Yoko Inai

Increased degradation of oxidized proteins in yeast defective in 26 S proteasome assembly.

An Rpn9-disrupted yeast strain, Delta rpn9, whose growth is temperature sensitive with defective assembly of the 26 S proteasome complex, was studied. This mutant yeast was more resistant to hydrogen peroxide treatment and able to degrade carbonylated proteins more efficiently than wild type. Nondenaturing gel electrophoresis followed by activity staining revealed that Delta rpn9 yeast cells had a higher activity of 20 S proteasome than wild type and that in both Delta rpn9 and wild-type cells treated with hydrogen peroxide, 20 S proteasome activity was increased with a concomitant decrease in 26 S proteasome activity. Protein multiubiquitination was not observed in the hydrogen peroxide-treated cells. Taken together, these results suggest that the 20 S proteasome degrades oxidized proteins without ubiquitination of target proteins.

Fragmentation and dimerization of copper-loaded prion protein by copper-catalysed oxidation

Prion protein consists of an N-terminal domain containing a series of octapeptide repeats with the consensus sequence PHGGGWGQ and a C-terminal domain composed of three alpha-helices and two short beta-strands. Several studies have shown that the N-terminal domain binds five Cu2+ ions. In the present study, we have investigated copper-catalysed oxidation of a recombinant mouse prion protein, PrP23-231. The copper-loaded PrP23-231 was found to be carbonylated by incubation with dopamine. Besides the formation of carbonyls, a cross-linked species with the dimeric size and C-terminally truncated species were generated. These reactions were retarded in the presence of Cu+- and Cu2+-specific copper chelators, catalase, and SOD (superoxide dismutase), but not in the presence of various bivalent metal ions. Together, these results indicate that the copper bound to prion protein undergoes catalytic cycling in the presence of catecholamines and causes the oxidation of the protein.

In vivo role of aldehyde reductase

BACKGROUND Aldehyde reductase (AKR1A; EC 1.1.1.2) catalyzes the reduction of various types of aldehydes. To ascertain the physiological role of AKR1A, we examined AKR1A knockout mice. METHODS Ascorbic acid concentrations in AKR1A knockout mice tissues were examined, and the effects of human AKR1A transgene were analyzed. We purified AKR1A and studied the activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis. Metabolomic analysis and DNA microarray analysis were performed for a comprehensive study of AKR1A knockout mice. RESULTS The levels of ascorbic acid in tissues of AKR1A knockout mice were significantly decreased which were completely restored by human AKR1A transgene. The activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis, were suppressed in AKR1A knockout mice. The accumulation of d-glucuronic acid and saccharate in knockout mice tissue and the expression of acute-phase proteins such as serum amyloid A2 are significantly increased in knockout mice liver. CONCLUSIONS AKR1A plays a predominant role in the reduction of both d-glucuronic acid and d-glucurono-γ-lactone in vivo. The knockout of AKR1A in mice results in accumulation of d-glucuronic acid and saccharate as well as a deficiency of ascorbic acid, and also leads to upregulation of acute phase proteins. GENERAL SIGNIFICANCE AKR1A is a major enzyme that catalyzes the reduction of d-glucuronic acid and d-glucurono-γ-lactone in vivo, besides acting as an aldehyde-detoxification enzyme. Suppression of AKR1A by inhibitors, which are used to prevent diabetic complications, may lead to the accumulation of d-glucuronic acid and saccharate.

