Han-Chul Kang

MARCH5-mediated quality control on acetylated Mfn1 facilitates mitochondrial homeostasis and cell survival

Mitochondrial dynamics and quality control have a central role in the maintenance of cellular integrity. Mitochondrial ubiquitin ligase membrane-associated RING-CH (MARCH5) regulates mitochondrial dynamics. Here, we show that mitochondrial adaptation to stress is driven by MARCH5-dependent quality control on acetylated Mfn1. Under mitochondrial stress conditions, levels of Mfn1 were elevated twofold and depletion of Mfn1 sensitized these cells to apoptotic death. Interestingly, overexpression of Mfn1 also promoted cell death in these cells, indicating that a fine tuning of Mfn1 levels is necessary for cell survival. MARCH5 binds Mfn1 and the MARCH5-dependent Mfn1 ubiquitylation was significantly elevated under mitochondrial stress conditions along with an increase in acetylated Mfn1. The acetylation-deficient K491R mutant of Mfn1 showed weak interaction with MARCH5 as well as reduced ubiquitylation. Neither was observed in the acetylation mimetic K491Q mutant. In addition, MARCH5-knockout mouse embryonic fibroblast and MARCH5H43W-expressing HeLa cells lacking ubiquitin ligase activity experienced rapid cell death upon mitochondrial stress. Taken together, a fine balance of Mfn1 levels is maintained by MARCH5-mediated quality control on acetylated Mfn1, which is crucial for cell survival under mitochondria stress conditions.

Growth inhibition of a phytopathogenic fungus, Colletotrichum species by acetic acid.

Acetic, oxalic, malic, and citric acids significantly inhibited the growth of Colletotrichurm, gloeosporioides, a phytopathogenic fungus, and acetic acid showed the strongest inhibition with no growth at 50 mM. The growth inhibition by these organic acids was closely related with the inhibition of respiration, as tested using three species, C. gloeosporioides, C. coccodes, and C. dermatium. Optimum growth of C. gloeosporioides was observed around pH 6.0. The inhibition of growth by acetic acid accelerated along with a decrease in pH from 6.0 to 4.0, suggesting that the inhibition might be more enhanced by undissociated form of acetic acid. Despite of growth inhibition by acetic acid, the fungus was able to grow in a normal medium when acetic acid was eliminated, implying that the growth inhibition may be resulted from an acetic acid-mediated inhibition of respiration than a structural damage of cell. Catalase activity of the fungus increased in response to 0.1% hydrogen peroxide, but addition of this together with 30 mM acetic acid brought about a decrease in the activity. The fungus which showed no grow at 30 mM acetic acid or 0.5% hydrogen peroxide began to grow after the elimination of these. But the fungus added simultaneously by these two compounds did not grow at all despite the elimination of these. Thus, controlling of Colletotrichum might be developed using acetic acid which is generally less dangerous than chemical reagents.

Characteristics of an α-galactosidase associated with grape flesh

Abstract α-Galactosidase activity in grape flesh (Vitis venifera L. Muscat of Alexandria) was characterized by a marked increase in its activity 4 weeks after fruit bearing. After 12 weeks the specific activity of the enzyme had increased 15-fold. Several other glycosidases were measured at different stages of fruit development but none showed the increased levels of activity displayed by this α-galactosidase. α-Galactosidase activity (unit/g·fresh wt) increased by 52% during postharvest storage, whereas the unripe grape showed a “stagnancy” for 10–15 days prior to the increase. An α-galactosidase was partially purified ca. 103-fold from grape flesh of Vitis labruscana Honey black, by a procedure involving ammonium sulfate fractionation, Biogel P-60, melibiose-agarose, and Sephacryl S-200 chromatographic separations. The enzyme was effectively separated by affinity chromatography on melibiose-agarose, and was a monomer of 40–45 kDa as determined by SDS-PAGE and Sephacryl S-200 chromatographic analysis. The hydrolysis rate of p-nitrophenyl-α- d -Gal (PNP-α- d -Gal) was 4.2 times higher than that of PNP-β- d -Gal, implying an apparent α-anomer specificity, and natural oligosaccharides such as melibiose, stachyose, and raffinose were also considerably hydrolyzed. The enzyme was active over a narrow pH range with an optimal hydrolysis of stachyose and PNP-α- d -Gal at pH 6.0 and 7.0, respectively. EDTA or 1,10-phenanthroline did not substantially affect enzyme activity.

Characterization of an Aminopeptidase from Grapes

The aminopeptidase activities from grapes, Vitis labruscana B. Takasumi, increased parallel to the developmental stages and were, in order of activity, high for Phe‐, Leu‐, and Met‐p‐nitroanilides (aminoacyl‐NAs). The activities were concentrated in the hypodermis from fully ripe grapes rather than in the flesh (mesocarp and placental tissue) or seeds. The rapid hydrolysis of the substrates was a common characteristic of the hypodermis of other strains, indicating that Phe‐specific aminopeptidase may play an important role in the hypodermis. An aminopeptidase was purified over 130‐fold from the hypodermis. The enzyme is a monomer of ca. 71–74 kDa as determined by SDS‐PAGE and Sephacryl S‐200 chromatography. The purified aminopeptidase, as well the crude extracts from whole grape and hypodermis, hydrolyzed aminoacyl‐NAs with nonpolar side chains such as Phe and Leu more efficiently. The order of activity was similar to the crude extract of the hypodermis, indicating that the enzyme may be a major aminopeptidase in the hypodermis. The enzyme had a pH optimum of 7.0. The increase of activity up to pH 7.0 seems to be correlated with the increase in pH in the hypodermis during ripening. N‐terminal‐blocked Phe‐ and some oligopeptidyl‐NAs as well as azocasein were not hydrolyzed. Phenylmethylsulfonyl fluoride and N‐ethylmaleimide inhibited the enzyme in concentration‐ and time‐dependent manners. To some extent, aprotinin selectively decreased the activity. The enzyme was neither significantly influenced by some metal ions nor inhibited by EDTA or 1,10‐phenanthroline.

