Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20

Nuclear factor κB (NF-κB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-κB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-κB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.     

Quercetin-induced ubiquitination and down-regulation of Her-2/neu

Her-2/neu (ErbB2) is a transmembrane tyrosine kinase and acts as a co-receptor for the other EGFR family members. It is well known that high expression of Her-2/neu is associated with a poor prognosis in breast cancer. Quercetin, a flavonoid present in many vegetables and fruits, has been studied extensively as a chemoprevention agent in several cancer models. In this study, we observed that quercetin decreased the level of Her-2/neu protein in time- and dose-dependent manners and also inhibited the downstream survival PI3K-Akt signaling pathway in Her-2/neu-overexpressing breast cancer SK-Br3 cells. We also observed that quercetin induced polyubiquitination of Her-2/neu. When the proteasome pathway was blocked by MG-132 during quercetin treatment, accumulation of the NP-40 insoluble form of Her-2/neu occurred. Interestingly, data from immunocomplex studies revealed that quercetin promoted interaction between Her-2/neu and Hsp90 which is a molecular chaperone involved in stabilization of Her-2/neu. In this condition, inhibition of Hsp90 activity by a specific inhibitor, geldanamycin (GA), or intracellular ATP depletion caused dissociation of Hsp90 from Her-2/neu and promoted ubiquitination and down-regulation of Her-2/neu protein. In addition, the carboxyl terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, played a crucial role in the quercetin-induced ubiquitination of Her-2/neu. Inhibition of tyrosine kinase activity of Her-2/neu by quercetin could indicate an lateration in the Her-2/neu structure which promotes CHIP recruitments and down-regulation of Her-2/neu. We believe that by using quercetin, new therapeutic strategies can be developed to treat Her-2/neu overexpressing cancers.     

High-affinity interaction between IKKbeta and NEMO

The Ser/Thr-specific IkappaB kinase (IKK), which comprises IKKalpha or IKKbeta and the regulatory protein NEMO, is at the bottleneck for NF-kappaB activation. IKK activity relies on interaction between NEMO and IKKalpha or IKKbeta. A conserved region in the C-terminal tail of IKKbeta or IKKalpha (NEMO-binding domain, NBD, residues 734-745 of IKKbeta) is important for interaction with NEMO. Here we show that the NBD peptide of IKKbeta is not sufficient for interaction with NEMO. Instead, a longer region of the IKKbeta C-terminal region provides high affinity for NEMO. Quantitative measurements using surface plasmon resonance and isothermal titration calorimetry confirm the differential affinities of these interactions and provide insight into the kinetic and thermodynamic behaviors of the interactions. Biochemical characterization using multiangle light scattering (MALS) coupled with refractive index shows that the longer IKKbeta C-terminal region forms a 2:2 stoichiometirc complex with NEMO.     

Disorder of trafficking system of plasma membrane and vacuole antiporter proteins causes hypersensitive response to salinity stress in <Emphasis Type="Italic">Arabidopsis Thaliana</Emphasis>

Rab GTPases play an important role in regulating intracellular vesicular trafficking in eukaryotic cells. Previously, we found that Oryza sativa rice Rab11 (OsRab11) is required for the regulation of vesicular trafficking from the trans- Golgi network (TGN) to the plasma membrane (PM) and/or vacuoles. To further elucidate the relationship between vesicular trafficking and abiotic and biotic stresses, we determined OsRab11 expression levels under several environmental stress conditions. OsRab11 expression was induced by pathogens, jasmonic acid (JA), and high salt treatment. Under high salt conditions, dominant negative OsRab11(S28N) mutant plants exhibited a hypersensitive phenotype similar to that of sos1-1, whereas overexpressed-OsRab11 plants showed resistance to high salt stress. When the expression of vacuolar and PM Na⁺/H⁺ antiporter genes such as AtNHX1, AtNHX2, and AtSOS1 was examined, there was no significant difference between the wild-type and OsRab11(S28N) mutant plants. However, PM trafficking of AtSOS1-green fluorescent protein (GFP) in 35S::AtSOS1-GFP sos1-1 plants was severely impaired by T7-OsRab11(S28N) expression. Similarly, vacuolar trafficking of AtNHX2-GFP was inhibited by T7-OsRab11 (S28N) expression. These results indicate that trafficking of PM and vacuolar antiporter proteins by OsRab11 is important for high salt stress resistance.     

Antimicrobial Peptide from <Emphasis Type="Italic">Bacillus</Emphasis> Strain K1R Exhibits Ameliorative Potential Against Vancomycin-Resistant <Emphasis Type="Italic">Enterococcus</Emphasis> Group of Organisms

Microorganisms secrete antimicrobial peptides (AMPs) which are part of the innate immune system and rapidly increase in concentration in the host upon challenge by pathogens, which they produce themselves. Kimchi, a traditional Korean food fermented by Bacillus organisms, is found to be ideal for AMP production. Our aim was to investigate the therapeutic potential of antimicrobial substances produced by Bacillus species. Peptide K1R was subjected to fermentation in a culture media containing carbon and nitrogen sources and metal ions. A protein band around 4.6 kDa was detected in tricine-SDS-PAGE and confirmed by in situ inhibitory activity of the gel. Peptide K1R was stable over a broad range of pH (6.5–9), thermo tolerant up to 60?°C and showed unaltered activity at low temperatures (0–4?°C). The complete amino acid sequence of peptide K1R was AVQGTLEDALNLSKGALNQVQKAIQNGDXLTVXGXLGTIXLAVSX. The antagonistic effect of peptide K1R against multiple drug resistant (MDR) pathogens such as Salmonella Typhimurium and Enterococcus sp. verified its potential application in treating MDR cases. The antioxidant activity of peptide K1R was also comparable to that of standard ascorbic acid.     

