of the 18 kilodalton translocator protein/peripheral-type benzodiazepine receptor (MBR family of proteins that is present in most organisms and is also referred to as Translocator protein 18 kDa. Although TSPO is associated with stress- and disease-related processes in organisms from bacteria to mammals, full elucidation of the functional role of the TSPO protein is lacking for most organisms in which it is found. In this study, we describe the regulation and function of a TSPO homolog in the cyanobacterium Fremyella diplosiphon, designated FdTSPO. Accumulation of the FdTSPO transcript is upregulated by green light and in response to nutrient deficiency and stress. A F. diplosiphon TSPO deletion mutant (i.e., ΔFdTSPO showed altered responses compared to the wild type strain under stress conditions, including salt treatment, osmotic stress and induced oxidative stress. Under salt stress, the FdTSPO transcript is upregulated and a ΔFdTSPO mutant accumulates lower levels of reactive oxygen species (ROS and displays increased growth compared to WT. In response to osmotic stress, FdTSPO transcript levels are upregulated and ΔFdTSPO mutant cells exhibit impaired growth compared to the wild type. By comparison, methyl viologen-induced oxidative stress results in higher ROS levels in the ΔFdTSPO mutant compared to the wild type strain. Taken together, our results provide support for the involvement of membrane-localized FdTSPO in mediating cellular responses to stress in F. diplosiphon and represent detailed functional analysis of a cyanobacterial TSPO. This study advances our understanding of the functional roles of TSPO homologs in vivo.

  18. Adaptation of Lactobacillus casei Zhang to Gentamycin Involves an Alkaline Shock Protein

    Directory of Open Access Journals (Sweden)

    Wenyi Zhang

    2017-11-01

    Full Text Available Lactobacillus (L. casei Zhang is a koumiss-originated probiotic strain, which was used as a model in a long-term antibiotics-driven evolution experiment to reveal bacterial evolutionary dynamics; and we isolated gentamycin-resistant L. casei Zhang descendents. To decipher the gentamycin resistance mechanism, here we cultivated the parental L. casei Zhang and its descendent cells in an antibiotics-containing environment to compare their global protein expression profiles using the iTRAQ-based proteomic approach. A total of 72 proteins were significantly up-regulated (>2.0-fold, P < 0.05, whilst 32 proteins were significantly down-regulated <−2.0-fold, P < 0.05 in the descendent line. The gentamycin-resistant descendent line showed elevated expression in some carbohydrates, amino acids, and purine metabolic pathways. Several stress-related proteins were also differentially expressed. Among them, one alkaline shock protein, asp23, was up-regulated most in the gentamycin-resistant strain (21.9-fold increase compared with the parental strain. The asp23 gene disruption mutant was significantly more sensitive to gentamycin compared with the wild type, suggesting an important role of this gene in developing the gentamycin-resistant phenotype in L. casei. Our report has described the adaptation of a probiotic strain that has acquired antibiotics resistance through long-term antibiotics exposure at the proteome level, and we revealed a novel mechanism of gentamycin resistance.

  19. MARS: A protein family involved in the formation of vertical skeletal elements.

    Science.gov (United States)

    Abehsera, Shai; Peles, Shani; Tynyakov, Jenny; Bentov, Shmuel; Aflalo, Eliahu D; Li, Shihao; Li, Fuhua; Xiang, Jianhai; Sagi, Amir

    2017-05-01

    Vertical organizations of skeletal elements are found in various vertebrate teeth and invertebrate exoskeletons. The molecular mechanism behind the development of such structural organizations is poorly known, although it is generally held that organic matrix proteins play an essential role. While most crustacean cuticular organizations exhibit horizontal chitinous layering, a typical vertical organization is found towards the surface of the teeth in the mandibles of the crayfish Cherax quadricarinatus. Candidate genes encoding for mandible-forming structural proteins were mined in C. quadricarinatus molt-related transcriptomic libraries by using a binary patterning approach. A new protein family, termed the Mandible Alanine Rich Structural (MARS) protein family, with a modular sequence design predicted to form fibers, was found. Investigations of spatial and temporal expression of the different MARS genes suggested specific expression in the mandibular teeth-forming epithelium, particularly during the formation of the chitinous vertical organization. MARS loss-of-function RNAi experiments resulted in the collapse of the organization of the chitin fibers oriented vertically to the surface of the crayfish mandibular incisor tooth. A general search of transcriptomic libraries suggested conservation of MARS proteins across a wide array of crustaceans. Our results provide a first look into the molecular mechanism used to build the complex crustacean mandible and into the specialized vertical structural solution that has evolved in skeletal elements. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. A Web server for predicting proteins involved in pluripotent network

    Indian Academy of Sciences (India)

    2016-11-04

    Nov 4, 2016 ... which are important in pluripotency from the existing knowledge about pluripotent ... proteins, we took 117 genes with gene ontology term developmental ... space to find a hyperplane which maximizes the margin between two ...

  1. Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression.

    Science.gov (United States)

    Sindarovska, Y R; Gerasymenko, I M; Sheludko, Y V; Olevinskaya, Z M; Spivak, N Y; Kuchuk, N V

    2010-01-01

    Human interferon alpha2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 x 10(3) IU/g fresh weight (FW) with an average of 2.1 +/- 0.8 x 10(3) IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN alpha2b.

  2. Arabidopsis thaliana FLA4 functions as a glycan-stabilized soluble factor via its carboxy-proximal Fasciclin 1 domain.

    Science.gov (United States)

    Xue, Hui; Veit, Christiane; Abas, Lindy; Tryfona, Theodora; Maresch, Daniel; Ricardi, Martiniano M; Estevez, José Manuel; Strasser, Richard; Seifert, Georg J

    2017-08-01

    Fasciclin-like arabinogalactan proteins (FLAs) are involved in numerous important functions in plants but the relevance of their complex structure to physiological function and cellular fate is unresolved. Using a fully functional fluorescent version of Arabidopsis thaliana FLA4 we show that this protein is localized at the plasma membrane as well as in endosomes and soluble in the apoplast. FLA4 is likely to be GPI-anchored, is highly N-glycosylated and carries two O-glycan epitopes previously associated with arabinogalactan proteins. The activity of FLA4 was resistant against deletion of the amino-proximal fasciclin 1 domain and was unaffected by removal of the GPI-modification signal, a highly conserved N-glycan or the deletion of predicted O-glycosylation sites. Nonetheless these structural changes dramatically decreased endoplasmic reticulum (ER)-exit and plasma membrane localization of FLA4, with N-glycosylation acting at the level of ER-exit and O-glycosylation influencing post-secretory fate. We show that FLA4 acts predominantly by molecular interactions involving its carboxy-proximal fasciclin 1 domain and that its amino-proximal fasciclin 1 domain is required for stabilization of plasma membrane localization. FLA4 functions as a soluble glycoprotein via its carboxy-proximal Fas1 domain and its normal cellular trafficking depends on N- and O-glycosylation. © 2017 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

  3. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins

    DEFF Research Database (Denmark)

    Weber, Daniela; Davies, Michael J.; Grune, Tilman

    2015-01-01

    Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple...

  4. Protein improvement in Gari by the use of pure cultures of microorganisms involved in the natural fermentation process.

    Science.gov (United States)

    Ahaotu, I; Ogueke, C C; Owuamanam, C I; Ahaotu, N N; Nwosu, J N

    2011-10-15

    The ability of microorganisms involved in cassava mash fermentation to produce and improve protein value by these microorganisms during fermentation was studied. Standard microbiological procedures were used to isolate, identify and determine the numbers of the organisms. Alcaligenes faecalis, Lactobacillus plantarum, Bacillus subtilis, Leuconostoc cremoris, Aspergillus niger, A. tamari, Geotrichum candidum and Penicillium expansum were isolated and identified from cassava waste water while standard analytical methods were used to determine the ability of the isolates to produce linamarase and the proximate composition, pH and titrable acidity of the fermenting mash. The linamarase activity of the isolates ranged from 0.0416 to 0.2618 micromol mL(-1) nmol(-1). Bacillus subtilis, A. niger, A. tamari and P. expansum did not express any activity for the enzyme. Protein content of mash fermented with mixed fungal culture had the highest protein value (15.4 mg/g/dry matter) while the raw cassava had the least value (2.37 mg/g/dry matter). The naturally fermented sample had the least value for the fermented samples (3.2 mg/g/dry matter). Carbohydrate and fat contents of naturally fermented sample were higher than values obtained from the other fermented samples. Microbial numbers of the sample fermented with mixed bacterial culture was highest and got to their peak at 48 h (57 x 10(8) cfu g(-1)). pH decreased with increase in fermentation time with the mash fermented by the mixed culture of fungi having the lowest pH of 4.05 at the end of fermentation. Titrable acidity increased with increase in fermentation time with the highest value of 1.32% at 96 h of fermentation produced by the mixed culture of fungi. Thus fermentation with the pure cultures significantly increased the protein content of mash.

  5. The interaction of protein S with the phospholipid surface is essential for the activated protein C-independent activity of protein S

    NARCIS (Netherlands)

    van Wijnen, M.; Stam, J. G.; van't Veer, C.; Meijers, J. C.; Reitsma, P. H.; Bertina, R. M.; Bouma, B. N.

    1996-01-01

    Protein S is a vitamin-K dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Recent data showed a direct anticoagulant role of protein S independent of APC, as demonstrated by the inhibition of prothrombinase and tenase activity both in

  6. Candida albicans Hom6 is a homoserine dehydrogenase involved in protein synthesis and cell adhesion

    Directory of Open Access Journals (Sweden)

    Pei-Wen Tsai

    2017-12-01

    Full Text Available Background/Purpose: Candida albicans is a common fungal pathogen in humans. In healthy individuals, C. albicans represents a harmless commensal organism, but infections can be life threatening in immunocompromised patients. The complete genome sequence of C. albicans is extremely useful for identifying genes that may be potential drug targets and important for pathogenic virulence. However, there are still many uncharacterized genes in the Candida genome database. In this study, we investigated C. albicans Hom6, the functions of which remain undetermined experimentally. Methods: HOM6-deleted and HOM6-reintegrated mutant strains were constructed. The mutant strains were compared with wild-type in their growth in various media and enzyme activity. Effects of HOM6 deletion on translation were further investigated by cell susceptibility to hygromycin B or cycloheximide, as well as by polysome profiling, and cell adhesion to polystyrene was also determined. Results: C. albicans Hom6 exhibits homoserine dehydrogenase activity and is involved in the biosynthesis of methionine and threonine. HOM6 deletion caused translational arrest in cells grown under amino acid starvation conditions. Additionally, Hom6 protein was found in both cytosolic and cell-wall fractions of cultured cells. Furthermore, HOM6 deletion reduced C. albicans cell adhesion to polystyrene, which is a common plastic used in many medical devices. Conclusion: Given that there is no Hom6 homologue in mammalian cells, our results provided an important foundation for future development of new antifungal drugs. Keywords: Candida albicans, cell adhesion, Hom6, homoserine dehydrogenase, protein synthesis

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

    Science.gov (United States)

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

    2009-06-10

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

  8. Protein-Protein Interactions of Viroporins in Coronaviruses and Paramyxoviruses: New Targets for Antivirals?

    Directory of Open Access Journals (Sweden)

    Jaume Torres

    2015-06-01

    Full Text Available Viroporins are members of a rapidly growing family of channel-forming small polypeptides found in viruses. The present review will be focused on recent structural and protein-protein interaction information involving two viroporins found in enveloped viruses that target the respiratory tract; (i the envelope protein in coronaviruses and (ii the small hydrophobic protein in paramyxoviruses. Deletion of these two viroporins leads to viral attenuation in vivo, whereas data from cell culture shows involvement in the regulation of stress and inflammation. The channel activity and structure of some representative members of these viroporins have been recently characterized in some detail. In addition, searches for protein-protein interactions using yeast-two hybrid techniques have shed light on possible functional roles for their exposed cytoplasmic domains. A deeper analysis of these interactions should not only provide a more complete overview of the multiple functions of these viroporins, but also suggest novel strategies that target protein-protein interactions as much needed antivirals. These should complement current efforts to block viroporin channel activity.

  9. The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

    Science.gov (United States)

    Kristó, Ildikó; Bajusz, Csaba; Borsos, Barbara N; Pankotai, Tibor; Dopie, Joseph; Jankovics, Ferenc; Vartiainen, Maria K; Erdélyi, Miklós; Vilmos, Péter

    2017-10-01

    Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. The neuroprotection of cannabidiol against MPP⁺-induced toxicity in PC12 cells involves trkA receptors, upregulation of axonal and synaptic proteins, neuritogenesis, and might be relevant to Parkinson's disease.

