Hie-Joon Kim

A new arabidopsis gene, FLK, encodes an RNA binding protein with K homology motifs and regulates flowering time via FLOWERING LOCUS C

Posttranscriptional RNA metabolism plays versatile roles in the regulation of gene expression during eukaryotic growth and development. It is mediated by a group of RNA binding proteins with distinct conserved motifs. In this study, an Arabidopsis (Arabidopsis thaliana) gene, designated FLK, was identified and shown to encode a putative RNA binding protein with K homology motifs. A mutant in which FLK was inactivated by T-DNA insertion exhibited a severe late flowering phenotype both in long and short days. The late flowering phenotype was reversed by gibberellin and vernalization treatments. The FLOWERING LOCUS C (FLC) transcription was greatly upregulated, whereas those of FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 decreased in the mutant. These observations demonstrate that FLK regulates the autonomous flowering pathway via FLC. It is now evident that a battery of different RNA binding proteins are involved in the posttranscriptional regulation of flowering time in Arabidopsis.

Proteomic Analysis of the Anti-Cancer Effect of 20S-Ginsenoside Rg3 in Human Colon Cancer Cell Lines

Ginseng is a well known herbal medicine in Asia, and ginsenoside Rg3 has anti-cancer and various pharmacological effects. In particular, 20S-ginsenoside Rg3 may increase the anti-proliferative effects of chemotherapy. The authors investigated the mechanism of the anti-proliferative effect of 20S-Rg3 at the protein level in HT29 colon cancer cells. MTT, caspase-3 assays, and flow cytometry analysis were performed to determine cytotoxicity and apoptosis, and proteomic analysis was performed by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS, and a database was used to identify protein changes in 20S-Rg3 treated HT29 cells. The proteins identified included down-regulated Rho GDP dissociation inhibitor, up-regulated tropomyosin1, and annexin5 and glutathione s-transferase p1, which are apoptosis associated proteins. The anti-proliferative mechanism of 20S-Rg3 was found to be involved in mitotic inhibition, DNA replication, and repair and growth factor signaling. The findings of this study suggest that the cytotoxicity of 20S-Rg3 in colon cancer is dependent on several mechanisms, including apoptosis.

Controlled nuclear import of the transcription factor NTL6 reveals a cytoplasmic role of SnRK2.8 in the drought-stress response

Controlled proteolytic activation of membrane-anchored transcription factors provides an adaptation strategy that guarantees rapid transcriptional responses to abrupt environmental stresses in both animals and plants. NTL6 is a plant-specific NAC [NAM/ATAF1/2/CUC2] transcription factor that is expressed as a dormant plasma membrane-associated form in Arabidopsis. Proteolytic processing of NTL6 is triggered by abiotic stresses and ABA (abscisic acid). In the present study, we show that NTL6 is linked directly with SnRK (Snf1-related protein kinase) 2.8-mediated signalling in inducing a drought-resistance response. SnRK2.8 phosphorylates NTL6 primarily at Thr142. NTL6 phosphorylation by SnRK2.8 is required for its nuclear import. Accordingly, a mutant NTL6 protein, in which Thr142 was mutated to an alanine, was poorly phosphorylated and failed to enter the nucleus. In accordance with the role of SnRK2.8 in drought-stress signalling, transgenic plants overproducing either NTL6 or its active form 6ΔC (35S:NTL6 and 35S:6ΔC) exhibited enhanced resistance to water-deficit conditions such as those overproducing SnRK2.8 (35S:SnRK2.8). In contrast, NTL6 RNAi (RNA interference) plants were susceptible to dehydration as observed in the SnRK2.8-deficient snrk2.8-1 mutant. Furthermore, the dehydration-resistant phenotype of 35S:NTL6 transgenic plants was compromised in 35S:NTL6 X snrk2.8-1 plants. These observations indicate that SnRK2.8-mediated protein phosphorylation, in addition to a proteolytic processing event, is important for NTL6 function in inducing a drought-resistance response.

Proteolytic processing of an Arabidopsis membrane-bound NAC transcription factor is triggered by cold-induced changes in membrane fluidity.

