A novel secreted protease from Pseudomonas aeruginosa activates NF-kappaB through protease-activated receptors

The Pseudomonas aeruginosa -derived alkaline protease (AprA), elastase A (LasA), elastase B (LasB) and protease IV are considered to play an important role in pathogenesis of this organism. Although the sequence analysis of P. aeruginosa genome predicts the presence of several genes encoding other potential proteases in the genome, little has been known about the proteases involving in pathogenesis. Recently, Porphyromonas gingivalis gingipains and Serratia marcescens serralysin have been shown to activate protease-activated receptor 2 (PAR-2), thereby modulating host inflammatory and immune responses. Accordingly, we hypothesized that unknown protease(s) from P. aeruginosa would also modulate such responses through PARs. In this study, we found that P. aeruginosa produces a novel l arge e xo p rotease (LepA) distinct from known proteases such as AprA, LasA, LasB and protease IV. Sequence analysis of LepA showed a molecular feature of the proteins transported by the two-partner secretion pathway. Our results indicated that LepA activates NF-κB-driven promoter through human PAR-1, -2 or -4 and cleaves the peptides corresponding to the tethered ligand region of human PAR-1, -2 and -4 at a specific site with exposure of their tethered ligands. Considered together, these results suggest that LepA would require PARs to modulate various host responses against bacterial infection.     

Proteomic and metabolomic analyses of the white-rot fungus Phanerochaete chrysosporium exposed to exogenous benzoic acid

Intracellular processes of the white-rot basidiomycete Phanerochaete chrysosporium involved in the metabolism of benzoic acid (BA) were investigated at the proteome and metabolome level. Up-regulation of aryl-alcohol dehydrogenase, arylaldehyde dehydrogenase, and cytochrome P450s was observed upon addition of exogenous BA, suggesting that these enzymes play key roles in its metabolism. Intracellular metabolic shifts from the short-cut TCA/glyoxylate bicycle system to the TCA cycle and an increased flux in the TCA cycle indicated activation of the heme biosynthetic pathway and the production of NAD(P)H. In addition, combined analyses of proteome and metabolome clearly indicated the role of trehalose as a storage disaccharide and that the mannitol cycle plays a role in an alternative energy-producing pathway.     

Evolution and control of imprinted FWA genes in the genus Arabidopsis

A central question in genomic imprinting is how a specific sequence is recognized as the target for epigenetic marking. In both mammals and plants, imprinted genes are often associated with tandem repeats and transposon-related sequences, but the role of these elements in epigenetic gene silencing remains elusive. FWA is an imprinted gene in Arabidopsis thaliana expressed specifically in the female gametophyte and endosperm. Tissue-specific and imprinted expression of FWA depends on DNA methylation in the FWA promoter, which is comprised of two direct repeats containing a sequence related to a SINE retroelement. Methylation of this element causes epigenetic silencing, but it is not known whether the methylation is targeted to the SINE-related sequence itself or the direct repeat structure is also necessary. Here we show that the repeat structure in the FWA promoter is highly diverse in species within the genus Arabidopsis. Four independent tandem repeat formation events were found in three closely related species. Another related species, A. halleri, did not have a tandem repeat in the FWA promoter. Unexpectedly, even in this species, FWA expression was imprinted and the FWA promoter was methylated. In addition, our expression analysis of FWA gene in vegetative tissues revealed high frequency of intra-specific variation in the expression level. In conclusion, we show that the tandem repeat structure is dispensable for the epigenetic silencing of the FWA gene. Rather, SINE-related sequence is sufficient for imprinting, vegetative silencing, and targeting of DNA methylation. Frequent independent tandem repeat formation events in the FWA promoter led us to propose that they may be a consequence, rather than cause, of the epigenetic control. The possible significance of epigenetic variation in reproductive strategies during evolution is also discussed.     

Lycoparins A-C, new alkaloids from Lycopodium casuarinoides inhibiting acetylcholinesterase

Three new Lycopodium alkaloids, lycoparins A-C (1-3), have been isolated from the club moss Lycopodium casuarinoides. Structures and stereochemistry of 1-3 were elucidated on the basis of 2D NMR correlations. Lycoparins C (3) exhibited an inhibitory activity against acetylcholinesterase, while lycoparins A (1) and B (2) did not show activity.     