    Science.gov (United States)

    Santos, Neife Aparecida Guinaim; Martins, Nádia Maria; Sisti, Flávia Malvestio; Fernandes, Laís Silva; Ferreira, Rafaela Scalco; Queiroz, Regina Helena Costa; Santos, Antônio Cardozo

    2015-12-25

    Cannabidiol (CBD) is a non-psychoactive constituent of Cannabis sativa with potential to treat neurodegenerative diseases. Its neuroprotection has been mainly associated with anti-inflammatory and antioxidant events; however, other mechanisms might be involved. We investigated the involvement of neuritogenesis, NGF receptors (trkA), NGF, and neuronal proteins in the mechanism of neuroprotection of CBD against MPP(+) toxicity in PC12 cells. CBD increased cell viability, differentiation, and the expression of axonal (GAP-43) and synaptic (synaptophysin and synapsin I) proteins. Its neuritogenic effect was not dependent or additive to NGF, but it was inhibited by K252a (trkA inhibitor). CBD did not increase the expression of NGF, but protected against its decrease induced by MPP(+), probably by an indirect mechanism. We also evaluated the neuritogenesis in SH-SY5Y cells, which do not express trkA receptors. CBD did not induce neuritogenesis in this cellular model, which supports the involvement of trkA receptors. This is the first study to report the involvement of neuronal proteins and trkA in the neuroprotection of CBD. Our findings suggest that CBD has a neurorestorative potential independent of NGF that might contribute to its neuroprotection against MPP(+), a neurotoxin relevant to Parkinson's disease. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Protein adhesives

    Science.gov (United States)

    Charles R. Frihart; Linda F. Lorenz

    2018-01-01

    Nature uses a wide variety of chemicals for providing adhesion internally (e.g., cell to cell) and externally (e.g., mussels to ships and piers). This adhesive bonding is chemically and mechanically complex, involving a variety of proteins, carbohydrates, and other compounds.Consequently,the effect of protein structures on adhesive properties is only partially...

  12. Pathway of phloem unloading in tobacco sink leaves

    International Nuclear Information System (INIS)

    Turgeon, R.

    1987-01-01

    Phloem unloading in transition sink leaves of tobacco (Nicotiana tabacum L.) was analyzed by quantitative autoradiography. Source leaves were labeled with 14 CO 2 and experimental treatments were begun approximately 1 h later when label had entered the sink leaves. Autoradiographs were prepared from rapidly frozen, lyophilized sink tissue at the beginning and end of the treatments and the amount of label in veins and in surrounding cells was determined by microdensitometry. Photoassimilate unloaded from third order and larger, but not smaller, veins. Long-distance import and unloading did not respond the same way to all experimental treatments. Import was completely inhibited by cold, anaerobiosis or steam girdling the sink leaf petiole. Unloading was inhibited by cold but continued in an anaerobic atmosphere and after steam girdling. Uptake of exogenous [ 14 C]sucrose was inhibited by anaerobiosis. Since an apoplastic pathway of phloem unloading would involve solute uptake from the apoplast the results are most consistent with passive symplastic unloading of photoassimilates from phloem to surrounding cells

  13. Non-structural proteins P17 and P33 are involved in the assembly of the internal membrane-containing virus PRD1

    Energy Technology Data Exchange (ETDEWEB)

    Karttunen, Jenni; Mäntynen, Sari [Centre of Excellence in Biological Interactions, Department of Biological and Environmental Science and Nanoscience Center, University of Jyväskylä, P.O. Box 35, 40014 Jyväskylä (Finland); Ihalainen, Teemu O. [Stem Cells in Neurological Applications Group, BioMediTech, University of Tampere, Tampere (Finland); Bamford, Jaana K.H. [Centre of Excellence in Biological Interactions, Department of Biological and Environmental Science and Nanoscience Center, University of Jyväskylä, P.O. Box 35, 40014 Jyväskylä (Finland); Oksanen, Hanna M., E-mail: hanna.oksanen@helsinki.fi [Institute of Biotechnology and Department of Biosciences, University of Helsinki, Biocenter 2, P.O. Box 56 (Viikinkaari 5), FIN-00014 Helsinki (Finland)

    2015-08-15

    Bacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diffusion of the P33 fluorescent fusion protein was substantially slower in E. coli than theoretically calculated, presumably resulting from intermolecular interactions. Our results indicate that P33 and P17 function in procapsid assembly, possibly in association with the host chaperonin complex GroEL/GroES. - Highlights: • Two non-structural proteins of PRD1 are involved in the virus assembly. • P17 and P33 complement the defect in GroES of Escherichia coli. • P33 co-localises with GroEL and P17 in the bacterium. • Slow motion of P33 in the bacterium suggests association with cellular components.

  14. Hepatitis C virus E2 protein involve in insulin resistance through an impairment of Akt/PKB and GSK3β signaling in hepatocytes

    Directory of Open Access Journals (Sweden)

    Hsieh Ming-Ju

    2012-06-01

    Full Text Available Abstract Background Hepatitis C virus (HCV infection may cause liver diseases of various severities ranging from primary acute infection to life-threatening diseases, such as cirrhosis or hepatocellular carcinoma with poor prognosis. According to clinical findings, HCV infection may also lead to some extra-hepatic symptoms, including type 2 diabetes mellitus (DM. Since insulin resistance is the major etiology for type 2 DM and numerous evidences showed that HCV infection associated with insulin resistance, the involvement of E2 in the pathogenesis of type 2 DM and underlying mechanisms were investigated in this study. Methods Reverse transcription and real-time PCR, Western blot assay, Immunoprecipitation, Glucose uptake assay and analysis of cellular glycogen content. Results Results showed that E2 influenced on protein levels of insulin receptor substrate-1 (IRS-1 and impaired insulin-induced Ser308 phosphorylation of Akt/PKB and Ser9 phosphorylation of GSK3β in Huh7 cells, leading to an inhibition of glucose uptake and glycogen synthesis, respectively, and eventually insulin resistance. Conclusions Therefore, HCV E2 protein indeed involved in the pathogenesis of type 2 DM by inducing insulin resistance.

  15. Characterization of Enzymatic Activity of MlrB and MlrC Proteins Involved in Bacterial Degradation of Cyanotoxins Microcystins.

    Science.gov (United States)

    Dziga, Dariusz; Zielinska, Gabriela; Wladyka, Benedykt; Bochenska, Oliwia; Maksylewicz, Anna; Strzalka, Wojciech; Meriluoto, Jussi

    2016-03-16

    Bacterial degradation of toxic microcystins produced by cyanobacteria is a common phenomenon. However, our understanding of the mechanisms of these processes is rudimentary. In this paper several novel discoveries regarding the action of the enzymes of the mlr cluster responsible for microcystin biodegradation are presented using recombinant proteins. In particular, the predicted active sites of the recombinant MlrB and MlrC were analyzed using functional enzymes and their inactive muteins. A new degradation intermediate, a hexapeptide derived from linearized microcystins by MlrC, was discovered. Furthermore, the involvement of MlrA and MlrB in further degradation of the hexapeptides was confirmed and a corrected biochemical pathway of microcystin biodegradation has been proposed.

  16. Characterization of Enzymatic Activity of MlrB and MlrC Proteins Involved in Bacterial Degradation of Cyanotoxins Microcystins

    Directory of Open Access Journals (Sweden)

    Dariusz Dziga

    2016-03-01

    Full Text Available Bacterial degradation of toxic microcystins produced by cyanobacteria is a common phenomenon. However, our understanding of the mechanisms of these processes is rudimentary. In this paper several novel discoveries regarding the action of the enzymes of the mlr cluster responsible for microcystin biodegradation are presented using recombinant proteins. In particular, the predicted active sites of the recombinant MlrB and MlrC were analyzed using functional enzymes and their inactive muteins. A new degradation intermediate, a hexapeptide derived from linearized microcystins by MlrC, was discovered. Furthermore, the involvement of MlrA and MlrB in further degradation of the hexapeptides was confirmed and a corrected biochemical pathway of microcystin biodegradation has been proposed.

  17. Sulfated glycopeptide nanostructures for multipotent protein activation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sungsoo S.; Fyrner, Timmy; Chen, Feng; Álvarez, Zaida; Sleep, Eduard; Chun, Danielle S.; Weiner, Joseph A.; Cook, Ralph W.; Freshman, Ryan D.; Schallmo, Michael S.; Katchko, Karina M.; Schneider, Andrew D.; Smith, Justin T.; Yun, Chawon; Singh, Gurmit; Hashmi, Sohaib Z.; McClendon, Mark T.; Yu, Zhilin; Stock, Stuart R.; Hsu, Wellington K.; Hsu, Erin L.; Stupp , Samuel I. (NWU)

    2017-06-19

    Biological systems have evolved to utilize numerous proteins with capacity to bind polysaccharides for the purpose of optimizing their function. A well-known subset of these proteins with binding domains for the highly diverse sulfated polysaccharides are important growth factors involved in biological development and tissue repair. We report here on supramolecular sulfated glycopeptide nanostructures, which display a trisulfated monosaccharide on their surfaces and bind five critical proteins with different polysaccharide-binding domains. Binding does not disrupt the filamentous shape of the nanostructures or their internal β-sheet backbone, but must involve accessible adaptive configurations to interact with such different proteins. The glycopeptide nanostructures amplified signalling of bone morphogenetic protein 2 significantly more than the natural sulfated polysaccharide heparin, and promoted regeneration of bone in the spine with a protein dose that is 100-fold lower than that required in the animal model. These highly bioactive nanostructures may enable many therapies in the future involving proteins.

  18. Kinases Involved in Both Autophagy and Mitosis.

    Science.gov (United States)

    Li, Zhiyuan; Zhang, Xin

    2017-08-31

    Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases), Aurora kinases, PLK-1 (polo-like kinase 1), BUB1 (budding uninhibited by benzimidazoles 1), MAPKs (mitogen-activated protein kinases), mTORC1 (mechanistic target of rapamycin complex 1), AMPK (AMP-activated protein kinase), PI3K (phosphoinositide-3 kinase) and protein kinase B (AKT). By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  19. Kinases Involved in Both Autophagy and Mitosis

    Directory of Open Access Journals (Sweden)

    Zhiyuan Li

    2017-08-01

    Full Text Available Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases, Aurora kinases, PLK-1 (polo-like kinase 1, BUB1 (budding uninhibited by benzimidazoles 1, MAPKs (mitogen-activated protein kinases, mTORC1 (mechanistic target of rapamycin complex 1, AMPK (AMP-activated protein kinase, PI3K (phosphoinositide-3 kinase and protein kinase B (AKT. By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  20. Microsecond molecular dynamics simulations of intrinsically disordered proteins involved in the oxidative stress response

    NARCIS (Netherlands)

    Cino, E.A.; Wong-ekkabut, J.; Karttunen, M.E.J.; Choy, W.-Y.

    2011-01-01

    Intrinsically disordered proteins (IDPs) are abundant in cells and have central roles in protein-protein interaction networks. Interactions between the IDP Prothymosin alpha (ProTa) and the Neh2 domain of Nuclear factor erythroid 2-related factor 2 (Nrf2), with a common binding partner, Kelch-like

  1. Cloning, crystallization and preliminary X-ray studies of XC2981 from Xanthomonas campestris, a putative CutA1 protein involved in copper-ion homeostasis

    International Nuclear Information System (INIS)

    Lin, Chien-Hung; Chin, Ko-Hsin; Gao, Fei Philip; Lyu, Ping-Chiang; Shr, Hui-Lin; Wang, Andrew H.-J.; Chou, Shan-Ho

    2006-01-01

    A probable copper-ion tolerance protein from the plant pathogen X. campestris has been overexpressed in E. coli, purified and crystallized. Divalent metal ions play key roles in all living organisms, serving as cofactors for many proteins involved in a variety of electron-transfer activities. However, copper ions are highly toxic when an excessive amount is accumulated in a cell. CutA1 is a protein found in all kingdoms of life that is believed to participate in copper-ion tolerance in Escherichia coli, although its specific function remains unknown. Several crystal structures of multimeric CutA1 with different rotation angles and degrees of interaction between trimer interfaces have been reported. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC2981, a possible CutA1 protein present in the plant pathogen Xanthomonas campestris, are reported. The XC2981 crystals diffracted to a resolution of 2.6 Å. They are cubic and belong to space group I23, with unit-cell parameters a = b = c = 130.73 Å

  2. Identification of structural protein-protein interactions of herpes simplex virus type 1.

    Science.gov (United States)

    Lee, Jin H; Vittone, Valerio; Diefenbach, Eve; Cunningham, Anthony L; Diefenbach, Russell J

    2008-09-01

    In this study we have defined protein-protein interactions between the structural proteins of herpes simplex virus type 1 (HSV-1) using a LexA yeast two-hybrid system. The majority of the capsid, tegument and envelope proteins of HSV-1 were screened in a matrix approach. A total of 40 binary interactions were detected including 9 out of 10 previously identified tegument-tegument interactions (Vittone, V., Diefenbach, E., Triffett, D., Douglas, M.W., Cunningham, A.L., and Diefenbach, R.J., 2005. Determination of interactions between tegument proteins of herpes simplex virus type 1. J. Virol. 79, 9566-9571). A total of 12 interactions involving the capsid protein pUL35 (VP26) and 11 interactions involving the tegument protein pUL46 (VP11/12) were identified. The most significant novel interactions detected in this study, which are likely to play a role in viral assembly, include pUL35-pUL37 (capsid-tegument), pUL46-pUL37 (tegument-tegument) and pUL49 (VP22)-pUS9 (tegument-envelope). This information will provide further insights into the pathways of HSV-1 assembly and the identified interactions are potential targets for new antiviral drugs.

  3. Identification of FUSE-binding proteins as interacting partners of TIA proteins

    International Nuclear Information System (INIS)

    Rothe, Francoise; Gueydan, Cyril; Bellefroid, Eric; Huez, Georges; Kruys, Veronique

    2006-01-01

    TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm

  4. Nitric oxide mediates the indole acetic acid induction activation of a mitogen-activated protein kinase cascade involved in adventitious root development.

