Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation

As an activator of adenylate cyclase, the neuropeptide Pituitary Adenylate Cyclase Activating Peptide (PACAP) impacts levels of cyclic AMP, a key second messenger available in brain cells. PACAP is involved in certain adult behaviors. To elucidate PACAP interactions, a compendium of microarrays representing mRNA expression in the adult mouse whole brain was pooled from the Phenogen database for analysis. A regulatory network was computed based on mutual information between gene pairs using gene expression data across the compendium. Clusters among genes directly linked to PACAP, and probable interactions between corresponding proteins were computed. Database “experts” affirmed some of the inferred relationships. The findings suggest ADCY7 is probably the adenylate cyclase isoform most relevant to PACAP's action. They also support intervening roles for kinases including GSK3B, PI 3-kinase, SGK3 and AMPK. Other high-confidence interactions are hypothesized for future testing. This new information has implications for certain behavioral and other disorders.     

Molecular cloning, characterization, and expression analysis of a cytosolic HSP90 gene from Haematococcus pluvialis

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that plays key roles in the folding, maintenance of structural integrity, and regulation of a subset of cytosolic proteins. In this study, the cDNA of Haematococcus pluvialis HSP90 (designated HpHSP90) was cloned by the combination of homology cloning and rapid amplification of cDNA ends approaches. The full-length cDNA of HpHSP90 was of 2,606 bp, including an open reading frame of 2,109 bp encoding a polypeptide of 702 amino acids with predicted molecular weight of 80.14 kDa and theoretical isoelectric point of 5.07. BLAST analysis revealed that HpHSP90 shared high similarity with other known HSP90s, and the five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in HpHSP90, which indicated that HpHSP90 should be a cytosolic member of the HSP90 family. Under different stress conditions, messenger RNA (mRNA) expression levels of HpHSP90 were quantified by quantitative RT-PCR. To H. pluvialis kept at different temperatures for 1 h, maximum HpHSP90 expression was observed in the range 5 to 10°C and 35 to 40°C and the expression level of HpHSP90 at 40°C was the highest (threefold compared with that at 25°C). In H. pluvialis kept at 35°C for different times, the mRNA expression level of HpHSP90 reached a maximum level after 7 h and then dropped progressively. The results indicate that HpHSP90 responded to cold and heat stresses with a temperature-dependent expression pattern as well as exposure time effect and could be used as a molecular biomarker in adverse stress environment.     

Thermo and pH stable ATP-independent chaperone activity of heat-inducible Hsp70 from Pennisetum glaucum

Heat shock proteins (Hsps) are a class of molecular chaperones that play an essential role in preserving cellular functions under stressful conditions. The over production of recombinant proteins often causes cellular stress that results in aggregation/misfolding of proteins, which sometimes leads to the formation of inclusion bodies. Here we report the cloning and characterization of heat-inducible PgHsp70 from Pennisetum glaucum, a heat and drought tolerant plant that showed stability and chaperone activity at elevated temperatures. The predicted amino acid sequence of PgHsp70 revealed a high homology with Hsp70 from other plants, and the overall 3D structure homology modeling is similar to that of the constitutively expressed bovine cytosolic Heat Shock Cognate (HSC)-70. The purified recombinant protein had an apparent molecular mass of 70 kDa and displayed optimal chaperone activity at 50°C, and pH 8.0. Under these conditions, the T_1/2 of PgHsp70 increased from 10 to 15 h in the presence of glycerol. The PgHsp70 exhibited a higher chaperone activity towards glutamate dehydrogenase than alcohol dehydrogenase. The expression of recombinant carbonic anhydrase (CA ) in E. coli in a catalytically active soluble form rather than in inclusion bodies was made feasible by co-expression of PgHsp70. Circular dichroism (CD) studies of the recombinant PgHsp70 did not reveal any discernible changes in the α-helix content, with increase in temperature from 35 to 85°C, thus suggesting a critical role of α-helix content in maintaining the chaperone activity.Key words: abiotic stress, carbonic anhydrase, chaperone, circular dichroism, heat shock proteins (Hsp70), Pennisetum glaucum, pH stable     

Molecular cloning of a cDNA encoding a high mobility group protein in Cucumis sativus and its expression by abiotic stress treatments