C-Mannosylated peptides derived from the thrombospondin type 1 repeat interact with Hsc70 to modulate its signaling in RAW264.7 cells

The thrombospondin type 1 repeat (TSR) is a functional module of proteins called TSR superfamily proteins (e.g., thrombospondin, F-spondin, mindin, etc.) and includes a conserved Trp-x-x-Trp (W-x-x-W) motif, in which the first Trp residue is preferably modified by C-mannosylation. We previously reported that synthesized C-mannosylated TSR-derived peptides (e.g., C-Man-WSPW) specifically enhanced lipopolysaccharide-induced signaling in macrophage-like RAW264.7 cells. In this study, we searched for the proteins that bind to C-mannosylated TSR-derived peptides in RAW264.7 cells and identified heat shock cognate protein 70 (Hsc70). The binding affinity of Hsc70 for C-mannosylated peptides in solution was higher than that for the peptides without C-mannose. The binding was influenced by a nucleotide-induced conformational change of Hsc70, and C-mannosylated peptides preferred the substrate-binding domain of Hsc70. Furthermore, in RAW264.7 cells, addition of Hsc70 stimulated cellular signaling to produce tumor necrosis factor-alpha, via transforming growth factor-beta-activated kinase 1, and the Hsc70-induced signaling was enhanced more in the presence of the peptides with C-mannose than that without C-mannose, suggesting functional interaction between Hsc70 and the C-mannosylated peptides in the cells. Together, these results demonstrate a novel function of the C-mannosylation of TSR-derived peptides in terms of interaction with Hsc70 to regulate cellular signaling.

Key odorants in cured Madagascar vanilla beans (Vanilla planiforia) of differing bean quality.

The odor-active volatiles in Madagascar vanilla beans (Vanilla planiforia) of two grades, red whole beans as standard quality and cuts beans as substandard quality, were characterized by instrumental and sensory analyses. The higher contents of vanillin and β-damascenone in red whole beans than in cuts beans respectively contributed to significant differences in the sweet and dried fruit-like notes, while the higher contents of guaiacol and 3-phenylpropanoic acid in cuts beans than in red whole beans respectively contributed to significant differences in the phenolic and metallic notes. A sensory evaluation to compare red whole beans and their reconstituted aroma characterized both samples as being similar, while in respect of the phenolic note, the reconstituted aroma significantly differed from the reconstituted aroma with guaiacol added at the concentration ratio of vanillin and guaiacol in cuts beans. It is suggested from these results that the concentration ratio of vanillin and guaiacol could be used as an index for the quality of Madagascar vanilla beans.

Identification of the Key Odorants in Tahitian Cured Vanilla Beans (Vanilla tahitensis) by GC-MS and an Aroma Extract Dilution Analysis

The key odorants of Tahitian vanilla beans (Vanilla tahitensis) were characterized by a sensory evaluation, aroma extract dilution analysis (AEDA), quantification, and aroma reconstitution. Vanillin and anisaldehyde were identified in the same highest flavor dilution (FD) factor as the most characteristic odor-active compounds in Tahitian vanilla beans, followed by anisyl alcohol and anisyl acetate. Vanillin and anisyl alcohol were by far the most abundant odorants present with the highest concentration in the beans, followed by acetic acid, anisaldehyde, and anisyl acetate. A sensory evaluation of Tahitian vanilla beans and its reconstitute aroma concentrate characterized both samples as similar. These results indicated vanillin, anisaldehyde, anisyl alcohol, and anisyl acetate to be the key odorants in Tahitian vanilla beans. 3-Methylnonane-2,4-dione were identified for the first time in vanilla beans. β-Damascenone and phenylacetic acid were identified for the first time in Tahitian vanilla beans.

Calreticulin inhibits prion protein PrP-(23-98) aggregation in vitro.

Because prion protein PrP-(23–98) was recently found to polymerize into amyloid-like and proteinase K-resistant spherical aggregates in the presence of NADPH plus copper ions, we tested to determine whether calreticulin (CRT) inhibits PrP-(23–98) aggregation in vitro. The results indicated that CRT suppressed PrP-(23–98) aggregation, and that CRT-mediated solubilization occurred in the aggregates.

Proteinase K-resistant aggregates of recombinant prion protein PrP-(23-98) are toxic to cultured cells.

Here, we show for the first time that non-fibrillar and spherical aggregates produced from PrP-(23-98) in the presence of NADPH plus copper ions are toxic to cultured cells and induce apoptotic signals. It is also confirmed that endogenous cellular PrP isoform is not required for toxicity to occur.