    Science.gov (United States)

    Pagnussat, Gabriela Carolina; Lanteri, María Luciana; Lombardo, María Cristina; Lamattina, Lorenzo

    2004-05-01

    Recently, it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002, 2003). However, not much is known about the complex molecular network operating during the cell proliferation and morphogenesis triggered by auxins and NO in that process. Anatomical studies showed that formation of adventitious root primordia was clearly detected in indole acetic acid (IAA)- and NO-treated cucumber explants, while neither cell proliferation nor differentiation into root primordia could be observed in control explants 3 d after primary root was removed. In order to go further with signal transduction mechanisms that operate during IAA- and NO-induced adventitious root formation, experiments were designed to test the involvement of a mitogen-activated protein kinase (MAPK) cascade in that process. Cucumber explants were treated with the NO-donor sodium nitroprusside (SNP) or with SNP plus the specific NO-scavenger cPTIO. Protein extracts from those explants were assayed for protein kinase (PK) activity by using myelin basic protein (MBP) as substrate in both in vitro and in-gel assays. The activation of a PK of approximately 48 kD could be detected 1 d after NO treatment with a maximal activation after 3 d of treatment. In control explants, a PK activity was detected only after 4 d of treatment. The MBP-kinase activity was also detected in extracts from IAA-treated explants, while no signal was observed in IAA + cPTIO treatments. The PK activity could be inhibited by the cell-permeable MAPK kinase inhibitor PD098059, suggesting that the NO-dependent MBP-kinase activity is a MAPK. Furthermore, when PD098059 was administered to explants treated with SNP or IAA, it produced a delay in root emergence and a dose-dependent reduction in root number. Altogether, our results suggest that a MAPK signaling cascade is activated during the adventitious rooting process

  5. Outer membrane protein A (OmpA of Shigella flexneri 2a induces TLR2-mediated activation of B cells: involvement of protein tyrosine kinase, ERK and NF-κB.

    Directory of Open Access Journals (Sweden)

    Rajsekhar Bhowmick

    Full Text Available B cells are critically important in combating bacterial infections and their differentiation into plasma cells and memory cells aids bacterial clearance and long-lasting immunity conferred by essentially all vaccines. Outer membrane protein A (OmpA of Shigella flexneri 2a has been demonstrated to induce the production of IgG and IgA in vivo following immunization of mice through intranasal route, but the direct involvement of B cells in OmpA-mediated immune regulation was not determined. Consequently, we investigated whether OmpA can modulate B cell functions and identified the molecular events involved in OmpA-induced B cell immune response in vitro. We show that OmpA of S. flexneri 2a activates B cells to produce protective cytokines, IL-6 and IL-10 as well as facilitates their differentiation into antibody secreting cells (ASCs. The immunostimulatory properties of OmpA are attributed to the increased surface expression of MHCII and CD86 on B cells. We also report here that B cell activation by OmpA is mediated strictly through recognition by TLR2, resulting in initiation of cascades of signal transduction events, involving increased phosphorylation of protein tyrosine kinases (PTKs, ERK and IκBα, leading to nuclear translocation of NF-κB. Importantly, a TLR2 antibody diminishes OmpA-induced upregulation of MHCII and CD86 on B cell surface as well as significantly inhibits B cell differentiation and cytokine secretion. Furthermore, we illustrate that B cell differentiation into ASCs and induction of cytokine secretion by OmpA are dependent on PTKs activity. Moreover, we identify that OmpA-induced B cell differentiation is entirely dependent on ERK pathway, whereas both NF-κB and ERK are essential for cytokine secretion by B cells. Overall, our data demonstrate that OmpA of S. flexneri 2a amplifies TLR signaling in B cells and triggers B cell immune response, which is critical for the development of an effective adaptive immunity to an

  6. Effect of elevated Al and pH on the growth and root morphology of Al-tolerant and Al-sensitive wheat seedlings in an acid soil

    Directory of Open Access Journals (Sweden)

    Md. Toufiq Iqbal

    2014-03-01

    Full Text Available Aluminium ion (Al3+ toxicity and hydrogen ion (H+ activity are the major constraints for plant growth in acid soil. This study was undertaken to determine the effect of pH and Al on the growth response and changes in root morphology of Al-tolerant (ET8 and Al-sensitive (ES8 wheat seedlings. Different levels of AlCl3 and CaCO3 were added to the soils to manipulate soil pH and extractable Al. The results showed that the bulk soil pH remained constant at pH 4.1 with further applications of AlCl3, and that the seedlings died at the 200 mg AlCl3/kg treatments. The ET8 seedlings responded better than the ES8 seedlings in both low and high Al and pH. The ET8 seedlings had higher root surface areas and root tip numbers than the ES8 seedlings in the Al treatment. In contrast, the ES8 had higher root diameters than the ET8 seedlings due to the elevated Al supply. Apoplast Al increased with the increase of soil available extractable Al, and declined with the decrease of soil extractable Al. The ET8 seedlings accumulated more Al in their apoplast than the ES8 seedlings. This study concluded that accumulation of Al in the apoplast is also involved in Al tolerance mechanism with the addition of organic acid exudation.

  7. GP-9s are ubiquitous proteins unlikely involved in olfactory mediation of social organization in the red imported fire ant, Solenopsis invicta.

    Directory of Open Access Journals (Sweden)

    Walter S Leal

    Full Text Available The red imported fire ant (RIFA, Solenopsis invicta, is an invasive species, accidentally introduced in the United States that can cause painful (sometimes life-threatening stings to human, pets, and livestock. Their colonies have two social forms: monogyne and polygyne that have a single and multiple functional queens, respectively. A major gene (Gp-9, identified as a putative pheromone-binding protein on the basis of a modest amino acid sequence identity, has been suggested to influence the expression of colony social organization. Monogyne queens are reported to possess only the GP-9B alleles, whereas polygyne queens possess both GP-9B and GP-9b. Thus, both social forms are reported to express GP-9B, with GP-9b being a marker expressed in polygynes but it is absent in monogynes. Here, we report two types of polygyne colonies, one that does not express GP-9b (monogyne-like and the other expressing both proteins, GP-9B and GP-9b. Given their expression pattern, GP-9s are hemolymph proteins, which are more likely to be involved in the transport of lipids and small ligands within the homocoel. GP-9B existed in two forms, one of them is phosphorylated. The helical-rich content of the protein resembles the secondary structures of a beetle hemolymph protein and moth pheromone-binding proteins. An olfactory role is unlikely given the lack of specific expression in the sensillar lymph. In marked contrast to GP-9s, a chemosensory protein, SinvCSP, is demonstrated to be specifically expressed in the antennae. Within the antennae, expression of SinvCSP is restricted to the last two segments, which are known to house olfactory sensilla.

  8. Evidence for proteins involved in prophenoloxidase cascade Eisenia fetida earthworms

    Czech Academy of Sciences Publication Activity Database

    Kohlerová, Petra; Šilerová, Marcela; Stijlemans, B.; Dieu, M.; Halada, Petr; Josková, Radka; Beschin, A.; De Baetselier, P.; Bilej, Martin

    2006-01-01

    Roč. 176, - (2006), s. 581-587 ISSN 0174-1578 R&D Projects: GA ČR GA310/04/0806; GA AV ČR KJB500200613 Institutional research plan: CEZ:AV0Z50200510 Keywords : protein * prophenoloxidase cascade * eisenia fetida Subject RIV: EE - Microbiology, Virology Impact factor: 1.740, year: 2006

  9. Altered Expression of a Malate-Permeable Anion Channel, OsALMT4, Disrupts Mineral Nutrition1[OPEN

    Science.gov (United States)

    Delhaize, Emmanuel

    2017-01-01

    Aluminum-activated malate transporters (ALMTs) form a family of anion channels in plants, but little is known about most of its members. This study examined the function of OsALMT4 from rice (Oryza sativa). We show that OsALMT4 is expressed in roots and shoots and that the OsALMT4 protein localizes to the plasma membrane. Transgenic rice lines overexpressing (OX) OsALMT4 released malate from the roots constitutively and had 2-fold higher malate concentrations in the xylem sap than nulls, indicating greater concentrations of malate in the apoplast. OX lines developed brown necrotic spots on the leaves that did not appear on nulls. These symptoms were not associated with altered concentrations of any mineral element in the leaves, although the OX lines had higher concentrations of Mn and B in their grain compared with nulls. While total leaf Mn concentrations were not different between the OX and null lines, Mn concentrations in the apoplast were greater in the OX plants. The OX lines also displayed increased expression of Mn transporters and were more sensitive to Mn toxicity than null plants. We showed that the growth of wild-type rice was unaffected by 100 µm Mn in hydroponics but, when combined with 1 mm malate, this concentration inhibited growth. We conclude that increasing OsALMT4 expression affected malate efflux and compartmentation within the tissues, which increased Mn concentrations in the apoplast of leaves and induced the toxicity symptoms. This study reveals new links between malate transport and mineral nutrition. PMID:29101278

  10. Benzoxazolinone detoxification by N-Glucosylation: The multi-compartment-network of Zea mays L.

    Science.gov (United States)

    Schulz, Margot; Filary, Barbara; Kühn, Sabine; Colby, Thomas; Harzen, Anne; Schmidt, Jürgen; Sicker, Dieter; Hennig, Lothar; Hofmann, Diana; Disko, Ulrich; Anders, Nico

    2016-01-01

    The major detoxification product in maize roots after 24 h benzoxazolin-2(3H)-one (BOA) exposure was identified as glucoside carbamate resulting from rearrangement of BOA-N-glucoside, but the pathway of N-glucosylation, enzymes involved and the site of synthesis were previously unknown. Assaying whole cell proteins revealed the necessity of H2O2 and Fe(2+) ions for glucoside carbamate production. Peroxidase produced BOA radicals are apparently formed within the extraplastic space of the young maize root. Radicals seem to be the preferred substrate for N-glucosylation, either by direct reaction with glucose or, more likely, the N-glucoside is released by glucanase/glucosidase catalyzed hydrolysis from cell wall components harboring fixed BOA. The processes are accompanied by alterations of cell wall polymers. Glucoside carbamate accumulation could be suppressed by the oxireductase inhibitor 2-bromo-4´-nitroacetophenone and by peroxidase inhibitor 2,3-butanedione. Alternatively, activated BOA molecules with an open heterocycle may be produced by microorganisms (e.g., endophyte Fusarium verticillioides) and channeled for enzymatic N-glucosylation. Experiments with transgenic Arabidopsis lines indicate a role of maize glucosyltransferase BX9 in BOA-N-glycosylation. Western blots with BX9 antibody demonstrate the presence of BX9 in the extraplastic space. Proteomic analyses verified a high BOA responsiveness of multiple peroxidases in the apoplast/cell wall. BOA incubations led to shifting, altered abundances and identities of the apoplast and cell wall located peroxidases, glucanases, glucosidases and glutathione transferases (GSTs). GSTs could function as glucoside carbamate transporters. The highly complex, compartment spanning and redox-regulated glucoside carbamate pathway seems to be mainly realized in Poaceae. In maize, carbamate production is independent from benzoxazinone synthesis.

  11. Phloem unloading in tomato fruit

    International Nuclear Information System (INIS)

    Damon, S.; Hewitt, J.; Bennett, A.B.

    1986-01-01

    To begin to identify those processes that contribute to the regulation of photosynthate partitioning in tomato fruit the path of phloem unloading in this tissue has been characterized. Assymetrically labelled sucrose ( 3 H-fructosyl sucrose) was applied to source leaves. Following translocation to the fruit the apoplast was sampled. The appearance of assymetric sucrose and 3 H-fructose in the apoplast indicates that phloem unloading is apoplastic and that extracellular invertase is active. Estimation of sucrose, glucose, and fructose concentrations in the apoplast were 1 mM, 40 mM, and 40 mM, respectively. Rates of uptake of sucrose, 1-fluorosucrose, glucose, and fructose across the plasma membrane were similar and non-saturating at physiological concentrations. These results suggest that, although extracellular invertase is present, sucrose hydrolysis is not required for uptake into tomato fruit pericarp cells. 1-fluorosucrose is used to investigate the role of sucrose synthase in hydrolysis of imported photosynthate

  12. Interrogating the architecture of protein assemblies and protein interaction networks by cross-linking mass spectrometry

    NARCIS (Netherlands)

    Liu, Fan; Heck, Albert J R

    2015-01-01

    Proteins are involved in almost all processes of the living cell. They are organized through extensive networks of interaction, by tightly bound macromolecular assemblies or more transiently via signaling nodes. Therefore, revealing the architecture of protein complexes and protein interaction

  13. Egfl6 is involved in zebrafish notochord development.

    Science.gov (United States)

    Wang, Xueqian; Wang, Xin; Yuan, Wei; Chai, Renjie; Liu, Dong

    2015-08-01

    The epidermal growth factor (EGF) repeat motif defines a superfamily of diverse protein involved in regulating a variety of cellular and physiological processes, such as cell cycle, cell adhesion, proliferation, migration, and neural development. Egfl6, an EGF protein, also named MAGE was first cloned in human tissue. Up to date, the study of zebrafish Egfl6 expression pattern and functional analysis of Egfl6 involved in embryonic development of vertebrate in vivo is thus far lacking. Here we reported that Egfl6 was involved in zebrafish notochord development. It was shown that Egfl6 mRNA was expressed in zebrafish, developing somites, fin epidermis, pharyngeal arches, and hindbrain region. Particularly the secreted Egfl6 protein was significantly accumulated in notochord. Loss of Egfl6 function in zebrafish embryos resulted in curved body with distorted notochord in the posterior trunk. It was observed that expression of all Notch ligand and receptors in notochord of 28 hpf Egfl6 morphants was not affected, except notch2, which was up-regulated. We found that inhibition of Notch signaling by DAPT efficiently rescued notochord developmental defect of Egfl6 deficiency embryos.