A cDNA encoding a high mobility group B (HMGB) protein was isolated from Cucumis sativus and characterized with respect to its sequence, expression and responses to various abiotic stress treatments. The predicted polypeptide of 146 amino acid residues contains characteristic features of HMGB family proteins including the N-terminal basic region, one HMG-box and a stretch of acidic amino acid residues at the C-terminus. In vitro nucleic acid-binding assay revealed that the HMGB protein bound to both single-stranded DNA and double-stranded DNA. DNA gel blot analysis indicated that the HMGB gene is a single copy gene in cucumber genome. RNA gel blot analysis showed that the cucumber HMGB was more abundantly expressed in the roots than in shoots and leaves. Various abiotic stresses, including cold, drought and high salinity, down regulated markedly the expression of the HMGB in cucumber. The present report identifies a novel gene encoding HMGB protein in cucumber that shows a significant response to abiotic stress treatments.     

Molecular characterization of soybean GmDjp1 encoding a type III J-protein induced by abiotic stress

Heat stress severely affects plant growth and development causing crop loss worldwide. Classical type I DnaJ proteins (also called as J-proteins, J-domain proteins or HSP40 proteins) function as molecular co-chaperones for the HSP70 proteins. In this study, we have cloned and characterized a novel gene GmDjp1 (G lycine m ax DnaJ protein 1) encoding a type III J-protein of which function has not been identified in plant. Deduced amino acid sequences of GmDjp1 show the highest homology with a J-protein from Medicago truncatula legume plant (83 %) and with Arabidopsis thaliana type III J-class proteins, atDjC53 (77 %) and atDjC32 (50 %). DNA blot analysis revealed that GmDjp1 exists as a 2-copy gene in soybean genome. GmDjp1 mRNA was induced by a broad spectrum of abiotic stresses, including wounding, heat-shock, dehydration, cold or high-salinity stress, suggesting its role in the signaling events in the abiotic stress-related defense response. Subcellular localization studies demonstrated that the GmDjp1-GFP fusion protein was localized in the nucleus. Differential RNA expression of GmDjp1 by heat-shock stress inspired us to test heat-shock tolerance of GmDjp1in E. coli. Heterologous expression of GmDjp1 conferred tolerance to high temperature stress in E. coli. This report provides strong evidence that GmDjp1 may play a critical role during heat-shock stress in cell.     

Molecular cloning and expression analysis of a cytosolic Hsp70 gene from Antarctic ice algae Chlamydomonas sp. ICE-L

A cDNA encoding heat shock protein 70 of Antarctic ice algae Chlamydomonas sp. ICE-L (designated as CiHsp70) was identified by RT-PCR and rapid amplification of cDNA ends approaches. The full-length cDNA of CiHsp70 was 2,232 bp, consisting of a 5′-terminal untranslated region (UTR) of 76 bp, a 3′-terminal UTR of 203 bp with a poly (A) tail, and an open reading frame of 1,953 bp. The CiHsp70 cDNA encoded a polypeptide of 651 amino acids with an ATPase domain of 388 amino acids, the substrate peptide binding domain of 246 amino acids and a C-terminus domain of 17 amino acids. The inducible CiHsp70 cDNA was highly homologous to other plant cytosolic Hsp70 genes and clustered together with green algae and higher plant rather than brown algae, diatom and Cryptophyta. Antarctic ice algae were treated with different stress conditions and messenger RNA (mRNA) expression levels of CiHsp70 were quantified by quantitative RT-PCR. The results showed that both cold and heat shock treatments could stimulate CiHsp70 mRNA expression. Meanwhile, CiHsp70 mRNA expression level increased 2.9-fold in response to UV-B radiation for 6 h, while the expression levels of CiHsp70 were remarkably increased after removing the UV-B radiation and immediately providing additional 6 h visible light. Furthermore, treating with 62 or 93‰ NaCl for 2 h, CiHsp70 mRNA expression level increased 3.0- and 2.1-fold, respectively. Together, our observations revealed that CiHsp70 as a molecular chaperone might play an important role in Antarctic ice algae Chlamydomonas sp. ICE-L acclimatizing to polar environment.