Eupalinin A isolated from Eupatorium chinense L. induces autophagocytosis in human leukemia HL60 cells

Eupalinin A, a natural phytoalexin included in Eupatorium chinense L., exhibited a marked inhibitory effect on cell growth in HL60 cells. The morphological aspects of eupalinin A-treated cells evaluated by Hoechst 33342 nuclear staining indicated cell death, only a small part of which showed a typical apoptosis with nuclear fragmentation and condensation. To determine what type of cell death is caused by eupalinin A, we examined the contribution of caspases, Bcl-2 family proteins, MAP kinase, and PI3K/Akt, and mitochondrial membrane potential to this cell death. As a result, most part of the cell death was not associated with apoptosis because of caspase independence and no death factor released from mitochondria. Electron microscopic study indicated a characteristic finding of autophagy such as the formation of autophagosomes. Furthermore, the level of microctubule-associated-protein light chain 3 (LC3) II protein and monodancylcanaverin (MDC) incorporation were gradually increased with reduction of mitochondrial membrane potential by the accumulation of intracellular ROS after eupalinin A treatment. From these results, we can conclude that eupalinin A-induced cell death was mainly due to autophagy, which was initiated by increased ROS, resulting in the perturbation of mitochondrial membrane potential. Since the class III PI3K inhibitor such as 3-MA or LY294002 did not inhibit the eupalinin A-induced type II programmed cell death (PCD II), it was suggested that the PCD II was executed by Beclin-1 independent pathway of damage-induced mitochondrial autophagy (mitophagy).     

Malondialdehyde generated from peroxidized linolenic acid causes protein modification in heat-stressed plants

When polyunsaturated fatty acids (PUFAs) in biomembrane are peroxidized, a great diversity of aldehydes is formed, and some of which are highly reactive. Thus they are thought to have biological impacts in stressed plants; however, the detailed mechanism of generation and biochemical effects are unknown. In this study, we show that chloroplasts are major organelles in which malondialdehyde (MDA) generated from peroxidized linolenic acid modifies proteins in heat-stressed plants. First, to clarify the biochemical process of MDA generation from PUFAs and its attachment to proteins, we carried out in vitro experiments using model proteins (BSA and Rubisco) and methylesters of C18 PUFAs that are major components of plant biomembrane. Protein modification was detected by Western blotting using monoclonal antibodies that recognize MDA binding to proteins. Results showed that peroxidation of linolenic acid methylester by reactive oxygen species was essential for protein modification by MDA, and the MDA modification was highly dependent on temperature, leading to a loss of Rubisco activity. When isolated spinach thylakoid membrane was peroxidized at 37 degrees C, oxygen-evolving complex 33kDa protein (OEC33) was modified by MDA. These model experiments suggest that protein modification by MDA preferentially occurs under higher temperatures and oxidative conditions, thus we examined protein modification in heat-stressed plants. Spinach plants were heat-stressed at 40 degrees C under illumination, and modification of OEC33 protein by MDA was detected. In heat-stressed Arabidopsis plants, light-harvesting complex protein was modified by MDA under illumination. This modification was not observed in linolenic acid-deficient mutants (fad3fad7fad8 triple mutant), suggesting that linolenic acid is a major source of protein modification by MDA in heat-stressed plants.     

NTH201, a novel class II KNOTTED1-like protein, facilitates the cell-to-cell movement of Tobacco mosaic virus in tobacco

NTH201, a novel class II KNOTTED1-like protein gene, was cloned from tobacco (Nicotiana tabacum cv. Xanthi) and its role in Tobacco mosaic virus (TMV) infection was analyzed. Virus-induced gene silencing of NTH201 caused a delay in viral RNA accumulation as well as virus spread in infected tobacco plants. Overexpression of the gene in a transgenic tobacco plant (N. tabacum cv. Xanthi nc) infected by TMV showed larger local lesions than those of the nontransgenic plant. NTH201 exhibited no intercellular trafficking ability but did exhibit colocalization with movement protein (MP) at the plasmodesmata. When NTH201-overexpressing tobacco BY-2 cultured cells were infected with TMV, the accumulation of MP but not of viral genomic and subgenomic RNA clearly was accelerated compared with those in nontransgenic cells at an early infection period. The formation of virus replication complexes (VRC) also was accelerated in these transgenic cells. Conversely, NTH201-silenced cells showed less MP accumulations and fewer VRC formations than did nontransgenic cells. These results suggested that NTH201 might indirectly facilitate MP accumulation and VRC formation in TMV-infected cells, leading to rapid viral cell-to-cell movement in plants at an early infection stage.     

Up-regulation of genes related to the ubiquitin-proteasome system in the brown adipose tissue of 24-h-fasted rats

The functional balance between brown adipose tissue (BAT) and white adipose tissue (WAT) is important for metabolic homeostasis. We compared the effects of fasting on the gene expression profiles in BAT, WAT and liver by using a DNA microarray analysis. Tissues were obtained from rats that had been fed or fasted for 24 h. Taking the false discovery rate into account, we extracted the top 1,000 genes that had been differentially expressed between the fed and fasted rats. In all three tissues, a Gene Ontology analysis revealed that the lipid and protein biosynthesis-related genes had been markedly down-regulated. The whole-body fuel shift from glucose to triacylglycerol and the induction of autophagy were also observed. There was marked up-regulation of genes in the 'protein ubiquitination' category particularly in BAT of the fasted rats, suggesting that the ubiquitin-proteasome system was involved in saving energy as an adaptation to food shortage.