  14. Yeast Interacting Proteins Database: YGL127C, YOR047C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ith protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; acts as a regula...rotein involved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sensors

  15. Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

    Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

  16. iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness.

    Directory of Open Access Journals (Sweden)

    Guangqi Li

    Full Text Available 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.

  17. Yeast Interacting Proteins Database: YOR047C, YKL038W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available racts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; acts as a...Bait description Protein involved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose senso...rs Snf3p and Rgt2p, and TATA-binding protein Spt15p; acts as a regulator of the tra

  18. Yeast Interacting Proteins Database: YFR049W, YOR047C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; acts as a regulator... (0) YOR047C STD1 Protein involved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sens...ors Snf3p and Rgt2p, and TATA-binding protein Spt15p; ac

  19. Accuracy issues involved in modeling in vivo protein structures using PM7.

    Science.gov (United States)

    Martin, Benjamin P; Brandon, Christopher J; Stewart, James J P; Braun-Sand, Sonja B

    2015-08-01

    Using the semiempirical method PM7, an attempt has been made to quantify the error in prediction of the in vivo structure of proteins relative to X-ray structures. Three important contributory factors are the experimental limitations of X-ray structures, the difference between the crystal and solution environments, and the errors due to PM7. The geometries of 19 proteins from the Protein Data Bank that had small R values, that is, high accuracy structures, were optimized and the resulting drop in heat of formation was calculated. Analysis of the changes showed that about 10% of this decrease in heat of formation was caused by faults in PM7, the balance being attributable to the X-ray structure and the difference between the crystal and solution environments. A previously unknown fault in PM7 was revealed during tests to validate the geometries generated using PM7. Clashscores generated by the Molprobity molecular mechanics structure validation program showed that PM7 was predicting unrealistically close contacts between nonbonding atoms in regions where the local geometry is dominated by very weak noncovalent interactions. The origin of this fault was traced to an underestimation of the core-core repulsion between atoms at distances smaller than the equilibrium distance. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published By Wiley Periodicals, Inc.

  20. Comparative proteomic analysis of oil palm leaves infected with Ganoderma boninense revealed changes in proteins involved in photosynthesis, carbohydrate metabolism, and immunity and defense.

    Science.gov (United States)

    Jeffery Daim, Leona Daniela; Ooi, Tony Eng Keong; Ithnin, Nalisha; Mohd Yusof, Hirzun; Kulaveerasingam, Harikrishna; Abdul Majid, Nazia; Karsani, Saiful Anuar

    2015-08-01

    The basidiomycete fungal pathogen Ganoderma boninense is the causative agent for the incurable basal stem rot (BSR) disease in oil palm. This disease causes significant annual crop losses in the oil palm industry. Currently, there is no effective method for disease control and elimination, nor is any molecular marker for early detection of the disease available. An understanding of how BSR affects protein expression in plants may help identify and/or assist in the development of an early detection protocol. Although the mode of infection of BSR disease is primarily via the root system, defense-related genes have been shown to be expressed in both the root and leafs. Thus, to provide an insight into the changes in the global protein expression profile in infected plants, comparative 2DE was performed on leaf tissues sampled from palms with and without artificial inoculation of the Ganoderma fungus. Comparative 2DE revealed that 54 protein spots changed in abundance. A total of 51 protein spots were successfully identified by LC-QTOF MS/MS. The majority of these proteins were those involved in photosynthesis, carbohydrate metabolism as well as immunity and defense. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. p44 and p34 subunits of the BTF2/TFIIH transcription factor have homologies with SSL1, a yeast protein involved in DNA repair.

    NARCIS (Netherlands)

    S. Humbert; H. van Vuuren; Y. Lutz; J.H.J. Hoeijmakers (Jan); J-M. Egly (Jean-Marc); V. Moncollin

    1994-01-01

    textabstractThe human BTF2 (TFIIH) transcription factor is a multisubunit protein involved in transcription initiation by RNA polymerase II (B) as well as in DNA repair. In addition to the previously characterized p62 and p89/ERCC3 subunits, we have cloned two other subunits of BTF2, p44 and p34.

  2. Structural analysis of intermolecular interactions in the kinesin adaptor complex fasciculation and elongation protein zeta 1/ short coiled-coil protein (FEZ1/SCOCO.

    Directory of Open Access Journals (Sweden)

    Marcos Rodrigo Alborghetti

    Full Text Available Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans, SCOCO (short coiled-coil protein / UNC-69 and kinesins (e.g. kinesin heavy chain / UNC116 are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth, we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance, cross-linking coupled with mass spectrometry (MS, SAXS (Small Angle X-ray Scattering and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance studies of the region involved in this process, corresponding to FEZ1 (92-194. Through studies involving the protein in its monomeric configuration (reduced and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.

  3. Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

    DEFF Research Database (Denmark)

    Ngo, HT; Pham, Long; Kim, JW

    2013-01-01

    Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...... increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation....

  4. Protein-protein interactions: an application of Tus-Ter mediated protein microarray system.

    Science.gov (United States)

    Sitaraman, Kalavathy; Chatterjee, Deb K

    2011-01-01

    In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.

  5. THERMODYNAMICS OF PROTEIN-LIGAND INTERACTIONS AND THEIR ANALYSIS

    Directory of Open Access Journals (Sweden)

    Rummi Devi Saini

    2017-11-01

    Full Text Available Physiological processes are controlled mainly by intermolecular recognition mechanisms which involve protein–protein and protein–ligand interactions with a high specificity and affinity to form a specific complex. Proteins being an important class of macromolecules in biological systems, it is important to understand their actions through binding to other molecules of proteins or ligands. In fact, the binding of low molecular weight ligands to proteins plays a significant role in regulating biological processes such as cellular metabolism and signal transmission. Therefore knowledge of the protein–ligand interactions and the knowledge of the mechanisms involved in the protein-ligand recognition and binding are key in understanding biology at molecular level which will facilitate the discovery, design, and development of drugs. In this review, the mechanisms involved in protein–ligand binding, the binding kinetics, thermodynamic concepts and binding driving forces are discussed. Thermodynamic mechanisms involved in a few important protein-ligand binding are described. Various spectroscopic, non-spectroscopic and computational method for analysis of protein–ligand binding are also discussed.

  6. A novel kinesin-like protein, KIF1Bbeta3 is involved in the movement of lysosomes to the cell periphery in non-neuronal cells.

    Science.gov (United States)

    Matsushita, Masafumi; Tanaka, Shingo; Nakamura, Norihiro; Inoue, Hiroki; Kanazawa, Hiroshi

    2004-03-01

    The kinesin superfamily protein, KIF1Bbeta, a splice variant of KIF1B, is involved in the transport of synaptic vesicles in neuronal cells, and is also expressed in various non-neuronal tissues. To elucidate the functions of KIF1Bbeta in non-neuronal cells, we analyzed the intracellular localization of KIF1Bbeta and characterized its isoform expression profile. In COS-7 cells, KIF1B colocalized with lysosomal markers and expression of a mutant form of KIF1Bbeta, lacking the motor domain, impaired the intracellular distribution of lysosomes. A novel isoform of the kinesin-like protein, KIF1Bbeta3, was identified in rat and simian kidney. It lacks the 5th exon of the KIF1Bbeta-specific tail region. Overexpression of KIF1Bbeta3 induced the translocation of lysosomes to the cell periphery. However, overexpression of KIF1Bbeta3-Q98L, which harbors a pathogenic mutation associated with a familial neuropathy, Charcot-Marie-Tooth disease type 2 A, resulted in the abnormal perinuclear clustering of lysosomes. These results indicate that KIF1Bbeta3 is involved in the translocation of lysosomes from perinuclear regions to the cell periphery.

  7. The involvement of wheat F-box protein gene TaFBA1 in the oxidative stress tolerance of plants.

    Directory of Open Access Journals (Sweden)

    Shu-Mei Zhou

    Full Text Available As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT. The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS accumulation, malondialdehyde (MDA content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD, catalase (CAT, ascorbate peroxidase (APX and peroxidase (POD, were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants' tolerance to multiple stress conditions.

  8. The light gene of Drosophila melanogaster encodes a homologue of VPS41, a yeast gene involved in cellular-protein trafficking.

    Science.gov (United States)

    Warner, T S; Sinclair, D A; Fitzpatrick, K A; Singh, M; Devlin, R H; Honda, B M

    1998-04-01

    Mutations in a number of genes affect eye colour in Drosophila melanogaster; some of these "eye-colour" genes have been shown to be involved in various aspects of cellular transport processes. In addition, combinations of viable mutant alleles of some of these genes, such as carnation (car) combined with either light (lt) or deep-orange (dor) mutants, show lethal interactions. Recently, dor was shown to be homologous to the yeast gene PEP3 (VPS18), which is known to be involved in intracellular trafficking. We have undertaken to extend our earlier work on the lt gene, in order to examine in more detail its expression pattern and to characterize its gene product via sequencing of a cloned cDNA. The gene appears to be expressed at relatively high levels in all stages and tissues examined, and shows strong homology to VPS41, a gene involved in cellular-protein trafficking in yeast and higher eukaryotes. Further genetic experiments also point to a role for lt in transport processes: we describe lethal interactions between viable alleles of lt and dor, as well as phenotypic interactions (reductions in eye pigment) between allels of lt and another eye-colour gene, garnet (g), whose gene product has close homology to a subunit of the human adaptor complex, AP-3.

  9. Hydrophobic patches on protein surfaces

    NARCIS (Netherlands)

    Lijnzaad, P.

    2007-01-01

    Hydrophobicity is a prime determinant of the structure and function of proteins. It is the driving force behind the folding of soluble proteins, and when exposed on the surface, it is frequently involved in recognition and binding of ligands and other proteins. The energetic cost of

  10. Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS.

    Science.gov (United States)

    Borsuk, Sibele; Newcombe, Jane; Mendum, Tom A; Dellagostin, Odir A; McFadden, Johnjoe

    2009-11-01

    The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.

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

    Science.gov (United States)

    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.

  12. Investigation the Response of Some Proteins That Involved in Cachexia Syndrome to Acute Resistance Exercise in Healthy Elderly People

    Directory of Open Access Journals (Sweden)

    Meysam Gholamali

    2015-01-01

    Full Text Available Objectives: The aim of this study was to investigate the response of plasma Myostatin and insulin growth factor like-1 (IGF-1, as two most important proteins that involved in Cachexia syndrome, to acute resistance exercise in healthy elderly people. Methods & Materials: Twelve healthy older men (Age=67±1.3 years, BMI=25±1.4 kg/m2 volunteered for participation in this study. 72 hours after the determination of muscular maximal strength (by 1-RM test, subjects participated in acute resistance exercises via 75% 1-RM. In this research, two blood samples were collected at before and immediately after the exercise from Antecubital vein. Plasma Myostatin and serum levels of IGF-1 were measured by ELISA methods. Paired T-Test used for statical analyses of research data. Significant level was set at P≤0.05. Results: The results of this study showed that plasma Myostatin significantly decreased in response to resistance exercise (P=0.0001. Also the serum levels of IGF-1 increased significantly in response to resistance exercise (P=0.0001. In turn, the results reveled that the IGF-1 to Myostatin ratio increased significantly in response to resistance exercise (P=0.001. Conclusion: The results of this study showed that resistance exercise through increases of IGF-1 and decreases of Myostatin causes increment of IGF-1 to Myostatin ratio. According to the results of this study it seems prescription of resistance exercise could positive changes in proteins that involved in Cachexia syndrome in elderly people. Presumably, through this way we can prevent from Cachexia and its many physiological and physical related dysfunctions in theses people. Although more study is needed to clear its mechanisms.

  13. Protein kinase C involvement in focal adhesion formation

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1992-01-01

    Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have...... still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form....... Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h...

  14. Yeast Interacting Proteins Database: YPR103W, YOR047C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available tein involved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sensors...gulated gene expression; interacts with protein kinase Snf1p, glucose sensors Snf

  15. Proteins involved in invasion of human red blood cells by malaria parasites

    Directory of Open Access Journals (Sweden)

    Ewa Jaśkiewicz

    2010-11-01

    Full Text Available Malaria is a disease caused by parasites of Plasmodium species. It is responsible for around 1-2 million deaths annually, mainly children under the age of 5. It occurs mainly in tropical and subtropical areas.Malaria is caused by five Plasmodium species:[i] P. falciparum, P. malariae, P. vivax, P. knowlesi[/i] and [i]P. ovale[/i]. Mosquitoes spread the disease by biting humans. The malaria parasite has two stages of development: the human stage and the mosquito stage. The first stage occurs in the human body and is divided into two phases: the liver phase and the blood phase.The invasion of erythrocytes by [i]Plasmodium[/i] merozoites is a multistep process of specific protein interactions between the parasite and red blood cell. The first step is the reversible merozoite attachment to the erythrocyte followed by its apical reorientation, then formation of an irreversible “tight” junction and finally entry into the red cell in a parasitophorous vacuole.The blood phase is supported by a number of proteins produced by the parasite. The merozoite surface GPI-anchored proteins (MSP-1, 2, 4, 5, 8 and 10 assist in the process of recognition of susceptible erythrocytes, apical membrane antigen (AMA-1 may be directly responsible for apical reorientation of the merozoite and apical proteins which function in tight junction formation. These ligands are members of two families: Duffy binding-like (DBL and reticulocyte binding-like (RBL proteins. In [i]Plasmodium[/i] [i]falciparum[/i] the DBL family includes: EBA-175, EBA-140 (BAEBL, EBA-181 (JESEBL, EBA-165 (PEBL and EBL-1 ligands.To date, no effective antimalarial vaccine has been developed, but there are several studies for this purpose. Therefore, it is crucial to understand the molecular basis of host cells invasion by parasites. Major efforts are focused on developing a multiantigenic and multiepitope vaccine preventing all steps of [i]Plasmodium[/i] invasion.

