161 research outputs found
Characterization of Two Soybean (Glycine max L.) LEA IV Proteins by Circular Dichroism and Fourier Transform Infrared Spectrometry
Late embryogenesis-abundant (LEA) proteins, accumulating to a high level during the late stages of seed development, may play a role as osmoprotectants. However, the functions and mechanisms of LEA proteins remained to be elucidated. Five major groups of LEA proteins have been described. In the present study, we report on the characterization of two members of soybean LEA IV proteins, basic GmPM1 and acidic GmPM28, by circular dichroism and Fourier transform infrared spectroscopy. The spectra of both proteins revealed limited defined secondary structures in the fully hydrated state. Thus, the soybean LEA IV proteins are members of ‘natively unfolded proteins’. GmPM1 or GmPM28 proteins showed a conformational change under hydrophobic or dry conditions. After fast or slow drying, the two proteins showed slightly increased proportions of defined secondary structures (α-helix and β-sheet), from 30 to 49% and from 34 to 42% for GmPM1 and GmPm28, respectively. In the dehydrated state, GmPM1 and GmPM28 interact with non-reducing sugars to improve the transition temperature of cellular glass, with poly-l-lysine to prevent dehydration-induced aggregation and with phospholipids to maintain the liquid crystal phase over a wide temperature range. Our work suggests that soybean LEA IV proteins are functional in the dry state. They are one of the important components in cellular glasses and may stabilize desiccation-sensitive proteins and plasma membranes during dehydration
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Genetic diversity at the Dhn3 locus in Turkish Hordeum spontaneum populations with comparative structural analyses
We analysed Hordeum spontaneum accessions from 21 different locations to understand the genetic diversity of HsDhn3 alleles and effects of single base mutations on the intrinsically disordered structure of the resulting polypeptide (HsDHN3). HsDHN3 was found to be YSK2-type with a low-frequency 6-aa deletion in the beginning of Exon 1. There is relatively high diversity in the intron region of HsDhn3 compared to the two exon regions. We have found subtle differences in K segments led to changes in amino acids chemical properties. Predictions for protein interaction profiles suggest the presence of a protein-binding site in HsDHN3 that coincides with the K1 segment. Comparison of DHN3 to closely related cereals showed that all of them contain a nuclear localization signal sequence flanking to the K1 segment and a novel conserved region located between the S and K1 segments [E(D/T)DGMGGR]. We found that H. vulgare, H. spontaneum, and Triticum urartu DHN3s have a greater number of phosphorylation sites for protein kinase C than other cereal species, which may be related to stress adaptation. Our results show that the nature and extent of mutations in the conserved segments of K1 and K2 are likely to be key factors in protection of cells
Computational and Statistical Analyses of Amino Acid Usage and Physico-Chemical Properties of the Twelve Late Embryogenesis Abundant Protein Classes
Late Embryogenesis Abundant Proteins (LEAPs) are ubiquitous proteins expected to play major roles in desiccation tolerance. Little is known about their structure - function relationships because of the scarcity of 3-D structures for LEAPs. The previous building of LEAPdb, a database dedicated to LEAPs from plants and other organisms, led to the classification of 710 LEAPs into 12 non-overlapping classes with distinct properties. Using this resource, numerous physico-chemical properties of LEAPs and amino acid usage by LEAPs have been computed and statistically analyzed, revealing distinctive features for each class. This unprecedented analysis allowed a rigorous characterization of the 12 LEAP classes, which differed also in multiple structural and physico-chemical features. Although most LEAPs can be predicted as intrinsically disordered proteins, the analysis indicates that LEAP class 7 (PF03168) and probably LEAP class 11 (PF04927) are natively folded proteins. This study thus provides a detailed description of the structural properties of this protein family opening the path toward further LEAP structure - function analysis. Finally, since each LEAP class can be clearly characterized by a unique set of physico-chemical properties, this will allow development of software to predict proteins as LEAPs
Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich domain
The cactus OpsDHN1 dehydrin belongs to a large family of disordered and highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins, which accumulate during the late stages of embryogenesis and in response to abiotic stresses. Herein, we present the in vivo OpsDHN1 subcellular localization by N-terminal GFP translational fusion; our results revealed a cytoplasmic and nuclear localization of the GFP::OpsDHN1 protein in Nicotiana benthamiana epidermal cells. In addition, dimer assembly of OpsDHN1 in planta using a Bimolecular Fluorescence Complementation (BiFC) approach was demonstrated. In order to understand the in vivo role of the histidine-rich motif, the OpsDHN1 - Delta His version was produced and assayed for its subcellular localization and dimer capability by GFP fusion and BiFC assays, respectively. We found that deletion of the OpsDHN1 histidine-rich motif restricted its localization to cytoplasm, but did not affect dimer formation. In addition, the deletion of the S-segment in the OpsDHN1 protein affected its nuclear localization. Our data suggest that the deletion of histidine-rich motif and S-segment show similar effects, preventing OpsDHN1 from getting into the nucleus. Based on these results, the histidine-rich motif is proposed as a targeting element for OpsDHN1 nuclear localization.This work was supported by the CONACYT (Investigacion Ciencia Basica CB-2013-221075) funding to JJ, NSERC Discovery Grant to SG, and funding from the Spanish MICINN/MINECO (BIO2011-23828) to AF and MICINN (BIO2011-23828) to JC. The authors acknowledge to Marisol Gascon Irian from Institut de Biologia Molecular y Celular de Plantas and Nydia Hernandez-Rios from Neurology Institute-UNAM for their technical assistance using the confocal laser-scanning microscope.Hernández-Sánchez, I.; Maruri-López, I.; Ferrando Monleón, AR.; Carbonell Gisbert, J.; Graether, S.; Jimenez-Bremont, J. (2015). Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich domain. Frontiers in Plant Science. 6(702):1-8. https://doi.org/10.3389/fpls.2015.00702S186702Alsheikh, M. K., Heyen, B. J., & Randall, S. K. (2003). Ion Binding Properties of the Dehydrin ERD14 Are Dependent upon Phosphorylation. Journal of Biological Chemistry, 278(42), 40882-40889. doi:10.1074/jbc.m307151200Belda-Palazón, B., Ruiz, L., Martí, E., Tárraga, S., Tiburcio, A. F., Culiáñez, F., … Ferrando, A. (2012). Aminopropyltransferases Involved in Polyamine Biosynthesis Localize Preferentially in the Nucleus of Plant Cells. PLoS ONE, 7(10), e46907. doi:10.1371/journal.pone.0046907Briesemeister, S., Rahnenf�hrer, J., & Kohlbacher, O. (2010). YLoc—an interpretable web server for predicting subcellular localization. Nucleic Acids Research, 38(suppl_2), W497-W502. doi:10.1093/nar/gkq477Carjuzaa, P., Castellión, M., Distéfano, A. J., del Vas, M., & Maldonado, S. (2008). 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The maize abscisic acid-responsive protein Rab17 is located in the nucleus and interacts with nuclear localization signals. The Plant Cell, 6(3), 351-360. doi:10.1105/tpc.6.3.351Godoy, J. A., Lunar, R., Torres-Schumann, S., Moreno, J., Rodrigo, R. M., & Pintor-Toro, J. A. (1994). Expression, tissue distribution and subcellular localization of dehydrin TAS14 in salt-stressed tomato plants. Plant Molecular Biology, 26(6), 1921-1934. doi:10.1007/bf00019503Graether, S. P., & Boddington, K. F. (2014). Disorder and function: a review of the dehydrin protein family. Frontiers in Plant Science, 5. doi:10.3389/fpls.2014.00576Hanin, M., Brini, F., Ebel, C., Toda, Y., Takeda, S., & Masmoudi, K. (2011). Plant dehydrins and stress tolerance. Plant Signaling & Behavior, 6(10), 1503-1509. doi:10.4161/psb.6.10.17088Hara, M. (2010). The multifunctionality of dehydrins: An overview. Plant Signaling & Behavior, 5(5), 503-508. doi:10.4161/psb.11085Hara, M., Fujinaga, M., & Kuboi, T. (2005). Metal binding by citrus dehydrin with histidine-rich domains. Journal of Experimental Botany, 56(420), 2695-2703. doi:10.1093/jxb/eri262Hara, M., Kondo, M., & Kato, T. (2013). A KS-type dehydrin and its related domains reduce Cu-promoted radical generation and the histidine residues contribute to the radical-reducing activities. Journal of Experimental Botany, 64(6), 1615-1624. doi:10.1093/jxb/ert016HARA, M., SHINODA, Y., TANAKA, Y., & KUBOI, T. (2009). DNA binding of citrus dehydrin promoted by zinc ion. Plant, Cell & Environment, 32(5), 532-541. doi:10.1111/j.1365-3040.2009.01947.xHara, M., Terashima, S., & Kuboi, T. (2001). Characterization