Pin1 promotes production of Alzheimer's amyloid beta from beta-cleaved amyloid precursor protein

Here we show that prolyl isomerase Pin1 is involved in the Abeta production central to the pathogenesis of Alzheimer's disease. Enzyme immunoassay of brains of the Pin1-deficient mice revealed that production of Abeta40 and Abeta42 was lower than that of the wild-type mice, indicating that Pin1 promotes Abeta production in the brain. GST-Pin1 pull-down and immunoprecipitation assay revealed that Pin1 binds phosphorylated Thr668-Pro of C99. In the Pin1-/- MEF transfected with C99, Pin1 co-transfection enhanced the levels of Abeta40 and Abeta42 compared to that without Pin1 co-transfection. In COS7 cells transfected with C99, Pin1 co-transfection enhanced the generation of Abeta40 and Abeta42, and reduced the expression level of C99, facilitating the C99 turnover. Thus, Pin1 interacts with C99 and promotes its gamma-cleavage, generating Abeta40 and Abeta42. Further, GSK3 inhibitor lithium blocked Pin1 binding to C99 by decreasing Thr668 phosphorylation and attenuated Abeta generation, explaining the inhibitory effect of lithium on Abeta generation.     

Use of GFP tags to monitor localization of different luciferases in E. coli.

The utility of the green fluorescent protein (GFP) as a probe to monitor protein localization in living cells is gaining a great deal of attention. In this study, to understand the localization of luciferases in E. coli, we have attached GFP tags at both the N- and the C-terminus of firefly luciferase (FF-Luc)(from Pyrocoelia miyako) and of red (RE-Luc) and green (GR-Luc) bioluminescence-emitting luciferases (from Phrixothrix railroad-worms), respectively. There was no significant change in the bioluminescence emission spectrum for any of the three luciferases following the tagging with GFP at either the N- or C-terminus, confirming the absence of energy transfer between one another. Using confocal imaging microscopy, we observed that all three luciferases expressed in the E.coli cultured at 37 degrees C tend to aggregate and are seen to localize in the poles, thus confirming their poor folding properties. In contrast, in the E.coli cultured at 18 degrees C FF-Luc was found to be highly expressed in the soluble form when compared to RE-Luc and GR-Luc. These results support our previous finding that the folding properties of FF-Luc and RE/GR-Luc are totally different.     

Partitioning of the F plasmid: Overproduction of an essential protein for partition inhibits plasmid maintenance

Summary Multicopy plasmids carrying the sopB gene of the F plasmid inhibit stable inheritance of a coexisting mini-F plasmid. This incompatibility, termed IncG, is found to be caused by excess amounts of the SopB protein, which is essential for accuratepartitioning of plasmid DNA molecules into daughter cells. A sopB-carrying multicopy plasmid that shows the IncG⁺ phenotype was mutagenized in vitro and IncG negative mutant plasmids were isolated. Among these amber and missense mutants of sopB, mutants with a low plasmid copy number and a mutant in the Shine-Dalgarno sequence for translation of the SopB protein were obtained. These results demonstrate that the IncG phenotype is caused by the SopB protein, and that the incompatibility is expressed only when the protein is overproduced. This suggests that the protein must be kept at appropriate concentrations to ensure stable maintenance of the plasmid.     

Possible involvement of the ugpA gene product in the stable maintenance of mini-F plasmid in Escherichia coli

Summary The seg-3 mutant Escherichia coli does not support the maintenance of mini-F plasmid at 42° C. We cloned the chromosomal DNA segment of the wild-type strain W3110 that complements the Seg^– phenotype of this mutant. Cleavage mapping of this segment showed that it was derived from the 76-min region of the E. coli chromosome map. Complementation tests using plasmids carrying subcloned DNA segments suggested that the seg-3 mutant carried two mutations that additively affected the maintenance of mini-F plasmid; one was in the ugpA gene and the other was presumably in the rpoH gene. We generated a disrupted ugpA null mutant and found that the mini-F plasmid was unstable in this ugpA null mutant even at 30° C. This suggests that the ugpA gene product is required for the stable maintenance of mini-F plasmid.     