  16. Identification of Proteins Involved in Salinity Tolerance in Salicornia bigelovii

    KAUST Repository

    Salazar Moya, Octavio Ruben

    2017-11-01

    With a global growing demand in food production, agricultural output must increase accordingly. An increased use of saline soils and brackish water would contribute to the required increase in world food production. Abiotic stresses, such as salinity and drought, are also major limiters of crop growth globally - most crops are relatively salt sensitive and are significantly affected when exposed to salt in the range of 50 to 200 mM NaCl. Genomic resources from plants that naturally thrive in highly saline environments have the potential to be valuable in the generation of salt tolerant crops; however, these resources have been largely unexplored. Salicornia bigelovii is a plant native to Mexico and the United States that grows in salt marshes and coastal regions. It can thrive in environments with salt concentrations higher than seawater. In contrast to most crops, S. bigelovii is able to accumulate very high concentrations (in the order of 1.5 M) of Na+ and Cl- in its photosynthetically active succulent shoots. Part of this tolerance is likely to include the storage of Na+ in the vacuoles of the shoots, making S. bigelovii a good model for understanding mechanisms of Na+ compartmentalization in the vacuoles and a good resource for gene discovery. In this research project, phenotypic, genomic, transcriptomic, and proteomic approaches have been used for the identification of candidate genes involved in salinity tolerance in S. bigelovii. The genomes and transcriptomes of three Salicornia species have been sequenced. This information has been used to support the characterization of the salt-induced transcriptome of S. bigelovii shoots and the salt-induced proteome of various organellar membrane enriched fractions from S. bigelovii shoots, which led to the creation of organellar membrane proteomes. Yeast spot assays at different salt concentrations revealed several proteins increasing or decreasing yeast salt tolerance. This work aims to create the basis for

  17. Ubiquitin domain proteins in disease

    DEFF Research Database (Denmark)

    Klausen, Louise Kjær; Schulze, Andrea; Seeger, Michael

    2007-01-01

    The human genome encodes several ubiquitin-like (UBL) domain proteins (UDPs). Members of this protein family are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells. Despite...... and cancer. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com)....

  18. The relationship between protein synthesis and protein degradation in object recognition memory.

    Science.gov (United States)

    Furini, Cristiane R G; Myskiw, Jociane de C; Schmidt, Bianca E; Zinn, Carolina G; Peixoto, Patricia B; Pereira, Luiza D; Izquierdo, Ivan

    2015-11-01

    For decades there has been a consensus that de novo protein synthesis is necessary for long-term memory. A second round of protein synthesis has been described for both extinction and reconsolidation following an unreinforced test session. Recently, it was shown that consolidation and reconsolidation depend not only on protein synthesis but also on protein degradation by the ubiquitin-proteasome system (UPS), a major mechanism responsible for protein turnover. However, the involvement of UPS on consolidation and reconsolidation of object recognition memory remains unknown. Here we investigate in the CA1 region of the dorsal hippocampus the involvement of UPS-mediated protein degradation in consolidation and reconsolidation of object recognition memory. Animals with infusion cannulae stereotaxically implanted in the CA1 region of the dorsal hippocampus, were exposed to an object recognition task. The UPS inhibitor β-Lactacystin did not affect the consolidation and the reconsolidation of object recognition memory at doses known to affect other forms of memory (inhibitory avoidance, spatial learning in a water maze) while the protein synthesis inhibitor anisomycin impaired the consolidation and the reconsolidation of the object recognition memory. However, β-Lactacystin was able to reverse the impairment caused by anisomycin on the reconsolidation process in the CA1 region of the hippocampus. Therefore, it is possible to postulate a direct link between protein degradation and protein synthesis during the reconsolidation of the object recognition memory. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Maternal protein restriction compromises myocardial contractility in the young adult rat by changing proteins involved in calcium handling.

    Science.gov (United States)

    de Belchior, Aucelia C S; Freire, David D; da Costa, Carlos P; Vassallo, Dalton V; Padilha, Alessandra S; Dos Santos, Leonardo

    2016-02-01

    Maternal protein restriction (MPR) during pregnancy is associated with increased cardiovascular risk in the offspring in adulthood. In this study we evaluated the cardiac function of young male rats born from mothers subjected to MPR during pregnancy, focusing on the myocardial mechanics and calcium-handling proteins. After weaning, rats received normal diet until 3 mo old, when the following parameters were assessed: arterial and left ventricular hemodynamics and in vitro cardiac contractility in isolated papillary muscles. The body weight was lower and arterial pressure higher in the MPR group compared with young adult offspring of female rats that received standard diet (controls); and left ventricle time derivatives increased in the MPR group. The force developed by the cardiac muscle was similar; but time to peak and relaxation time were longer, and the derivatives of force were depressed in the MPR. In addition, MPR group exhibited decreased post-pause potentiation of force, suggesting reduced reuptake function of the sarcoplasmic reticulum. Corroborating, the myocardial content of SERCA-2a and phosphorylated PLB-Ser16/total PLB ratio was decreased and sodium-calcium exchanger was increased in the MPR group. The contraction dependent on transsarcolemmal influx of calcium was higher in MPR if compared with the control group. In summary, young rats born from mothers subjected to protein restriction during pregnancy exhibit changes in the myocardial mechanics with altered expression of calcium-handling proteins, reinforcing the hypothesis that maternal malnutrition is related to increased cardiovascular risk in the offspring, not only for hypertension, but also cardiac dysfunction. Copyright © 2016 the American Physiological Society.

  20. Protein disulfide isomerase-like protein 1-1 controls endosperm development through regulation of the amount and composition of seed proteins in rice.

    Directory of Open Access Journals (Sweden)

    Yeon Jeong Kim

    Full Text Available Protein disulfide isomerase (PDI is a chaperone protein involved in oxidative protein folding by acting as a catalyst and assisting folding in the endoplasmic reticulum (ER. A genome database search showed that rice contains 19 PDI-like genes. However, their functions are not clearly identified. This paper shows possible functions of rice PDI-like protein 1-1 (PDIL1-1 during seed development. Seeds of the T-DNA insertion PDIL1-1 mutant, PDIL1-1Δ, identified by genomic DNA PCR and western blot analysis, display a chalky phenotype and a thick aleurone layer. Protein content per seed was significantly lower and free sugar content higher in PDIL1-1Δ mutant seeds than in the wild type. Proteomic analysis of PDIL1-1Δ mutant seeds showed that PDIL1-1 is post-translationally regulated, and its loss causes accumulation of many types of seed proteins including glucose/starch metabolism- and ROS (reactive oxygen species scavenging-related proteins. In addition, PDIL1-1 strongly interacts with the cysteine protease OsCP1. Our data indicate that the opaque phenotype of PDIL1-1Δ mutant seeds results from production of irregular starch granules and protein body through loss of regulatory activity for various proteins involved in the synthesis of seed components.

  1. Involvement of protein kinase B and mitogen-activated protein kinases in experimental normothermic liver ischaemia-reperfusion injury.

    Science.gov (United States)

    Cursio, R; Filippa, N; Miele, C; Van Obberghen, E; Gugenheim, J

    2006-06-01

    This study evaluated the role of protein kinase B (PKB), phosphatidylinositol 3-kinase (PI3-K), Bcl-2-associated death protein (BAD) and mitogen-activated protein kinases (MAPKs) in normothermic ischaemia-reperfusion (IR)-induced apoptosis in rat liver. Rats were divided into two groups that received either phosphate-buffered saline (control) or the caspase inhibitor Z-Asp-2,6-dichorobenzoyloxymethylketone (Z-Asp-cmk), injected intravenously 2 min before the induction of 120 min of normothermic liver ischaemia. Liver apoptosis was assessed by the terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) method. PI3-K, PKB, BAD and MAPK activities were measured in ischaemic and non-ischaemic lobes at various times after reperfusion. The number of TUNEL-positive cells was significantly decreased after pretreatment with Z-Asp-cmk. In controls, PI3-K and PKB activities and BAD phosphorylation were inhibited in ischaemic liver lobes. The MAPKs (extracellular signal-regulated kinases, c-Jun N-terminal kinase and p38) showed different patterns of activation during IR. PKB activity was not modified by pretreatment with Z-Asp-cmk. Induction of apoptosis during IR liver injury might be triggered by inactivation of the antiapoptotic PI3-K-PKB pathway and activation of the proapoptotic MAPKs. Copyright (c) 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

  2. Biallelic mutation of UNC50, encoding a protein involved in AChR trafficking, is responsible for arthrogryposis.

    Science.gov (United States)

    Abiusi, Emanuela; D'Alessandro, Manuela; Dieterich, Klaus; Quevarec, Loic; Turczynski, Sandrina; Valfort, Aurore-Cecile; Mezin, Paulette; Jouk, Pierre Simon; Gut, Marta; Gut, Ivo; Bessereau, Jean Louis; Melki, Judith

    2017-10-15

    Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Homozygosity mapping of disease loci combined with whole exome sequencing in a consanguineous family presenting with lethal AMC allowed the identification of a homozygous frameshift deletion in UNC50 gene (c.750_751del:p.Cys251Phefs*4) in the index case. To assess the effect of the mutation, an equivalent mutation in the Caenorhabditis elegans orthologous gene was created using CRISPR/Cas9. We demonstrated that unc-50(kr331) modification caused the loss of acetylcholine receptor (AChR) expression in C. elegans muscle. unc-50(kr331) animals were as resistant to the cholinergic agonist levamisole as unc-50 null mutants suggesting that AChRs were no longer expressed in this animal model. This was confirmed by using a knock-in strain in which a red fluorescent protein was inserted into the AChR locus: no signal was detected in unc-50(kr331) background, suggesting that UNC-50, a protein known to be involved in AChR trafficking, was no longer functional. These data indicate that biallelic mutation in the UNC50 gene underlies AMC through a probable loss of AChR expression at the neuromuscular junction which is essential for the cholinergic transmission during human muscle development. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  3. Whereas Short-Term Facilitation Is Presynaptic, Intermediate-Term Facilitation Involves Both Presynaptic and Postsynaptic Protein Kinases and Protein Synthesis

    Science.gov (United States)

    Jin, Iksung; Kandel, Eric R.; Hawkins, Robert D.

    2011-01-01

    Whereas short-term plasticity involves covalent modifications that are generally restricted to either presynaptic or postsynaptic structures, long-term plasticity involves the growth of new synapses, which by its nature involves both pre- and postsynaptic alterations. In addition, an intermediate-term stage of plasticity has been identified that…

  4. Regulation of heterotrimeric G-protein signaling by NDPK/NME proteins and caveolins: an update.

    Science.gov (United States)

    Abu-Taha, Issam H; Heijman, Jordi; Feng, Yuxi; Vettel, Christiane; Dobrev, Dobromir; Wieland, Thomas

    2018-02-01

    Heterotrimeric G proteins are pivotal mediators of cellular signal transduction in eukaryotic cells and abnormal G-protein signaling plays an important role in numerous diseases. During the last two decades it has become evident that the activation status of heterotrimeric G proteins is both highly localized and strongly regulated by a number of factors, including a receptor-independent activation pathway of heterotrimeric G proteins that does not involve the classical GDP/GTP exchange and relies on nucleoside diphosphate kinases (NDPKs). NDPKs are NTP/NDP transphosphorylases encoded by the nme/nm23 genes that are involved in a variety of cellular events such as proliferation, migration, and apoptosis. They therefore contribute, for example, to tumor metastasis, angiogenesis, retinopathy, and heart failure. Interestingly, NDPKs are translocated and/or upregulated in human heart failure. Here we describe recent advances in the current understanding of NDPK functions and how they have an impact on local regulation of G-protein signaling.

  5. Microdomain forming proteins in oncogenesis

    Directory of Open Access Journals (Sweden)

    I. B. Zborovskaya

    2016-01-01

    Full Text Available Lipid rafts are lateral assembles of cholesterol, sphingomyelin, glicosphingolipids and specific proteins within cell plasma membrane. These microdomains are involved into a number of important cellular processes including membrane rearrangement, protein internalization, signal transduction, entry of viruses into the cell. Some of lipid rafts are stabilized by special microdomain-forming proteins such as caveolins, SPFH domain containing superfamily, tetraspanins, galectins, which maintain integrity of rafts and regulate signal transduction via forming of “signalosomes”. Involvement of the different lipid rafts is necessary in many situations such as binding of growth factors with their receptors, integrin regulation, cytoskeleton and extracellular matrix rearrangements, vesicular transport, etc. However, such classes of microdomain-forming proteins are still considered separately from each other. In this review we tried to perform complex analysis of microdomain-forming proteins in regulation of cancer assotiated processes.