Identification and functional characterization of polyunsaturated fatty acid elongase (McELOVL5) gene from pike eel (Muraenesox cinereus)

The cDNA coding for a polyunsaturated fatty acid elongase (McELOVL5) was isolated from the brain of the pike eel (Muraenesox cinereus) being based on available sequences in 23 types of fish. Four sequence variants were identified with different amino acid substitutions as compared with two clones of McELOVL5 gene (McELOVL5 11.7 and McELOVL5 12.4). When the two variants of McELOVL5 were expressed in Saccharomyces cerevisiae, the two recombinant yeasts elongated γ-linolenic acid (GLA, 18:3n-6) to di-homo-γ-linolenic acid (DGLA, 20:3n-6) but differed in the rate of GLA conversion to DGLA. Cells transformed with McELOVL5 12.4 also converted arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3) to docosatetraenoic acid (22:4n-6) and docosapentaenoic acid (22:5n-3), respectively. However McELOVL5 11.7 lost its function for the elongation of C20 fatty acids. The four sequence variants have changed substrate specificities. Three-dimensional models of the McELOVL5 proteins are suggested.

Metabolic engineering to produce γ-linolenic acid in Brassica napus using a Δ6-desaturase from pike eel

We generated γ-linolenic acid (GLA, C18:3Δ6,9,12)-producing transgenic Brassica napus transformed with McD6DES, the Δ6-desaturase gene identified from pike eel (Muraenesox cinereus) under the control of the seed-specific vicilin promoter. Seed-specific expression of McD6DES in B. napus produced up to 8.4% of GLA by creating a double bond at the sixth position from the carboxyl end of linoleic acid (LA, C18:2Δ9,12) in seeds. These results demonstrate that McD6DES expression enables to reconstitute in polyunsaturated fatty acid biosynthetic pathways, highlighting the potential of GLA biosynthesis as a target for metabolic engineering of oilseed crops.

Heterologous Reconstitution of Omega-3 Polyunsaturated Fatty Acids in Arabidopsis

Reconstitution of nonnative, very-long-chain polyunsaturated fatty acid (VLC-PUFA) biosynthetic pathways in Arabidopsis thaliana was undertaken. The introduction of three primary biosynthetic activities to cells requires the stable coexpression of multiple proteins within the same cell. Herein, we report that C22 VLC-PUFAs were synthesized from C18 precursors by reactions catalyzed by Δ6-desaturase, an ELOVL5-like enzyme involved in VLC-PUFA elongation, and Δ5-desaturase. Coexpression of the corresponding genes (McD6DES, AsELOVL5, and PtD5DES) under the control of the seed-specific vicilin promoter resulted in production of docosapentaenoic acid (22:5 n-3) and docosatetraenoic acid (22:4 n-6) as well as eicosapentaenoic acid (20:5 n-3) and arachidonic acid (20:4 n-6) in Arabidopsis seeds. The contributions of the transgenic enzymes and endogenous fatty acid metabolism were determined. Specifically, the reasonable synthesis of omega-3 stearidonic acid (18:4 n-3) could be a useful tool to obtain a sustainable system for the production of omega-3 fatty acids in seeds of a transgenic T3 line 63-1. The results indicated that coexpression of the three proteins was stable. Therefore, this study suggests that metabolic engineering of oilseed crops to produce VLC-PUFAs is feasible.

Isolation and functional characterization of a PISTILLATA -1 gene promoter from Brassica napus

The PISTILLATA is a floral organ identity gene required for the flower development. To gain a better understanding of the BnPI-1 promoter function, the promoter 5′ deletion analysis was conducted, both in transgenic Brassica napus and transgenic Arabidopsis. In the β-glucuronidase (GUS) expression assay of transgenic B. napus, most of the 5′ deletion constructs of the BnPI-1 promoter expressed the GUS gene strongly in all organs of the flower except the style, and deletion up to −326 bp region (removed up to the G-box located near the TATA-box) reduced dramatically the GUS expression, and deletion up to −150 bp region (removed up to the TATA-box) abolished the GUS expression. In the GUS fluorometric assay, GUS activity in the flower was about 4-fold higher than that in seed or silique, and GUS activity in the leaf was not detected. The GUS expression patterns in transgenic Arabidopsis were similar to those in transgenic B. napus. These results suggest that the BnPI-1 promoter can lead the foreign gene expression mainly in the floral-tissue, which is shown to be regulated by the G-box element responsive to basic leucine-zipper transcription factors.

Isolation and Functional Characterization of the Brassica napus Cruciferin Gene cru4 promoter

The 12S globulin protein cruciferin is main seed storage protein in Brassica napus. To gain a better understanding of the Bncru4 promoter function, we conducted the promoter 5′ deletion analysis in transgenic Arabidopsis. In the β-glucuronidase (GUS) expression assay, Bncru4 promoter was strongly active in transgenic seeds. In addition, deletion of RY-elements (−236 bp region) dramatically decreased the promoter activity in seed embryos; however, the GUS expression could be observed in seed coat. Further deletion up to −113 bp region (removed up to the CAAT and TATA box), GUS expression was completely abolished in all tissues. These results were consistent with that of the GUS activity in transgenic seeds. Therefore, we consider that RYelement is crucial to the seed-specific expression of Bncru4 promoter