Aralin,a type II ribosome-inactivating protein from Aralia elata, exhibits selective anticancer activity through the processed form of a 110-kDa high-density lipoprotein-binding protein: A promising anticancer drug

Aralin from Aralia elata is a newly identified type II ribosome- inactivating protein, which preferentially induces apoptosis in cancer cells. In this study, we identified that the aralin receptor is a 110-kDa high-density lipoprotein-binding protein (HDLBP), which functions as a HDL receptor. The sensitivities of tumor cell lines to aralin were dependent on the expression levels of the 110-kDa HDLBP and its forced expression in aralin-resistant Huh7 cells conferred aralin sensitivity. HDLBP-knockdown HeLa cells showed a significant aralin resistance in vitro and in vivo. Conversely, ectopic expression of the 150-kDa HDLBP resulted in increased aralin sensitivity in vivo, accompanying enhanced expression of the 110-kDa HDLBP. Thus, these results showed that the110-kDa HDLBP in lipid rafts acted as an aralin receptor and that its expression levels determined aralin sensitivity, suggesting that aralin could be a promising anticancer drug for HDLBP-overexpressing tumors.     

Metabolomics approach for enzyme discovery

The search for novel enzymes is an important but difficult task in functional genomics. Here, we present a systematic method based on in vitro assays in combination with metabolite profiling to discover novel enzymatic activities. A complex mixture of metabolites is incubated with purified candidate proteins and the reaction mixture is subsequently profiled by capillary electrophoresis electrospray ionization mass spectrometry (CE-MS). Specific changes in the metabolite composition can directly suggest the presence of an enzymatic activity while subsequent identification of the compounds whose level changed specifically can pinpoint the actual substrate(s) and product(s) of the reaction. We first evaluated the method using several Escherichia coli metabolic enzymes and then applied it to the functional screening of uncharacterized proteins. In this manner, YbhA and YbiV proteins were found to display both phosphotransferase and phosphatase activity toward different sugars/sugar phosphates. Our approach should be broadly applicable and useful for enzyme discovery in any system.     

Carbonic anhydrase II regulates differentiation of ameloblasts via intracellular pH‐dependent JNK signaling pathway

Differentiation of ameloblasts from undifferentiated epithelial cells is controlled by diverse growth factors, as well as interactions between epithelium and mesenchyme. However, there is a considerable lack of knowledge regarding the precise mechanisms that control ameloblast differentiation and enamel biomineralization. We found that the expression level of carbonic anhydrase II (CAII) is strongly up‐regulated in parallel with differentiation of enamel epithelium tissues, while the enzyme activity of CA was also increased along with differentiation in ameloblast primary cultures. The expression level of amelogenin, a marker of secretory‐stage ameloblasts, was enhanced by ethoxzolamide (EZA), a CA inhibitor, as well as CAII antisense (CAIIAS), whereas the expression of enamel matrix serine proteinase‐1 (EMSP‐1), a marker for maturation‐stage ameloblasts, was suppressed by both. These agents also promoted ameloblast proliferation. In addition, inhibition of ameloblast differentiation by EZA and CAIIAS was confirmed using tooth germ organ cultures. Furthermore, EZA and CAIIAS elevated intracellular pH in ameloblasts, while experimental decreases in intracellular pH abolished the effect of CAIIAS on ameloblasts and triggered the activation of c‐Jun N‐terminal kinase (JNK). SP600125, a JNK inhibitor, abrogated the response of ameloblasts to an experimental decrease in intracellular pH, while the inhibition of JNK also impaired ameloblast differentiation. These results suggest a novel role for CAII during amelogenesis, that is, controlling the differentiation of ameloblasts. Regulation of intracellular pH, followed by activation of the JNK signaling pathway, may be responsible for the effects of CAII on ameloblasts. J. Cell. Physiol. 225: 709–719, 2010. © 2010 Wiley‐Liss, Inc.