  6. Comparative Proteomic Analysis Provides insight into the Key Proteins as Possible Targets Involved in Aspirin Inhibiting Biofilm Formation of Staphylococcus xylosus

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    Chang-Geng Xu

    2017-08-01

    Full Text Available Staphylococcus xylosus is an opportunistic pathogen that causes infection in humans and cow mastitis. And S. xylosus possesses a strong ability to form biofilms in vitro. As biofilm formation facilitates resistance to antimicrobial agents, the discovery of new medicinal properties for classic drugs is highly desired. Aspirin, which is the most common active component of non-steroidal anti-inflammatory compounds, affects the biofilm-forming capacity of various bacterial species. We have found that aspirin effectively inhibits biofilm formation of S. xylosus by Crystal violet (CV staining and scanning electron microscopy analyses. The present study sought to elucidate possible targets of aspirin in suppressing S. xylosus biofilm formation. Based on an isobaric tag for relative and absolute quantitation (iTRAQ fold-change of >1.2 or <0.8 (P-value < 0.05, 178 differentially expressed proteins, 111 down-regulated and 67 up-regulated, were identified after application of aspirin to cells at a 1/2 minimal inhibitory concentration. Gene ontology analysis indicated enrichment in metabolic processes for the majority of the differentially expressed proteins. We then used the Kyoto Encyclopedia of Genes and Genomes (KEGG pathway database to analyze a large number of differentially expressed proteins and identified genes involved in biosynthesis of amino acids pathway, carbon metabolism (pentose phosphate and glycolytic pathways, tricarboxylic acid cycle and nitrogen metabolism (histidine metabolism. These novel proteins represent candidate targets in aspirin-mediated inhibition of S. xylosus biofilm formation at sub-MIC levels. The findings lay the foundation for further studies to identify potential aspirin targets.

  7. Enhanced expression of WD repeat-containing protein 35 (WDR35 stimulated by domoic acid in rat hippocampus: involvement of reactive oxygen species generation and p38 mitogen-activated protein kinase activation

    Directory of Open Access Journals (Sweden)

    Tsunekawa Koji

    2013-01-01

    Full Text Available Abstract Background Domoic acid (DA is an excitatory amino acid analogue of kainic acid (KA that acts via activation of glutamate receptors to elicit a rapid and potent excitotoxic response, resulting in neuronal cell death. Recently, DA was shown to elicit reactive oxygen species (ROS production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK in vitro. We have reported that WDR35, a WD-repeat protein, may mediate apoptosis in several animal models. In the present study, we administered DA to rats intraperitoneally, then used liquid chromatography/ion trap tandem mass spectrometry (LC-MS/MS to identify and quantify DA in the brains of the rats and performed histological examinations of the hippocampus. We further investigated the potential involvement of glutamate receptors, ROS, p38 MAPK, and WDR35 in DA-induced toxicity in vivo. Results Our results showed that intraperitoneally administered DA was present in the brain and induced neurodegenerative changes including apoptosis in the CA1 region of the hippocampus. DA also increased the expression of WDR35 mRNA and protein in a dose- and time-dependent manner in the hippocampus. In experiments using glutamate receptor antagonists, the AMPA/KA receptor antagonist NBQX significantly attenuated the DA-induced increase in WDR35 protein expression, but the NMDA receptor antagonist MK-801 did not. In addition, the radical scavenger edaravone significantly attenuated the DA-induced increase in WDR35 protein expression. Furthermore, NBQX and edaravone significantly attenuated the DA-induced increase in p38 MAPK phosphorylation. Conclusion In summary, our results indicated that DA activated AMPA/KA receptors and induced ROS production and p38 MAPK phosphorylation, resulting in an increase in the expression of WDR35 in vivo.

  8. Dendrimers destabilize proteins in a generation-dependent manner involving electrostatic interactions

    DEFF Research Database (Denmark)

    Gichm, Lise; Christensen, Casper; Boas, Ulrik

    2008-01-01

    Dendrimers are well-defined chemical polymers with a characteristic branching pattern that gives rise to attractive features such as antibacterial and antitumor activities as well as drug delivery properties. In addition, dendrimers can solubilize prion protein aggregates at very low concentratio...

  9. One of the Two Genes Encoding Nucleoid-Associated HU Proteins in Streptomyces coelicolor Is Developmentally Regulated and Specifically Involved in Spore Maturation▿ †

    Science.gov (United States)

    Salerno, Paola; Larsson, Jessica; Bucca, Giselda; Laing, Emma; Smith, Colin P.; Flärdh, Klas

    2009-01-01

    Streptomyces genomes encode two homologs of the nucleoid-associated HU proteins. One of them, here designated HupA, is of a conventional type similar to E. coli HUα and HUβ, while the other, HupS, is a two-domain protein. In addition to the N-terminal part that is similar to that of HU proteins, it has a C-terminal domain that is similar to the alanine- and lysine-rich C termini of eukaryotic linker histones. Such two-domain HU proteins are found only among Actinobacteria. In this phylum some organisms have only a single HU protein of the type with a C-terminal histone H1-like domain (e.g., Hlp in Mycobacterium smegmatis), while others have only a single conventional HU. Yet others, including the streptomycetes, produce both types of HU proteins. We show here that the two HU genes in Streptomyces coelicolor are differentially regulated and that hupS is specifically expressed during sporulation, while hupA is expressed in vegetative hyphae. The developmental upregulation of hupS occurred in sporogenic aerial hyphal compartments and was dependent on the developmental regulators whiA, whiG, and whiI. HupS was found to be nucleoid associated in spores, and a hupS deletion mutant had an average nucleoid size in spores larger than that in the parent strain. The mutant spores were also defective in heat resistance and spore pigmentation, although they possessed apparently normal spore walls and displayed no increased sensitivity to detergents. Overall, the results show that HupS is specifically involved in sporulation and may affect nucleoid architecture and protection in spores of S. coelicolor. PMID:19717607

  10. Quality control methodology for high-throughput protein-protein interaction screening.

    Science.gov (United States)

    Vazquez, Alexei; Rual, Jean-François; Venkatesan, Kavitha

    2011-01-01

    Protein-protein interactions are key to many aspects of the cell, including its cytoskeletal structure, the signaling processes in which it is involved, or its metabolism. Failure to form protein complexes or signaling cascades may sometimes translate into pathologic conditions such as cancer or neurodegenerative diseases. The set of all protein interactions between the proteins encoded by an organism constitutes its protein interaction network, representing a scaffold for biological function. Knowing the protein interaction network of an organism, combined with other sources of biological information, can unravel fundamental biological circuits and may help better understand the molecular basics of human diseases. The protein interaction network of an organism can be mapped by combining data obtained from both low-throughput screens, i.e., "one gene at a time" experiments and high-throughput screens, i.e., screens designed to interrogate large sets of proteins at once. In either case, quality controls are required to deal with the inherent imperfect nature of experimental assays. In this chapter, we discuss experimental and statistical methodologies to quantify error rates in high-throughput protein-protein interactions screens.

  11. The Orosomucoid 1 protein is involved in the vitamin D – mediated macrophage de-activation process

    Energy Technology Data Exchange (ETDEWEB)

    Gemelli, Claudia, E-mail: claudia.gemelli@unimore.it [Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41125 Modena (Italy); Center for Regenerative Medicine, University of Modena and Reggio Emilia, Via Gottardi 100, 41125 Modena (Italy); Martello, Andrea; Montanari, Monica; Zanocco Marani, Tommaso; Salsi, Valentina; Zappavigna, Vincenzo; Parenti, Sandra; Vignudelli, Tatiana; Selmi, Tommaso; Ferrari, Sergio; Grande, Alexis [Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41125 Modena (Italy)

    2013-12-10

    Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1 kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3 – VDR – ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling. - Highlights: • ORM1 is a Vitamin D primary response gene. • VD and its receptor VDR are involved in the de-activation process mediated by human resident macrophages. • The signaling pathway VD-VDR-ORM1 plays an important role in the control of macrophage de-activation process. • ORM1 may be defined as a signaling molecule implicated in the maintenance of tissue homeostasis and remodeling.

  12. The Orosomucoid 1 protein is involved in the vitamin D – mediated macrophage de-activation process

    International Nuclear Information System (INIS)

    Gemelli, Claudia; Martello, Andrea; Montanari, Monica; Zanocco Marani, Tommaso; Salsi, Valentina; Zappavigna, Vincenzo; Parenti, Sandra; Vignudelli, Tatiana; Selmi, Tommaso; Ferrari, Sergio; Grande, Alexis

    2013-01-01

    Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1 kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3 – VDR – ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling. - Highlights: • ORM1 is a Vitamin D primary response gene. • VD and its receptor VDR are involved in the de-activation process mediated by human resident macrophages. • The signaling pathway VD-VDR-ORM1 plays an important role in the control of macrophage de-activation process. • ORM1 may be defined as a signaling molecule implicated in the maintenance of tissue homeostasis and remodeling

  13. Interaction between plate make and protein in protein crystallisation screening.

    Directory of Open Access Journals (Sweden)

    Gordon J King

    Full Text Available BACKGROUND: Protein crystallisation screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate. METHODOLOGY/PRINCIPAL FINDINGS: We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised. CONCLUSIONS/SIGNIFICANCE: Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallize, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make.

  14. DNA-Damage Response RNA-Binding Proteins (DDRBPs): Perspectives from a New Class of Proteins and Their RNA Targets.

    Science.gov (United States)

    Dutertre, Martin; Vagner, Stéphan

    2017-10-27

    Upon DNA damage, cells trigger an early DNA-damage response (DDR) involving DNA repair and cell cycle checkpoints, and late responses involving gene expression regulation that determine cell fate. Screens for genes involved in the DDR have found many RNA-binding proteins (RBPs), while screens for novel RBPs have identified DDR proteins. An increasing number of RBPs are involved in early and/or late DDR. We propose to call this new class of actors of the DDR, which contain an RNA-binding activity, DNA-damage response RNA-binding proteins (DDRBPs). We then discuss how DDRBPs contribute not only to gene expression regulation in the late DDR but also to early DDR signaling, DNA repair, and chromatin modifications at DNA-damage sites through interactions with both long and short noncoding RNAs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Seasonal patterns of ascorbate in the needles of Scots Pine (Pinus sylvestris L.) trees: Correlation analyses with atmospheric O3 and NO2 gas mixing ratios and meteorological parameters

    International Nuclear Information System (INIS)

    Haberer, Kristine; Jaeger, Lutz; Rennenberg, Heinz

    2006-01-01

    In the present field study the role of ascorbate in scavenging the harmful atmospheric trace gases O 3 and NO 2 was examined. For this purpose ascorbate contents were determined in needles of adult Scots pine trees (Pinus sylvestris L.) during three consecutive years. Ascorbate contents were correlated with ambient tropospheric O 3 and NO 2 concentrations and with meteorological parameters. The results showed a strong correlation of atmospheric O 3 but not of atmospheric NO 2 concentrations with the apoplastic content of ascorbate during the seasonal course. Ascorbate contents in needle extracts did not correlate with ambient trace gas concentrations. In the apoplastic space, but not in needle extracts ascorbate contents correlate highly significantly with global radiation. From these results it is assumed that apoplastic ascorbate in Scots pine needles is adapted to the actual atmospheric O 3 concentration to mediate immediate detoxification of O 3 , while the atmospheric O 3 concentration itself is largely dependent on light intensity. - Contents of apoplastic but not symplastic ascorbate correlate significantly with atmospheric ozone concentrations

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Benjamin Clémençon

    2012-02-01

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

  18. Comparative Analyses of Tomato yellow leaf curl virus C4 Protein-Interacting Host Proteins in Healthy and Infected Tomato Tissues

    Directory of Open Access Journals (Sweden)

    Namgyu Kim

    2016-10-01

    Full Text Available Tomato yellow leaf curl virus (TYLCV, a member of the genus Begomovirus, is one of the most important viruses of cultivated tomatoes worldwide, mainly causing yellowing and curling of leaves with stunting in plants. TYLCV causes severe problems in sub-tropical and tropical countries, as well as in Korea. However, the mechanism of TYLCV infection remains unclear, although the function of each viral component has been identified. TYLCV C4 codes for a small protein involved in various cellular functions, including symptom determination, gene silencing, viral movement, and induction of the plant defense response. In this study, through yeast-two hybrid screenings, we identified TYLCV C4-interacting host proteins from both healthy and symptom-exhibiting tomato tissues, to determine the role of TYLCV C4 proteins in the infection processes. Comparative analyses of 28 proteins from healthy tissues and 36 from infected tissues showing interactions with TYLCV C4 indicated that TYLCV C4 mainly interacts with host proteins involved in translation, ubiquitination, and plant defense, and most interacting proteins differed between the two tissues but belong to similar molecular functional categories. Four proteins—two ribosomal proteins, S-adenosyl-L-homocysteine hydrolase, and 14-3-3 family protein—were detected in both tissues. Furthermore, the identified proteins in symptom-exhibiting tissues showed greater involvement in plant defenses. Some are key regulators, such as receptor-like kinases and pathogenesis-related proteins, of plant defenses. Thus, TYLCV C4 may contribute to the suppression of host defense during TYLCV infection and be involved in ubiquitination for viral infection.

  19. Characterization of Carbohydrate Active Enzymes Involved in Arabinogalactan Protein Metabolism

    DEFF Research Database (Denmark)

    Knoch, Eva

    and tissues, their functions and synthesis are still poorly understood. The aim of the research presented in the thesis was to characterize carbohydrate active enzymes involved in AGP biosynthesis and modification to gain insights into the biosynthesis of the glycoproteins in plants. Candidate...... glycosyltransferases and glycoside hydrolases were selected based on co-expression profiles from a transcriptomics analysis. Reverse genetics approach on a novel glucuronosyltransferase involved in AGP biosynthesis has revealed that the enzyme activity is required for normal cell elongation in etiolated seedlings....... The enzymatic activity of a hydrolase from GH family 17 was investigated, without successful determination of the activity. Members of hydrolase family 43 appeared to be localized in the Golgi-apparatus, which is also the compartment for glycan biosynthesis. The localization of these glycoside hydrolases...

