Protein and Peptide Letters - Volume 21, Issue 9, 2014

Flooding is one of the serious problems for soybean plants because it inhibits growth. Proteomic and metabolomic techniques were used to determine whether proteins and metabolites are altered in the root tips of soybeans under flooding stress. Two-day-old soybean plants were flooded for 2 days, and proteins and metabolites were extracted from root tips. Flooding-responsive proteins were identified using two-dimensional- or SDS-polyacrylamide gel electrophoresis- based proteomics techniques. Using both techniques, 172 proteins increased and 105 proteins decreased in abundance in the root tips of flood-stressed soybean. The abundance of methionine synthase, heat shock cognate protein, urease, and phosphoenol pyruvate carboxylase was significantly increased by flooding stress. Furthermore, 73 floodingresponsive metabolites were identified using capillary electrophoresis-mass spectrometry. The levels of gammaaminobutyric acid, glycine, NADH2, and phosphoenol pyruvate were increased by flooding stress. Taken together, these results suggest that synthesis of phosphoenol pyruvate by way of oxaloacetate produced in the tricarboxylic acid cycle is activated in soybean root tips in response to flooding stress, and that flooding stress also leads to modulation of the urea cycle in the root tips.

Bacillus subtilis HSO121 was previously isolated from the formation water of an oil field and found to produce surfactin lipopeptides. Effects of 10 different components on surfactin production have been studied by Plackett- Burman design (PBD). Each component was screened on the basis of P-value, which was at 92% or above of confidence level. In PBD, significant factors influencing the surfactin production were selected for further optimization via response surface methodology approach (RSM). Four significant variables (calcium chloride, ferrous sulfate, maltose, and L-arginine) were selected for the optimization studies, and constructed via central composite design (CCD). Calcium chloride, maltose and L-arginine showed a significant positive effect on surfactin production, while ferrous sulfate had no significant effect. An overall 38.06-fold increase in surfactin production yield was achieved in the optimized medium as compared with the unoptimized basal medium. Surfactin production yield increased significantly with optimized medium (47.58 g/l) when compared with the unoptimized one (1.25 g/l).

7α-hydroxysteroid dehydrogenase (7α-HSDH, EC 1.1.1.159), one of the short-chain dehydrogenase/reductase superfamily, catalyzes the dehydrogenation of C7 hydroxyl group of the steroid skeleton of bile acids. Clostridium absonum 7α-HSDH (Ca 7α-HSDH) was cloned and heterologously expressed in Escherichia coli. The function of carboxyterminal (C-terminal) and Arg38 of Ca 7α-HSDH was investigated through truncations and site-directed mutagenesis, respectively. When 2 and 6 amino acids of C-terminal were removed, the catalytic efficiency (kcat/Km) of Ca 7α-HSDH remained 19.1% and 2.5%, respectively. Furthermore, the activity could not be detected after 8, 14 and 17 amino acids were deleted. No activity could be detected with coenzyme either NADP+ or NAD+ after replacement of arginine at position 38 by aspartic acid. The metal ions Mg2+ (50 mM), Na+ (200 mM) and K+ (500 mM) could maximally improve the activity of Ca 7α-HSDH by 61.4%, 64.7% and 105.7%, respectively. The activity had no significant change after incubation at 4 or 25 °C for 108 h, but decreased dramatically at 37 °C. Our study confirmed that C-terminal and Arg38 were essential for the catalytic function of Ca 7α-HSDH and the enzyme activity can be improved by metal ions.

WH1fungin (WF), a lipopeptide surfactin, has been verified as an immunoadjuvant previously. In this study, mice were intranasally or parenterally immunized with WF plus Hepatitis B surface antigen (HBsAg), then the immune responses were detected. The results showed 50 µg WF plus 20 μg HBsAg for intranasal and 10 μg WF plus 1 µg HBsAg for parenteral immunization was efficient inducing strong immune response against HBsAg in mice. A high titer and longterm anti-HBsAg IgG was observed for more than 19 weeks in intranasal or parenteral immunizations, much higher than that induced by CpG or Alum adjuvant. The anti-HBsAg IgA was also induced in intestine and lung, indicating that mucosal as well as systemic immune response was elicited for intranasal immunization. The IgG isotype in serum revealed WF induced a Th2-bias immune response with a higher titer of anti-HBsAg IgG1 than IgG2a in mice. Moreover, WF also induced more Th1 cells producing interferon (IFN)-γ and stronger cytotoxic T lymphocytes response than controls for intramuscular administration. These data further confirmed that WF induced Th1- as well as Th2- type immune response toward HBsAg. Taken together, WF-adjuvanted HBsAg elicits more effective immune response than that adjuvanted by Alum or CpG, suggesting its potential for development of more efficient HBV vaccines in the future.