Cooperation between p21 and Akt is required for p53‐dependent cellular senescence

Cellular senescence has been implicated in normal aging, tissue homeostasis, and tumor suppression. Although p53 has been shown to be a central mediator of cellular senescence, the signaling pathway by which it induces senescence remains incompletely understood. In this study, we have shown that both Akt and p21 are required to induce cellular senescence in response to p53 expression. In a p53‐induced senescence model, we found that Akt activation was essential for inducing a cellular senescence phenotype. Surprisingly, Akt inhibition did not abolish p53‐induced cell cycle arrest, but it suppressed the increase in intracellular reactive oxygen species (ROS) levels. The results of the cell cycle and morphological analysis suggest that p53 induced quiescence, not senescence, following Akt inhibition. Conversely, the inhibition of p21 induction abolished cell cycle arrest but did not affect the p53‐induced increase in ROS levels. Additionally, p21 and Akt separately controlled cell cycle arrest and ROS levels, respectively, during H‐Ras‐induced senescence in human normal fibroblasts. The mechanistic analysis revealed that Akt increased ROS levels through NOX4 induction, and increased Akt‐dependent NF‐κB binding to the NOX4 promoter is responsible for NOX4 induction upon p53 expression. We further showed that Akt activation upon p53 expression is mediated by mammalian target of rapamycin complex 2. In addition, p53‐mediated IL6 and IL8 induction was abrogated by Akt inhibition, suggesting that Akt activation is also required for the senescence‐associated secretory phenotype. Collectively, these results suggest that p53 simultaneously controls multiple pathways to induce cellular senescence through p21 and Akt.     

The Korea brassica genome project: a glimpse of the brassica genome based on comparative genome analysis with Arabidopsis

A complete genome sequence provides unlimited information in the sequenced organism as well as in related taxa. According to the guidance of the Multinational Brassica Genome Project (MBGP), the Korea Brassica Genome Project (KBGP) is sequencing chromosome 1 (cytogenetically oriented chromosome #1) of Brassica rapa. We have selected 48 seed BACs on chromosome 1 using EST genetic markers and FISH analyses. Among them, 30 BAC clones have been sequenced and 18 are on the way. Comparative genome analyses of the EST sequences and sequenced BAC clones from Brassica chromosome 1 revealed their homeologous partner regions on the Arabidopsis genome and a syntenic comparative map between Brassica chromosome 1 and Arabidopsis chromosomes. In silico chromosome walking and clone validation have been successfully applied to extending sequence contigs based on the comparative map and BAC end sequences. In addition, we have defined the (peri)centromeric heterochromatin blocks with centromeric tandem repeats, rDNA and centromeric retrotransposons. In-depth sequence analyses of five homeologous BAC clones and an Arabidopsis chromosomal region reveal overall co-linearity, with 82% sequence similarity. The data indicate that the Brassica genome has undergone triplication and subsequent gene losses after the divergence of Arabidopsis and Brassica. Based on in-depth comparative genome analyses, we propose a comparative genomics approach for conquering the Brassica genome. In 2005 we intend to construct an integrated physical map, including sequence information from 500 BAC clones and integration of fingerprinting data and end sequence data of more than 100 000 BAC clones. The sequences have been submitted to GenBank with accession numbers: 10 204 BAC ends of the KBrH library (CW978640-CW988843); KBrH138P04, AC155338; KBrH117N09, AC155337; KBrH097M21, AC155348; KBrH093K03, AC155347; KBrH081N08, AC155346; KBrH080L24, AC155345; KBrH077A05, AC155343; KBrH020D15, AC155340; KBrH015H17, AC155339; KBrH001H24, AC155335; KBrH080A08, AC155344; KBrH004D11, AC155341; KBrH117M18, AC146875; KBrH052O08, AC155342.     

Biochemical changes as enzymatic defense system of phenanthrene exposure in olive flounder, Paralichthys olivaceus

The objective of this study was to investigate early biological response in olive flounder exposed to sub-lethal concentrations of waterborne phenanthrene (0.5, 1 or 2 microM). The fish were exposed for 4 weeks and we analyzed their enzymatic defense system, antioxidant and phase II enzyme activities, to evaluate the chronic exposure toxicity of phenanthrene. Waterborne phenanthrene affected antioxidant enzymes and glutathione-mediated detoxification as enzyme defense system. Hepatic, gill and kidney glutathione reductase as well as glutathione S-transferase, and catalase activities were markedly elevated after two or four weeks of exposure. These enzymes activities of olive flounder, Paralichthys olivaceus seem to be a convenient tool for monitoring pollution in coastal areas against PAHs pollution including phenanthrene.