Regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) by Src involves tyrosine phosphorylation of PDK1 and Src homology 2 domain binding

3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr(9) and Tyr(373/376)) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1-Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti-phospho-Tyr(9) antibodies showed that the level of Tyr(9) phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr(9), distinct from Tyr(373/376), is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr(9) phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.     

Crystal structure of Escherichia coli MazG, the regulator of nutritional stress response

MazG is a nucleoside triphosphate pyrophosphohydrolase that hydrolyzes all canonical nucleoside triphosphates. The mazG gene located downstream from the chromosomal mazEF "addiction module," that mediated programmed cell death in Escherichia coli. MazG activity is inhibited by the MazEF complex both in vivo and in vitro. Enzymatic activity of MazG in vivo affects the cellular level of guanosine 3',5'-bispyrophosphate (ppGpp), synthesized by RelA under amino acid starvation. The reduction of ppGpp, caused by MazG, may extend the period of cell survival under nutritional stress. Here we describe the first crystal structure of active MazG from E. coli, which is composed of two similarly folded globular domains in tandem. Among the two putative catalytic domains, only the C-terminal domain has well ordered active sites and exhibits an NTPase activity. The MazG-ATP complex structure and subsequent mutagenesis studies explain the peculiar active site environment accommodating all eight canonical NTPs as substrates. In vivo nutrient starvation experiments show that the C terminus NTPase activity is responsible for the regulation of bacterial cell survival under nutritional stress.     

Phytochelatin is not a primary factor in determining copper tolerance

Phytochelatin (PC) is involved in the detoxification of harmful, non-essential heavy metals and the homeostasis of essential heavy metals in plants. Its synthesis can be induced by either cadmium (Cd) or copper (Cu), and can form stable complexes with either element. This might suggest that PC has an important role in determining plant tolerance to both. However, this is not clearly apparent, as evidenced by a PC-deficient and Cd-sensitiveArabidopsis mutant (cad1-3) that shows no significant increase in its sensitivity to copper. Therefore, we investigated whether the mechanism for Cu tolerance differed from that for Cd by analyzing copper sensitivity in Cd-tolerant transgenics and Cd-sensitive mutants ofArabidopsis. Cadmium-tolerant transgenic plants that over-expressedA. thaliana phytochelatin synthase 1 (AtPCS1) were not tolerant of copper stress, thereby supporting the hypothesis that PC is not primarily involved in this tolerance mechanism. We also investigated Cu tolerance incad2-1, a Cd-sensitive and glutathione (GSH)-deficientArabidopsis mutant. Paradoxically,cad2-1 was more resistant to copper stress than were wild-type plants. This was likely due to the high level of cysteine present in that mutant. However, when the growth medium was supplemented with cysteine, the wild types also exhibited copper tolerance. Moreover,Saccharomyces cerevisiae that expressedAtPCS1 showed tolerance to Cd but hypersensitivity to Cu. All these results indicate that PC is not a major factor in determining copper tolerance in plants.     

Characteristics of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production byRalstonia eutropha NCIMB 11599 and ATCC 17699

Ralstonia eutropha NCIMB 11599 and ATCC 17699 were grown, and their productions of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] compared. In flask cultures ofR. eutropha NCIMB 11599, cell concentration, P(3HB-co-4HB) concentration and polymer content decreased considerably with increases in the γ-butyrolactone concentration, and the 4HB fraction was also very low (maximum 1.74 mol%). In fed-batch cultures ofR. eutropha NCIMB 11599, glucose and γ-butyrolactone were fed as the carbon sources, under a phosphate limitation strategy. When glucose was fed as the sole carbon source, with its concentration controlled using an on-line glucose analyzer, 86% of the P(3HB) homopolymer was obtained from 201 g/L of cells. In a two-stage fed-batch culture, where the cell concentration was increased to 104 g/L, with glucose fed in the first step and constant feeding of γ-butyrolactone, at 6 g/h, in the second, final cell concentration at 67 h was 106 g/L, with a polymer content of 82%, while the 4HB fraction was only 0.7 mol%. When the same feeding strategy was applied to the fedbatch culture ofR. eutropha ATCC 17699, where the cell concentration was increased to 42 g/L, by feeding fructose in the first step and γ-butyrolactone (1.5 g/h) in the second, the final cell concentration, polymer content and 4HB fraction at 74 h were 51 g/L, 35% and 32 mol%, respectively. In summary,R. eutropha ATCC 17699 was better thanR. eutropha NCIMB 11599 in terms of P(3HB-co-4HB) production with various 4HB fractions.     

Chlorophyll-deficient gene and morphological variations in Korean populations of maize (Zea mays)

We studied cultivated and naturalized Korean maize populations to determine the extent to which the chlorophylldeficient mutation and the phenotypic variations of two morphological characters (i.e., red coleoptiles and epicotyls, and the number of the first root hairs) are maintained. The frequency of the chlorophyll-deficient mutant gene (2.73% on average) was highly variable. Frequencies of red coleoptiles and epicotyls also were higher than expected from a mutation-selection balance. The average number of hairy phenotypes within populations was 1.8, ranging from 0.0 to 4.0. Naturalized populations were closely related to with cultivated communities. Most striking, however, was the more significant difference among populations than within populations with regard to both the frequency of chlorophyll-deficient mutant genes and the phenotypic variations of our two morphological characters. On a per-gene basis, the majority of the phenotypic variation (mean of 73.3%) resided among populations.     

Optimization of culture conditions and continuous production of chitosan by the fungi,Absidia coerulea

The production of chitosan from the mycelia ofAbsidia coerulea was studied to improve cell growth and chitosan productivity. Culture conditions were optimized in batch cultivation (pH 4.5 agitator speed of 250 rpm, and aeration rate of, 2 vvm) and the maximum chitosan concentration achieved was 2.3 g/L under optimized conditions. Continuous culture was carried out successfully by the formation of new growth spots under optimized conditions, with a chitosan productivity of 0.052 gL⁻¹ h⁻¹, which is the highest value to date, and was obtained at a dilution rate of 0.05 h⁻¹. Cell chitosan concentrations reached about 14% in the steady state, which is similar to that achieved in batch culture. This study shows that for the continuous culture ofAbsidia coerulea it is vital to control the medium composition.     

Biological control of bacterial spot disease and plant growth-promoting effects of lactic acid bacteria on pepper

In our previous studies, we observed the biological control effect of lactic acid bacteria strains (LABs) KLF01, KLC02 and KPD03 against different plant pathogenic bacteria in vitro against Ralstonia solanacearum, and strains KLF01 and KLC02 against Pectobacterium carotovorum under greenhouse and field experiments, respectively. In this study, we observed the efficacy of these bacteria against bacterial spot pathogen (Xanthomonas campestris pv. vesicatoria) and their plant growth-promoting activities in pepper (Capsicum annuum L. var. annuum), under greenhouse and field conditions. LABs significantly (P < 0.05) reduced bacterial spot on pepper plants in comparison to untreated plants in both the greenhouse and the field experiments. The plant growth-promoting effect of LABs on pepper varied; some strains had a significant effect on growth promotion (P < 0.05) compared with untreated plants, while some showed no significant effect in the greenhouse and field experiments. Additionally, LABs were able to colonise roots, produce indole-3-acetic acid (IAA), siderophores and solubilise phosphate. These findings indicate that application of LABs could provide a promising alternative for the management of bacterial spot disease in pepper plants and could therefore be used as a healthy plant growth-promoting agent.     

Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein

The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme’s inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4–5 and 8–9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8–9 position was necessary to significantly improve the inhibitory potency.