Dab1 binds to Fe65 and diminishes the effect of Fe65 or LRP1 on APP processing

Fe65 and Dab1 are adaptor proteins that interact with the cytoplasmic domain of amyloid precursor protein (APP) via phosphotyrosine‐binding (PTB) domain and that affect APP processing and Aβ production. Co‐expression of Dab1 with Fe65 and APP resumed nuclear translocation of Fe65 despite of its cytoplasmic anchor, APP. The decreased amount of Fe65 bound to APP was shown in co‐immunoprecipitation assay from the cells with Dab1 which also displayed the effect on APP processing. These data suggested that Fe65 and Dab1 compete for binding to APP. Surprisingly, we found that Fe65 interacts with Dab1 via C‐terminal region of Dab1 and unphosphorylated Dab1 is capable of binding Fe65. Dab1 interacts with the low‐density lipoprotein receptor‐related protein (LRP) as well as APP through its PTB domain. Dab1 significantly decreased the amount of APP bound to LRP and the level of secreted APP and APP‐CTF in LRP expressing cells, unlike Fe65. It implies that overexpression of Dab1 diminish LRP–APP complex formation, resulting in altered APP processing. The competition for overlapped binding site among adaptor proteins may be related to the regulation mechanism of APP metabolism in various conditions. J. Cell. Biochem. 111: 508–519, 2010. © 2010 Wiley‐Liss, Inc.     

AtCML8, a calmodulin-like protein, differentially activating CaM-dependent enzymes in Arabidopsis thaliana

Plants express many calmodulins (CaMs) and calmodulin-like (CML) proteins that sense and transduce different Ca²⁺ signals. Previously, we reported divergent soybean (Glycine max) CaM isoforms (GmCaM4/5) with differential abilities to activate CaM-dependent enzymes. To elucidate biological functions of divergent CaM proteins, we isolated a cDNA encoding a CML protein, AtCML8, from Arabidopsis. AtCML8 shows highest identity with GmCaM4 at the protein sequence level. Expression of AtCML8 was high in roots, leaves, and flowers but low in stems. In addition, the expression of AtCML8 was induced by exposure to salicylic acid or NaCl. AtCML8 showed typical characteristics of CaM such as Ca²⁺-dependent electrophoretic mobility shift and Ca²⁺ binding ability. In immunoblot analyses, AtCML8 was recognized only by antiserum against GmCaM4 but not by GmCaM1 antibodies. Interestingly, AtCML8 was able to activate phosphodiesterase (PDE) but did not activate NAD kinase. These results suggest that AtCML8 acts as a CML protein in Arabidopsis with characteristics similar to soybean divergent GmCaM4 at the biochemical levels.     

Isolation and characterization of enantioselective DNA aptamers for ibuprofen

Single stranded DNA aptamers that can bind to ibuprofen, a widely used anti-inflammation drug, were selected from random DNA library of 10¹⁵ nucleotides by FluMag-SELEX process. Five different sequences were selected and their enantioselectivity and affinity were characterized. Three out of five aptamer candidates did not show any affinity to (S)-ibuprofen, but only to racemic form of ibuprofen, suggesting that they are (R)-ibuprofen specific aptamers. Another two aptamer candidates showed affinity to both racemic form and (S)-ibuprofen, which were considered as (S)-ibuprofen specific aptamers. The affinity of five ssDNA aptamers isolated was in a range of 1.5–5.2 μM. In addition, all of these five aptamers did not show any affinity to analogues of ibuprofen in its profen’s group (fenoprofen, flubiprofen, and naproxen) and the antibiotics of oxytetracycline, another control.     

Synthesis and evaluation of benzoxazole derivatives as 5-lipoxygenase inhibitors

5-Lipoxygenase (5-LOX) is important enzyme in the biosynthesis of leukotrienes, and is a potential target in the treatment of asthma and allergy. We designed and synthesized a series of benzoxazoles and benzothiazoles as 5-LOX inhibitors. Fourteen compounds prepared showed the inhibition of LTC4 formation with IC₅₀ value of 0.12–23.88 μM. Also two compounds 2d and 2g showed improved airway hypersensitiveness.     