and cryoprotective activity of cold-responsive dehydrin from Citrus unshiu. Journal of Plant Physiology, 158(10), 1333-1339. doi:10.1078/0176-1617-00600Hernández-Sánchez, I. E., Martynowicz, D. M., RodrÃguez-Hernández, A. A., Pérez-Morales, M. B., Graether, S. P., & Jiménez-Bremont, J. F. (2014). A dehydrin-dehydrin interaction: the case of SK3 from Opuntia streptacantha. Frontiers in Plant Science, 5. doi:10.3389/fpls.2014.00520Heyen, B. J., Alsheikh, M. K., Smith, E. A., Torvik, C. F., Seals, D. F., & Randall, S. K. (2002). The Calcium-Binding Activity of a Vacuole-Associated, Dehydrin-Like Protein Is Regulated by Phosphorylation. Plant Physiology, 130(2), 675-687. doi:10.1104/pp.002550Houde, M., Daniel, C., Lachapelle, M., Allard, F., Laliberte, S., & Sarhan, F. (1995). Immunolocalization of freezing-tolerance-associated proteins in the cytoplasm and nucleoplasm of wheat crown tissues. The Plant Journal, 8(4), 583-593. doi:10.1046/j.1365-313x.1995.8040583.xHwang, I. S., Choi, D. S., Kim, N. H., Kim, D. S., & Hwang, B. K. (2013). The pepper cysteine/histidine-rich DC1 domain protein CaDC1 binds both RNA and DNA and is required for plant cell death and defense response. New Phytologist, 201(2), 518-530. doi:10.1111/nph.12521Jensen, A. B., Goday, A., Figueras, M., Jessop, A. 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Hv-CBF2A overexpression in barley accelerates COR gene transcript accumulation and acquisition of freezing tolerance during cold acclimation
Abstract C-Repeat Binding Factors (CBFs) are DNAbinding
transcriptional activators of gene pathways imparting
freezing tolerance. Poaceae contain three CBF subfamilies,
two of which, HvCBF3/CBFIII and HvCBF4/CBFIV,
are unique to this taxon. To gain mechanistic insight into
HvCBF4/CBFIV CBFs we overexpressed Hv-CBF2A in
spring barley (Hordeum vulgare) cultivar ‘Golden Promise’.
The Hv-CBF2A overexpressing lines exhibited stunted
growth, poor yield, and greater freezing tolerance compared
to non-transformed ‘Golden Promise’. Differences in
freezing tolerance were apparent only upon cold acclimation.
During cold acclimation freezing tolerance of the
Hv-CBF2A overexpressing lines increased more rapidly
than that of ‘Golden Promise’ and paralleled the freezing
tolerance of the winter hardy barley ‘Dicktoo’. Transcript
levels of candidate CBF target genes, COR14B and DHN5
were increased in the overexpressor lines at warm temperatures,
and at cold temperatures they accumulated to much
higher levels in the Hv-CBF2A overexpressors than in
‘Golden Promise’. Hv-CBF2A overexpression also
increased transcript levels of other CBF genes at FROST
RESISTANCE-H2-H2 (FR-H2) possessing CRT/DRE sites
in their upstream regions, the most notable of which was
CBF12. CBF12 transcript levels exhibited a relatively constant
incremental increase above levels in ‘Golden Promise’
both at warm and cold. These data indicate that Hv-CBF2A
activates target genes at warm temperatures and that transcript
accumulation for some of these targets is greatly
enhanced by cold temperatures
Promutagenicity of 8-Chloroguanine, A Major Inflammation-Induced Halogenated DNA Lesion
Chronic inflammation is closely associated with cancer development. One possible mechanism for inflammation-induced carcinogenesis is DNA damage caused by reactive halogen species, such as hypochlorous acid, which is released by myeloperoxidase to kill pathogens. Hypochlorous acid can attack genomic DNA to produce 8-chloro-2′-deoxyguanosine (ClG) as a major lesion. It has been postulated that ClG promotes mutagenic replication using its syn conformer; yet, the structural basis for ClG-induced mutagenesis is unknown. We obtained crystal structures and kinetics data for nucleotide incorporation past a templating ClG using human DNA polymerase β (polβ) as a model enzyme for high-fidelity DNA polymerases. The structures showed that ClG formed base pairs with incoming dCTP and dGTP using its anti and syn conformers, respectively. Kinetic studies showed that polβ incorporated dGTP only 15-fold less efficiently than dCTP, suggesting that replication across ClG is promutagenic. Two hydrogen bonds between syn-ClG and anti-dGTP and a water-mediated hydrogen bond appeared to facilitate mutagenic replication opposite the major halogenated guanine lesion. These results suggest that ClG in DNA promotes G to C transversion mutations by forming Hoogsteen base pairing between syn-ClG and anti-G during DNA synthesis
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