  20. Protein Thiols as an Indication of Oxidative Stress

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    Yousef Rezaei Chianeh

    2014-06-01

    Full Text Available Thiol is an organic compound that contain sulphhydryl group that have a critical role in preventing any involvement of oxidative stress in the cell. These defensive functions are generally considered to be carried out by the low molecular weight thiol glutathione and by cysteine residues in the active sites of proteins such as thioredoxin and peroxiredoxin. In addition, there are thiols exposed on protein surfaces that are not directly involved with protein function, although they can interact with the intracellular environment.The process of protection of the cell against an oxidative damage occur by thiol and cystein residue that has a low molecular weight. These residue are present in the active sites of a protein like, peroxiredoxin and thioredoxin. Apart from intracellular antioxidant defense mechanism by protein thiol, there are presence of thiol in outer surface of protein that are not involved with the function of protein, even though they can interact with intracellular part of the cell. [Archives Medical Review Journal 2014; 23(3.000: 443-456

  1. Prioritizing disease candidate proteins in cardiomyopathy-specific protein-protein interaction networks based on "guilt by association" analysis.

    Directory of Open Access Journals (Sweden)

    Wan Li

    Full Text Available The cardiomyopathies are a group of heart muscle diseases which can be inherited (familial. Identifying potential disease-related proteins is important to understand mechanisms of cardiomyopathies. Experimental identification of cardiomyophthies is costly and labour-intensive. In contrast, bioinformatics approach has a competitive advantage over experimental method. Based on "guilt by association" analysis, we prioritized candidate proteins involving in human cardiomyopathies. We first built weighted human cardiomyopathy-specific protein-protein interaction networks for three subtypes of cardiomyopathies using the known disease proteins from Online Mendelian Inheritance in Man as seeds. We then developed a method in prioritizing disease candidate proteins to rank candidate proteins in the network based on "guilt by association" analysis. It was found that most candidate proteins with high scores shared disease-related pathways with disease seed proteins. These top ranked candidate proteins were related with the corresponding disease subtypes, and were potential disease-related proteins. Cross-validation and comparison with other methods indicated that our approach could be used for the identification of potentially novel disease proteins, which may provide insights into cardiomyopathy-related mechanisms in a more comprehensive and integrated way.

  2. Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

    International Nuclear Information System (INIS)

    Yu, P.

    2007-01-01

    The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities) in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behavior and nutrient utilization and availability. The emphasis of this review was on (1) using the newly advanced synchrotron technology (S-FTIR) as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2) revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3) prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4) obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.

  3. Targeted proteins involved in the neuroprotective effects of lithium citrate

    OpenAIRE

    I. Yu. Torshin; O. A. Gromova; L. A. Mayorova; A. Yu. Volkov

    2017-01-01

    Preparations based on organic lithium salts are promising neuroprotective agents that are effective just in the micromolar concentration range and, at the same time, have high safety (Toxicity Class V).Objective: to elucidate more detailed mechanisms responsible for the biological and pharmacological effects of lithium citrate, by analyzing the possible interactions of lithium ion with human proteome proteins that are also represented in the rat proteome.Material and methods. The targets of l...

  4. Involvement of a velvet protein FgVeA in the regulation of asexual development, lipid and secondary metabolisms and virulence in Fusarium graminearum.

    Directory of Open Access Journals (Sweden)

    Jinhua Jiang

    Full Text Available The velvet protein, VeA, is involved in the regulation of diverse cellular processes. In this study, we explored functions of FgVeA in the wheat head blight pathogen, Fusarium graminearum,using a gene replacement strategy. The FgVEA deletion mutant exhibited a reduction in aerial hyphae formation, hydrophobicity, and deoxynivalenol (DON biosynthesis. Deletion of FgVEA gene led to an increase in conidial production, but a delay in conidial germination. Pathogencity assays showed that the mutant was impaired in virulence on flowering wheat head. Sensitivity tests to various stresses exhibited that the FgVEA deletion mutant showed increased resistance to osmotic stress and cell wall-damaging agents, but increased sensitivity to iprodione and fludioxonil fungicides. Ultrastructural and histochemical analyses revealed that conidia of FgVeA deletion mutant contained an unusually high number of large lipid droplets, which is in agreement with the observation that the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Serial analysis of gene expression (SAGE in the FgVEA mutant confirmed that FgVeA was involved in various cellular processes. Additionally, six proteins interacting with FgVeA were identified by yeast two hybrid assays in current study. These results indicate that FgVeA plays a critical role in a variety of cellular processes in F. graminearum.

  5. BraLTP1, a lipid transfer protein gene involved in epicuticular wax deposition, cell proliferation and flower development in Brassica napus.

    Directory of Open Access Journals (Sweden)

    Fang Liu

    Full Text Available Plant non-specific lipid transfer proteins (nsLTPs constitute large multigene families that possess complex physiological functions, many of which remain unclear. This study isolated and characterized the function of a lipid transfer protein gene, BraLTP1 from Brassica rapa, in the important oilseed crops Brassica napus. BraLTP1 encodes a predicted secretory protein, in the little known VI Class of nsLTP families. Overexpression of BnaLTP1 in B. napus caused abnormal green coloration and reduced wax deposition on leaves and detailed wax analysis revealed 17-80% reduction in various major wax components, which resulted in significant water-loss relative to wild type. BnaLTP1 overexpressing leaves exhibited morphological disfiguration and abaxially curled leaf edges, and leaf cross-sections revealed cell overproliferation that was correlated to increased cytokinin levels (tZ, tZR, iP, and iPR in leaves and high expression of the cytokinin biosynthsis gene IPT3. BnaLTP1-overexpressing plants also displayed morphological disfiguration of flowers, with early-onset and elongated carpel development and outwardly curled stamen. This was consistent with altered expression of a a number of ABC model genes related to flower development. Together, these results suggest that BraLTP1 is a new nsLTP gene involved in wax production or deposition, with additional direct or indirect effects on cell division and flower development.

  6. Effect of initial protein concentration and pH on in vitro gastric digestion of heated whey proteins.

    Science.gov (United States)

    Zhang, Sha; Vardhanabhuti, Bongkosh

    2014-02-15

    The in vitro digestion of heated whey protein aggregates having different structure and physicochemical properties was evaluated under simulated gastric conditions. Aggregates were formed by heating whey protein isolates (WPI) at 3-9% w/w initial protein concentration and pH 3.0-7.0. Results showed that high protein concentration led to formation of larger WPI aggregates with fewer remaining monomers. Aggregates formed at high protein concentrations showed slower degradation rate compared to those formed at low protein concentration. The effect of initial protein concentration on peptide release pattern was not apparent. Heating pH was a significant factor affecting digestion pattern. At pH above the isoelectric point, the majority of the proteins involved in the aggregation, and aggregates formed at pH 6.0 were more susceptible to pepsin digestion than at pH 7.0. At acidic conditions, only small amount of proteins was involved in the aggregation and heated aggregates were easily digested by pepsin, while the remaining unaggregated proteins were very resistant to gastric digestion. The potential physiological implication of these results on satiety was discussed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin.

    Science.gov (United States)

    Küberl, Andreas; Polen, Tino; Bott, Michael

    2016-04-26

    The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded by a dedicated AAA+ ATPase (Mycobacterium proteasomal AAA+ ATPase; ATPase forming ring-shaped complexes). In Mycobacteria, degradation of pupylated proteins by the proteasome serves as a protection mechanism against several stress conditions. Other bacterial genera capable of pupylation such as Corynebacterium lack a proteasome, and the fate of pupylated proteins is unknown. We discovered that Corynebacterium glutamicum mutants lacking components of the pupylation machinery show a strong growth defect under iron limitation, which was caused by the absence of pupylation and unfolding of the iron storage protein ferritin. Genetic and biochemical data support a model in which the pupylation machinery is responsible for iron release from ferritin independent of degradation.

  8. Towards a map of the Populus biomass protein-protein interaction network

    Energy Technology Data Exchange (ETDEWEB)

    Beers, Eric [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Brunner, Amy [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Helm, Richard [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Dickerman, Allan [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States)

    2015-07-31

    Biofuels can be produced from a variety of plant feedstocks. The value of a particular feedstock for biofuels production depends in part on the degree of difficulty associated with the extraction of fermentable sugars from the plant biomass. The wood of trees is potentially a rich source fermentable sugars. However, the sugars in wood exist in a tightly cross-linked matrix of cellulose, hemicellulose, and lignin, making them largely recalcitrant to release and fermentation for biofuels production. Before breeders and genetic engineers can effectively develop plants with reduced recalcitrance to fermentation, it is necessary to gain a better understanding of the fundamental biology of the mechanisms responsible for wood formation. Regulatory, structural, and enzymatic proteins are required for the complicated process of wood formation. To function properly, proteins must interact with other proteins. Yet, very few of the protein-protein interactions necessary for wood formation are known. The main objectives of this project were to 1) identify new protein-protein interactions relevant to wood formation, and 2) perform in-depth characterizations of selected protein-protein interactions. To identify relevant protein-protein interactions, we cloned a set of approximately 400 genes that were highly expressed in the wood-forming tissue (known as secondary xylem) of poplar (Populus trichocarpa). We tested whether the proteins encoded by these biomass genes interacted with each other in a binary matrix design using the yeast two-hybrid (Y2H) method for protein-protein interaction discovery. We also tested a subset of the 400 biomass proteins for interactions with all proteins present in wood-forming tissue of poplar in a biomass library screen design using Y2H. Together, these two Y2H screens yielded over 270 interactions involving over 75 biomass proteins. For the second main objective we selected several interacting pairs or groups of interacting proteins for in

  9. Prediction of protein–protein interactions: unifying evolution and structure at protein interfaces

    International Nuclear Information System (INIS)

    Tuncbag, Nurcan; Gursoy, Attila; Keskin, Ozlem

    2011-01-01

    The vast majority of the chores in the living cell involve protein–protein interactions. Providing details of protein interactions at the residue level and incorporating them into protein interaction networks are crucial toward the elucidation of a dynamic picture of cells. Despite the rapid increase in the number of structurally known protein complexes, we are still far away from a complete network. Given experimental limitations, computational modeling of protein interactions is a prerequisite to proceed on the way to complete structural networks. In this work, we focus on the question 'how do proteins interact?' rather than 'which proteins interact?' and we review structure-based protein–protein interaction prediction approaches. As a sample approach for modeling protein interactions, PRISM is detailed which combines structural similarity and evolutionary conservation in protein interfaces to infer structures of complexes in the protein interaction network. This will ultimately help us to understand the role of protein interfaces in predicting bound conformations

  10. The cation diffusion facilitator proteins MamB and MamM of Magnetospirillum gryphiswaldense have distinct and complex functions, and are involved in magnetite biomineralization and magnetosome membrane assembly

    DEFF Research Database (Denmark)

    Uebe, René; Junge, Katja; Henn, Verena

    2011-01-01

    Magnetotactic bacteria form chains of intracellular membrane‐enclosed, nanometre‐sized magnetite crystals for navigation along the earth's magnetic field. The assembly of these prokaryotic organelles requires several specific polypeptides. Among the most abundant proteins associated with the magn......Magnetotactic bacteria form chains of intracellular membrane‐enclosed, nanometre‐sized magnetite crystals for navigation along the earth's magnetic field. The assembly of these prokaryotic organelles requires several specific polypeptides. Among the most abundant proteins associated...... with the magnetosome membrane of Magnetospirillum gryphiswaldense are MamB and MamM, which were implicated in magnetosomal iron transport because of their similarity to the cation diffusion facilitator family. Here we demonstrate that MamB and MamM are multifunctional proteins involved in several steps of magnetosome...

  11. Microsecond molecular dynamics simulations of intrinsically disordered proteins involved in the oxidative stress response.

    Directory of Open Access Journals (Sweden)

    Elio A Cino

    Full Text Available Intrinsically disordered proteins (IDPs are abundant in cells and have central roles in protein-protein interaction networks. Interactions between the IDP Prothymosin alpha (ProTα and the Neh2 domain of Nuclear factor erythroid 2-related factor 2 (Nrf2, with a common binding partner, Kelch-like ECH-associated protein 1(Keap1, are essential for regulating cellular response to oxidative stress. Misregulation of this pathway can lead to neurodegenerative diseases, premature aging and cancer. In order to understand the mechanisms these two disordered proteins employ to bind to Keap1, we performed extensive 0.5-1.0 microsecond atomistic molecular dynamics (MD simulations and isothermal titration calorimetry experiments to investigate the structure/dynamics of free-state ProTα and Neh2 and their thermodynamics of bindings. The results show that in their free states, both ProTα and Neh2 have propensities to form bound-state-like β-turn structures but to different extents. We also found that, for both proteins, residues outside the Keap1-binding motifs may play important roles in stabilizing the bound-state-like structures. Based on our findings, we propose that the binding of disordered ProTα and Neh2 to Keap1 occurs synergistically via preformed structural elements (PSEs and coupled folding and binding, with a heavy bias towards PSEs, particularly for Neh2. Our results provide insights into the molecular mechanisms Neh2 and ProTα bind to Keap1, information that is useful for developing therapeutics to enhance the oxidative stress response.

  12. Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly.