Flooding is a serious abiotic stress for soybean because it restricts growth and reduces grain yields. To investigate the effect of gibberellic acid (GA) on soybean under flooding stress, root proteins were analyzed using a gel-free proteomic technique. Proteins were extracted from the roots of 4-days-old soybean seedlings exposed to flooding stress in the presence and absence of exogenous GA3 for 2 days. A total of 307, 324, and 250 proteins were identified from untreated, and flooding-treated soybean seedlings without or with GA3, respectively. Secondary metabolism- and cell-related proteins, and proteins involved in protein degradation/synthesis were decreased by flooding stress; however, the levels of these proteins were restored by GA3 supplementation under flooding. Fermentation- and cell wall-related proteins were not affected by GA3 supplementation. Furthermore, putative GA-responsive proteins, which were identified by the presence of a GA-responsive element in the promoter region, were less abundant by flooding stress; however, these proteins were more abundant by GA3 supplementation under flooding. Taken together, these results suggest that GA3 affects the abundance of proteins involved in secondary metabolism, cell cycle, and protein degradation/synthesis in soybeans under flooding stress.

α-Amylases hydrolyze α- 1,4-glycosidic bonds during assimilation of biological macromolecules. The amino acid sequences of these enzymes in thousands of diverse organisms are known and the 3D structures of several proteins have been solved. The 3D structure analysis of these universal enzymes from diverse organisms has been studied by the generation of phylogenetic trees and structure based sequence analysis to generate a metric for the degree of conservation that is responsible for individual speciation. Greater similarities are observed between reference NCBI tree and structure based phylogenetic tree compared to sequence based phylogenetic tree indicating that structures truly represent the functional aspects of proteins than from the sequence information alone. We report differences in the profile specific conserved and insertion/deletion regions, factors responsible for the Ca2+ and Cl- ion binding and the disulfide connectivity pattern that discriminate the enzymes over evolution.

Cystatin F is a unique member of the cystatin family of cysteine protease inhibitors, which is synthesized as an inactive dimer and it is activated by N-terminal cleavage in the endolysosomes. It is expressed in the cells of the immune system: myeloid cells and the cells involved in target cell killing: natural killer (NK) cells and cytotoxic T cells (CTLs). Upon activation of the NK cells with interleukin 2 (IL-2), cystatin F was found upregulated and co-localized in cytotoxic granules with cathepsin C (CatC) and CatV. However, cystatin F inhibits the CatC in cells only when its N-terminal part is processed. Although cystatin F could inhibit both CatV and CatC, the IL-2 stimulation of the YT cells resulted in an increased CatV activity, while the CatC activity was unchanged. The incubation of IL-2 activated NK cells with a cysteine proteinase inhibitor E-64d increased the cystatin F dimer formation. Our results suggest that cystatin F not only inhibits CatV, but it is processed by the CatV in order to inhibit the CatC activity in cytotoxic granules. The regulation of the CatC activity in the cytotoxic granules of the NK cells by the cystatin F could be important for the processing and activation of granule-associated serine proteases - granzymes.

Protein hydrolysates are widely applied as antioxidants in nutrition, but the potential antioxidant activities of small peptides remain unknown. Therefore, we investigated the antioxidant activities of small peptides isolated from solid-state fermented sesame meal via Sephadex G-15 chromotography. The scavenging capacities for 2, 2- diphenylpicrylhydrazyl (DPPH) and hydroxyl (•OH) radicals as well as the total reducing capacity were determined. The in vivo antioxidant activity was determined upon 30-d intragastric administration of the isolated small peptides (tripeptide, tetrapeptide, and hexapeptide) at different doses (0.1, 0.2, and 0.4 g/kg•d) in healthy Kunming mice. The results showed that the DPPH and •OH scavenging rates of the three peptides exceeded 80%. The total reducing activities of 4 mg/mL tetrapeptide or hexapeptide and 2 mg/mL tripeptide were comparable to that of 0.5 mg/mL glutathione. In mice fed sesame peptides, malondialdehyde levels in the serum and liver were lower than those in controls, whereas the activities of liver superoxide dismutase and glutathione peroxidase were significantly higher than those in controls (P<0.05). The antioxidant activity of tripeptide was significantly higher than those of tetrapeptide and hexapeptide (P<0.05). In conclusion, small peptides extracted from solid-state fermented sesame meal possess strong antioxidant activities that increase with decreasing peptide molecular weight.