E6AP ubiquitin ligase mediates ubiquitin‐dependent degradation of peroxiredoxin 1

E6‐associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of tumor suppressor p53 in conjunction with the high‐risk human papillomavirus E6 protein. We previously reported that E6AP targets annexin A1 protein for ubiquitin‐dependent proteasomal degradation. To gain a better understanding of the physiological function of E6AP, we have been seeking to identify novel substrates of E6AP. Here, we identified peroxiredoxin 1 (Prx1) as a novel E6AP‐binding protein using a tandem affinity purification procedure coupled with mass spectrometry. Prx1 is a 25‐kDa member of the Prx family, a ubiquitous family of antioxidant peroxidases that regulate many cellular processes through intracellular oxidative signal transduction pathways. Immunoprecipitation analysis showed that E6AP binds Prx1 in vivo. Pull‐down experiments showed that E6AP binds Prx1 in vitro. Ectopic expression of E6AP enhanced the degradation of Prx1 in vivo. In vivo and in vitro ubiquitylation assays revealed that E6AP promoted polyubiquitylation of Prx1. RNAi‐mediated downregulation of endogenous E6AP increased the level of endogenous Prx1 protein. Taken together, our data suggest that E6AP mediates the ubiquitin‐dependent proteasomal degradation of Prx1. Our findings raise a possibility that E6AP may play a role in regulating Prx1‐dependent intracellular oxidative signal transduction pathways. J. Cell. Biochem. 111: 676–685, 2010. © 2010 Wiley‐Liss, Inc.     

Taurine prevents the ischemia-induced apoptosis in cultured neonatal rat cardiomyocytes through Akt/caspase-9 pathway

Activated Akt kinase has been proposed as a central role in suppressing apoptosis by modulating the activities of Bcl-2 family proteins and/or caspase-9. To study the mechanism underlying the anti-apoptotic effect of taurine, the interaction between taurine and Akt/caspase-9 pathway was examined using a simulated ischemia model with cultured rat neonatal cardiomyocytes sealed in closed flasks. Taurine (20mM) treatment attenuated simulated ischemia-induced decline in the activity of Akt. Although taurine treatment had no effect on the expression of Bcl-2 in mitochondria and the level of cytosolic cytochrome c, it inhibited ischemia-induced cleavage of caspases 9 and 3. Moreover, adenovirus transfer of the dominant negative form of Akt objected taurine-mediated anti-apoptotic effects, cancelling the suppression of caspase-9 and caspase-3 activities by taurine. These findings provide the first evidence that taurine inhibits ischemia-induced apoptosis in cardiac myocytes with the increase in Akt activities, by inactivating caspase-9.     

ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell

We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.     

Isolation of putative glycoprotein gene from early somatic embryo of carrot and its possible involvement in somatic embryo development

Somatic embryogenesis is a unique process in plant cells. For example, embryogenic cells (EC) of carrot (Daucus carota) maintained in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) regenerate whole plants via somatic embryogenesis after the depletion of 2,4-D. Although some genes such as C-ABI3 and C-LEC1 have been found to be involved in somatic embryogenesis, the critical molecular and cellular mechanisms for somatic embryogenesis are unknown. To characterize the early mechanism in the induction of somatic embryogenesis, we isolated genes expressed during the early stage of somatic embryogenesis after 2,4-D depletion. Subtractive hybridization screening and subsequent RNA gel blot analysis suggested a candidate gene, Carrot Early Somatic Embryogenesis 1 (C-ESE1). C-ESE1 encodes a protein that has agglutinin and S-locus-glycoprotein domains and its expression is highly specific to primordial cells of somatic embryo. Transgenic carrot cells with reduced expression of C-ESE1 had wide intercellular space and decreased polysaccharides on the cell surface and showed delayed development in somatic embryogenesis. The importance of cell-to-cell attachment in somatic embryogenesis is discussed.     

Chiral discrimination of multiple profens as diastereomeric (R)-(+)-1-phenylethylamides by achiral dual-column gas chromatography

Profens were converted into diastereomeric (R)-(+)-1-phenylethylamides using ethyl chloroformate and triethylamine in dichloromethane. Gas chromatographic analysis on dual-columns with different polarities provided complete enantioresolution of eight profens, facilitating chiral discrimination based on matching with retention index sets characteristic of each enantiomer. The present method was linear (r >/= 0.9992) with good precision (0.8-6.0%) and accuracy (-9.3 to 0.003%), allowing detection of trace (R)-profens in optical purity test on four (S)-profen mixture in a single run. And the method allowed simultaneous enantiomeric screening for ibuprofen enantiomers and their chiral metabolites excreted in urine following administration of racemic ibuprofen.     

Nitric oxide inhibits ultraviolet B-induced murine keratinocyte apoptosis by regulating apoptotic signaling cascades

Cytotoxic effects of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) are considered to be one of the major causes of inflammatory diseases. On the other hand, protective effects of NO on toxic insults-induced cellular damage/apoptosis have been demonstrated recently. Ultraviolet B (UVB)-induced apoptosis of epidermal keratinocytes leads to skin inflammation and photoageing. However, it has not been elucidated what kind of effects NO has on UVB-induced keratinocyte apoptosis. Thus, in the present study, we investigated the problem and demonstrated that NO from NO donor suppressed UVB-induced apoptosis of murine keratinocytes. In addition, NO significantly suppressed activities of caspase 3, caspase 8 and caspase 9 that had been upregulated by UVB radiation. NO also suppressed p53 expression that had been upregulated by UVB radiation and upregulated Bcl-2 expression that had been downregulated by UVB radiation. These findings suggested that NO might suppress UVB-induced keratinocyte apoptosis by regulating apoptotic signaling cascades in p53, Bcl-2, caspase3, caspase 8 and caspase 9.     

Apoptotic cascade initiated by angiotensin II in neonatal cardiomyocytes: role of DNA damage

Angiotensin II contributes to ventricular remodeling by promoting both cardiac hypertrophy and apoptosis; however, the mechanism underlying the latter phenomenon is poorly understood. One possibility that has been advanced is that angiotensin II activates NADPH oxidase, generating free radicals that trigger apoptosis. In apparent support of this notion, it was found that angiotensin II-mediated apoptosis in the cardiomyocyte is blocked by the NADPH oxidase inhibitor diphenylene iodonium. However, three lines of evidence suggest that peroxynitrite, rather than superoxide, is responsible for angiotensin II-mediated DNA damage and apoptosis. First, the inducible nitric oxide inhibitor aminoguanidine prevents angiotensin II-induced DNA damage and apoptosis. Second, based on ligation-mediated PCR, the pattern of angiotensin II-induced DNA damage resembles peroxynitritemediated damage rather than damage caused by either superoxide or nitric oxide. Third, angiotensin II activates p53 through the phosphorylation of Ser15 and Ser20, residues that are commonly phosphorylated in response to DNA damage. It is proposed that angiotensin II promotes the oxidation of DNA, which in turn activates p53 to mediate apoptosis.