Hypermethylation of growth Arrest DNA-damage-inducible gene 45 in non-small cell lung cancer and its relationship with clinicopathologic features

The growth arrest DNA-damage-inducible protein 45 (GADD45) can serve as a key coordinator of the stress response by regulating cell cycle progression, genomic stability, DNA repair, and other stress-related responses. Although deregulation of GADD45 expression has been reported in several types of human tumors, its role in lung cancer is still unknown. DNA hypermethylation of promoter CpG islands is known to be a major mechanism for epigenetic inactivation of tumor suppressor genes. We investigated the methylation status of GADD45 family genes (GADD45A, B, and G) in 139 patients with non-small cell lung cancer (NSCLC) using methylation-specific PCR (MSP) and correlated the results with clinicopathologic features of the patients. Methylation frequencies in tumors were 1.4% for GADD45A, 7.2% for GADD45B, and 31.6% for GADD45G. RT-PCR and MSP analysis showed that promoter methylation of the GADD45G gene resulted in downregulation of its mRNA expression. GADD45G methylation was significantly more frequent in female patients than male patients (P = 0.035). This finding suggests that methylation-associated down-regulation of the GADD45G gene may be involved in lung tumorigenesis.     

A cyclophilin A CPR1 overexpression enhances stress acquisition in Saccharomyces cerevisiae

Cyclophilins are conserved cis-trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. We found the expression of cyclophilin A, Cpr1, changes in response to exposure to yeast Saccharomyces cerevisiae to abiotic stress conditions. The effect of Cpr1 overexpression in stress responses was therefore examined. The CPR1 gene was cloned to the yeast expression vector pVTU260 under regulation of an endogenous alcohol dehydrogenase (ADH) promoter. The overexpression of Cpr1 drastically increased cell viability of yeast in the presence of stress inducers, such as cadmium, cobalt, copper, hydrogen peroxide, tert-butyl hydroperoxide (t-BOOH), and sodium dodecyl sulfate (SDS). The Cpr1 expression also enhanced the cell rescue program resulting in a variety of antioxidanr enzymes including thioredoxin system (particularly, thioredoxin peroxidase), metabolic enzymes (glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase), and molecular chaperones (Hsp104, Hsp90, Hsp60 and Hsp42). Thus, our study illustrates the importance of Cpr1 as a molecular chaperone that improves cellular stress responses through collaborative relationships with other proteins when yeast cells are exposed to adverse conditions, and it also premises the improvement of yeast strains.     

Biosynthesis of dihydrochalcomycin: Characterization of a deoxyallosyltransferase (gerGTI)

Through an inactivation experiment followed by complementation, the gerGTII gene was previously characterized as a chalcosyltransferase gene involved in the biosynthesis of dihydochalcomycin. The glycosyltransferase gerGTI was identified as a deoxyallosyltransferase required for the glycosylation of D-mycinose sugar. This 6-deoxyhexose sugar was converted to mycinose, via bis-O-methylation, following attachment to the polyketide lactone during dihydrochalcomycin biosynthesis. Gene sequence alignment of gerGTI to several glycosyltransferases revealed a consensus sequence motif that appears to be characteristic of the enzymes in this sub-group of the glycosyltransferase family. To characterize its putative function, genetic disruption of gerGTI in the wild-type strain Streptomyces sp. KCTC 0041BP and in the gerGTII-deleted mutant (S. sp. ΔgerGTII), as well as complementation of gerGTII in S. sp. ΔgerGTII-GTI, were carried out, and the products were analyzed by LC/MS. S. sp. ΔgerGTII-GTI mutant produced dihydrochalconolide macrolide. S. sp. ΔgerGTI and S. sp. ΔgerGTII-GTI complementation of gerGTII yielded dihydrochalconolide without the mycinose sugar. The intermediate shows that gerGTI encodes a deoxyallosyltransferase that acts after gerGTII.     

Identification and characterization of a pantothenate kinase (PanK-sp) from streptomyces peucetius ATCC 27952

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Here, we report the identification, cloning, and characterization of panK-sp from Streptomyces peucetius ATCC 27952. The gene encoded a protein of 332 amino acids with a calculated molecular mass of 36.8 kDa and high homology with PanK from S. avermitilis and S. coelicolor A3(2). To elucidate the putative function of PanK-sp, it was cloned into pET32a(+) to construct pPKSP32, and the PanK-sp was then expressed in E. coli BL21(DE3) as a His-tag fusion protein and purified by immobilized metal affinity chromatography. The enzyme assay of PanK-sp was carried out as a coupling assay. The gradual decrease in NADH concentration with time clearly indicated the phosphorylating activity of PanK-sp. Furthermore, the ca. 1.4-fold increase of DXR and the ca. 1.5-fold increase of actinorhodin by in vivo overexpression of panK-sp, constructed in pIBR25 under the control of a strong ermE* promoter, established its positive role in secondary metabolite production from S. peucetius and S. coelicolor, respectively.