    Science.gov (United States)

    Chereji, Razvan V; Bharatula, Vasudha; Elfving, Nils; Blomberg, Jeanette; Larsson, Miriam; Morozov, Alexandre V; Broach, James R; Björklund, Stefan

    2017-09-06

    Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Saccharomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3'-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. The Fas-associated death domain protein/caspase-8/c-FLIP signaling pathway is involved in TNF-induced activation of ERK

    International Nuclear Information System (INIS)

    Lueschen, Silke; Falk, Markus; Scherer, Gudrun; Ussat, Sandra; Paulsen, Maren; Adam-Klages, Sabine

    2005-01-01

    The cytokine TNF activates multiple signaling pathways leading to cellular responses ranging from proliferation and survival to apoptosis. While most of these pathways have been elucidated in detail over the past few years, the molecular mechanism leading to the activation of the MAP kinases ERK remains ill defined and is controversially discussed. Therefore, we have analyzed TNF-induced ERK activation in various human and murine cell lines and show that it occurs in a cell-type-specific manner. In addition, we provide evidence for the involvement of the signaling components Fas-associated death domain protein (FADD), caspase-8, and c-FLIP in the pathway activating ERK in response to TNF. This conclusion is based on the following observations: (I) Overexpression of FADD, caspase-8, or a c-FLIP protein containing the death effector domains only leads to enhanced and prolonged ERK activation after TNF treatment. (II) TNF-induced ERK activation is strongly diminished in the absence of FADD. Interestingly, the enzymatic function of caspase-8 is not required for TNF-induced ERK activation. Additional evidence suggests a role for this pathway in the proliferative response of murine fibroblasts to TNF

  14. Determination of subcellular concentrations of soluble carbohydrates in rose petals during opening by nonaqueous fractionation method combined with infiltration-centrifugation method.

    Science.gov (United States)

    Yamada, Kunio; Norikoshi, Ryo; Suzuki, Katsumi; Imanishi, Hideo; Ichimura, Kazuo

    2009-11-01

    Petal growth associated with flower opening depends on cell expansion. To understand the role of soluble carbohydrates in petal cell expansion during flower opening, changes in soluble carbohydrate concentrations in vacuole, cytoplasm and apoplast of petal cells during flower opening in rose (Rosa hybrida L.) were investigated. We determined the subcellular distribution of soluble carbohydrates by combining nonaqueous fractionation method and infiltration-centrifugation method. During petal growth, fructose and glucose rapidly accumulated in the vacuole, reaching a maximum when petals almost reflected. Transmission electron microscopy showed that the volume of vacuole and air space drastically increased with petal growth. Carbohydrate concentration was calculated for each compartment of the petal cells and in petals that almost reflected, glucose and fructose concentrations increased to higher than 100 mM in the vacuole. Osmotic pressure increased in apoplast and symplast during flower opening, and this increase was mainly attributed to increases in fructose and glucose concentrations. No large difference in osmotic pressure due to soluble carbohydrates was observed between the apoplast and symplast before flower opening, but total osmotic pressure was much higher in the symplast than in the apoplast, a difference that was partially attributed to inorganic ions. An increase in osmotic pressure due to the continued accumulation of glucose and fructose in the symplast may facilitate water influx into cells, contributing to cell expansion associated with flower opening under conditions where osmotic pressure is higher in the symplast than in the apoplast.

  15. Interaction of translationally controlled tumor protein with Apaf-1 is involved in the development of chemoresistance in HeLa cells

    International Nuclear Information System (INIS)

    Jung, Jaehoon; Kim, Hyo Young; Maeng, Jeehye; Kim, Moonhee; Shin, Dong Hae; Lee, Kyunglim

    2014-01-01

    Translationally controlled tumor protein (TCTP), alternatively called fortilin, is believed to be involved in the development of the chemoresistance of tumor cells against anticancer drugs such as etoposide, taxol, and oxaliplatin, the underlying mechanisms of which still remain elusive. Cell death analysis of TCTP-overexpressing HeLa cells was performed following etoposide treatment to assess the mitochondria-dependent apoptosis. Apoptotic pathway was analyzed through measuring the cleavage of epidermal growth factor receptor (EGFR) and phospholipase C-γ (PLC-γ), caspase activation, mitochondrial membrane perturbation, and cytochrome c release by flow cytometry and western blotting. To clarify the role of TCTP in the inhibition of apoptosome, in vitro apoptosome reconstitution and immunoprecipitation was used. Pull-down assay and silver staining using the variants of Apaf-1 protein was applied to identify the domain that is responsible for its interaction with TCTP. In the present study, we confirmed that adenoviral overexpression of TCTP protects HeLa cells from cell death induced by cytotoxic drugs such as taxol and etoposide. TCTP antagonized the mitochondria-dependent apoptotic pathway following etoposide treatment, including mitochondrial membrane damage and resultant cytochrome c release, activation of caspase-9, and -3, and eventually, the cleavage of EGFR and PLC-γ. More importantly, TCTP interacts with the caspase recruitment domain (CARD) of Apaf-1 and is incorporated into the heptameric Apaf-1 complex, and that C-terminal cleaved TCTP specifically associates with Apaf-1 of apoptosome in apoptosome-forming condition thereby inhibiting the amplification of caspase cascade. TCTP protects the cancer cells from etoposide-induced cell death by inhibiting the mitochondria-mediated apoptotic pathway. Interaction of TCTP with Apaf-1 in apoptosome is involved in the molecular mechanism of TCTP-induced chemoresistance. These findings suggest that TCTP may serve

  16. Glucagon-Like Peptide 2 Stimulates Postresection Intestinal Adaptation in Preterm Pigs by Affecting Proteins Related to Protein, Carbohydrate, and Sulphur Metabolism

    DEFF Research Database (Denmark)

    Jiang, Pingping; Vegge, Andreas; Thymann, Thomas

    2017-01-01

    cellular structural proteins, while the added GLP-2 treatment affected proteins involved in protein processing and the metabolism of protein, carbohydrate, and sulphur. CONCLUSION: In the first days following resection, proteins affected by resection plus GLP-2 treatment differed markedly from those...

  17. A member of a new plant gene family encoding a meprin and TRAF homology (MATH) domain-containing protein is involved in restriction of long distance movement of plant viruses

    Science.gov (United States)

    Cosson, Patrick; Sofer, Luc; Schurdi-Levraud, Valérie

    2010-01-01

    Restriction of long distance movement of several potyviruses in Arabidopsis thaliana is controlled by at least three dominant restricted TEV movement (RTM) genes, named RTM1, RTM2 and RTM3 and acts as a non-conventional resistance. RTM1 encodes a protein belonging to the jacalin family and RTM2 encodes a protein which has similarities to small heat shock proteins. The recent cloning of RTM3 which encodes a protein belonging to an unknown protein family of 29 members that has a meprin and TRAF homology (MATH) domain in its N-terminal region and a coiled-coil (CC) domain at its C-terminal end is an important breakthrough for a better understanding of this resistance process. Not only the third gene involved in this resistance has been identified and has allowed revealing a new gene family in plant but the discovery that the RTM3 protein interacts directly with RTM1 strongly suggests that the RTM proteins form a multimeric complex. However, these data also highlight striking similarities of the RTM resistance with the well known R-gene mediated resistance. PMID:20930558

  18. Tritium Suicide Selection Identifies Proteins Involved in the Uptake and Intracellular Transport of Sterols in Saccharomyces cerevisiae▿

    Science.gov (United States)

    Sullivan, David P.; Georgiev, Alexander; Menon, Anant K.

    2009-01-01

    Sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) occurs by a nonvesicular mechanism that is poorly understood. To identify proteins required for this process, we isolated Saccharomyces cerevisiae mutants with defects in sterol transport. We used Upc2-1 cells that have the ability to take up sterols under aerobic conditions and exploited the observation that intracellular accumulation of exogenously supplied [3H]cholesterol in the form of [3H]cholesteryl ester requires an intact PM-ER sterol transport pathway. Upc2-1 cells were mutagenized using a transposon library, incubated with [3H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [3H]cholesterol. Many of the mutants had defects in the expression and trafficking of Aus1 and Pdr11, PM-localized ABC transporters that are required for sterol uptake. Through characterization of one of the mutants, a new role was uncovered for the transcription factor Mot3 in controlling expression of Aus1 and Pdr11. A number of mutants had transposon insertions in the uncharacterized Ydr051c gene, which we now refer to as DET1 (decreased ergosterol transport). These mutants expressed Aus1 and Pdr11 normally but were severely defective in the ability to accumulate exogenously supplied cholesterol. The transport of newly synthesized sterols from the ER to the PM was also defective in det1Δ cells. These data indicate that the cytoplasmic protein encoded by DET1 is involved in intracellular sterol transport. PMID:19060182

  19. Involvement of C-Terminal Histidines in Soybean PM1 Protein Oligomerization and Cu2+ Binding.

    Science.gov (United States)

    Liu, Guobao; Liu, Ke; Gao, Yang; Zheng, Yizhi

    2017-06-01

    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. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  20. Small G proteins Rac1 and Ras regulate serine/threonine protein phosphatase 5 (PP5)·extracellular signal-regulated kinase (ERK) complexes involved in the feedback regulation of Raf1.

    Science.gov (United States)

    Mazalouskas, Matthew D; Godoy-Ruiz, Raquel; Weber, David J; Zimmer, Danna B; Honkanen, Richard E; Wadzinski, Brian E

    2014-02-14

    Serine/threonine protein phosphatase 5 (PP5, PPP5C) is known to interact with the chaperonin heat shock protein 90 (HSP90) and is involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, such as cell growth, differentiation, proliferation, motility, and apoptosis. Here, we identify PP5 in stable complexes with extracellular signal-regulated kinases (ERKs). Studies using mutant proteins reveal that the formation of PP5·ERK1 and PP5·ERK2 complexes partially depends on HSP90 binding to PP5 but does not require PP5 or ERK1/2 activity. However, PP5 and ERK activity regulates the phosphorylation state of Raf1 kinase, an upstream activator of ERK signaling. Whereas expression of constitutively active Rac1 promotes the assembly of PP5·ERK1/2 complexes, acute activation of ERK1/2 fails to influence the phosphatase-kinase interaction. Introduction of oncogenic HRas (HRas(V12)) has no effect on PP5-ERK1 binding but selectively decreases the interaction of PP5 with ERK2, in a manner that is independent of PP5 and MAPK/ERK kinase (MEK) activity, yet paradoxically requires ERK2 activity. Additional studies conducted with oncogenic variants of KRas4B reveal that KRas(L61), but not KRas(V12), also decreases the PP5-ERK2 interaction. The expression of wild type HRas or KRas proteins fails to reduce PP5-ERK2 binding, indicating that the effect is specific to HRas(V12) and KRas(L61) gain-of-function mutations. These findings reveal a novel, differential responsiveness of PP5-ERK1 and PP5-ERK2 interactions to select oncogenic Ras variants and also support a role for PP5·ERK complexes in regulating the feedback phosphorylation of PP5-associated Raf1.

  1. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

    Science.gov (United States)

    Zeke, András; Misheva, Mariya

    2016-01-01

    SUMMARY The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

  2. An ontology-based search engine for protein-protein interactions.

    Science.gov (United States)

    Park, Byungkyu; Han, Kyungsook

    2010-01-18

    Keyword matching or ID matching is the most common searching method in a large database of protein-protein interactions. They are purely syntactic methods, and retrieve the records in the database that contain a keyword or ID specified in a query. Such syntactic search methods often retrieve too few search results or no results despite many potential matches present in the database. We have developed a new method for representing protein-protein interactions and the Gene Ontology (GO) using modified Gödel numbers. This representation is hidden from users but enables a search engine using the representation to efficiently search protein-protein interactions in a biologically meaningful way. Given a query protein with optional search conditions expressed in one or more GO terms, the search engine finds all the interaction partners of the query protein by unique prime factorization of the modified Gödel numbers representing the query protein and the search conditions. Representing the biological relations of proteins and their GO annotations by modified Gödel numbers makes a search engine efficiently find all protein-protein interactions by prime factorization of the numbers. Keyword matching or ID matching search methods often miss the interactions involving a protein that has no explicit annotations matching the search condition, but our search engine retrieves such interactions as well if they satisfy the search condition with a more specific term in the ontology.

  3. Antigenic proteins involved in occupational rhinitis and asthma caused by obeche wood (Triplochiton scleroxylon.

    Directory of Open Access Journals (Sweden)

    Ana Aranda

    Full Text Available BACKGROUND: Obeche wood dust is a known cause of occupational asthma where an IgE-mediated mechanism has been demonstrated. OBJECTIVE: To characterize the allergenic profile of obeche wood dust and evaluate the reactivity of the proteins by in vitro, ex vivo and in vivo assays in carpenters with confirmed rhinitis and/or asthma MATERIALS AND METHODS: An in-house obeche extract was obtained, and two IgE binding bands were purified (24 and 12 kDa and sequenced by N-terminal identity. Specific IgE and IgG, basophil activation tests and skin prick tests (SPTs were performed with whole extract and purified proteins. CCD binding was analyzed by ELISA inhibition studies. RESULTS: Sixty-two subjects participated: 12 with confirmed occupational asthma/rhinitis (ORA+, 40 asymptomatic exposed (ORA-, and 10 controls. Of the confirmed subjects, 83% had a positive SPT to obeche. There was a 100% recognition by ELISA in symptomatic subjects vs. 30% and 10% in asymptomatic exposed subjects and controls respectively (p<0.05. Two new proteins were purified, a 24 kDa