Changes in membrane fluidity are the earliest cellular events that occur in plant cells upon exposure to cold. This subsequently triggers physiological processes, such as calcium influx and reorganization of actin cytoskeletons, and induces expression of cold-responsive genes. The plasma-membrane-anchored NAC (NAM/ATAF/CUC) transcription factor NTL6 is of particular interest. Cold triggers proteolytic activation of the dormant NTL6 protein, which in turn elicits pathogen-resistance responses by inducing a small group of cold-inducible PR (pathogenesis-related) genes in Arabidopsis. In the present study, we show that proteolytic processing of NTL6 is regulated by cold-induced remodelling of membrane fluidity. NTL6 processing was stimulated rapidly by cold. The protein stability of NTL6 was also enhanced by cold. The effects of cold on NTL6 processing and protein stability were significantly reduced in cold-acclimatized plants, supporting the regulation of NTL6 processing by membrane fluidity. Consistent with this, although NTL6 processing was stimulated by pharmacological agents that reduce membrane fluidity and thus mimic cold, it was inhibited when plants were treated with a 18:3 unsaturated fatty acid, linolenic acid. In addition, the pattern of NTL6 processing was changed in Arabidopsis mutants with altered membrane lipid compositions. Assays employing chemicals that inhibit activities of the proteasome and proteases showed that NTL6 processing occurs via the regulated intramembrane proteolysis mechanism. Interestingly, a metalloprotease inhibitor blocked the NTL6 processing. These observations indicate that a metalloprotease activity is responsible for NTL6 processing in response to cold-induced changes in membrane fluidity.

Determination of 5-hydroxymethylfurfural by ion-exclusion chromatography with UV detection

Abstract 5-Hydroxymethylfurfural (HMF) was determined without interferences in juices, honey, syrup, tomato paste, grape juice concentrate and dehydrated pear by anion-exclusion chromatographic separation and UV detection at 285 nm. The samples were mixed with water, filtered and injected without extensive sample treatment, which is normally required in conventional spectrophotometric or reversed-phase high-performance liquid chromatographic methods, HMF at the 50 ppb level was determined with a signal-to-noise ratio of 6. The recovery of HMF added to honey was 98% at the 10 ppm level and 91% at 30 ppm.

Tyrosinase-induced cross-linking of tyrosine-containing peptides investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Tyrosinase-induced oxidation of tyrosine is known to lead to melanin by cross-linking of 5,6-dihydroxyindole (DHI) and indole-5,6-quinone intermediates. However, tyrosinase-induced cross-linking of tyrosine-containing peptides has not been reported. We observed tyrosinase-induced adducts of tyrosine-containing peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). MALDI-TOFMS was also used to observe tyrosine adducts at various levels of oxidation derived from acid hydrolysis of the peptide adducts. The rate of tyrosinase-induced browning of lys-tyr-lys was about half of that of tyrosine. These results indicate that tyrosinase-induced browning of tyrosine-containing peptides via direct oxidation and cross-linking of the benzene ring of the tyrosine residue occurs at a significant rate and needs to be considered in melanogenesis.

Chiral separation of homocysteine by derivatization with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole followed by capillary electrophoresis using γ-cyclodextrin

Homocysteine was derivatized with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F) to form an inclusion complex with cyclodextrin and to facilitate UV detection. ABD-homocysteine showed interaction with beta- and gamma-cyclodextrin in capillary electrophoresis at pH 2.25 as indicated by the decreased migration time. However, chiral separation of D,L-ABD-homocysteine was observed using gamma-CD only. Optimal separation was obtained at pH 2.25, 50 mM gamma-CD concentration, and 20 kV applied voltage. L-ABD-Homocysteine migrated faster than the D-isomer as demonstrated by a spiking experiment using dithiothreitol-reduced L-homocystine.

De novo analysis of protein N-terminal sequence utilizing MALDI signal enhancing derivatization with Br signature

De novo analysis of protein N-terminal sequence is important for identification of N-terminal proteolytic processing such as N-terminal methionine or signal peptide removal, or for the genome annotation of uncharacterized proteins. We introduce a de novo sequencing method of protein N terminus utilizing matrix-assisted laser desorption/ionization (MALDI) signal enhancing picolinamidination with bromine isotopic tag incorporated to the N terminus. The doublet signature of bromine in the tandem mass (MS/MS) spectrum distinguished N-terminal ion series from C-terminal ion series, facilitating de novo N-terminal sequencing of protein. The dual advantage of MALDI signal enhancement by the basic picolinamidine and b-ion selection aided by Br signature is demonstrated using a variety of peptides. The N-terminal sequences of myoglobin and hemoglobin as model proteins were determined by incorporating the Br tag to the N terminus of the proteins and obtaining a series of b-ions with Br signature by MS/MS analysis after chymotryptic digestion of the tagged proteins. The N-terminal peptide was selected for MS/MS analysis from the chymotryptic digest based on the Br signature in the mass spectrum. Identification of phosphorylation site as well as N-terminal sequencing of a phosphopeptide was straightforward.

Matrix-assisted laser desorption/ionization signal enhancement of peptides by picolinamidination of amino groups

Picolinamidination of amino groups in peptides was carried out using ethyl picolinimidate tetrafluoroborate synthesized from picolinamide and triethyloxonium tetrafluoroborate. The N-terminal amino group as well as the epsilon-amino group of lysine were derivatized. The matrix-assisted laser desorption/ionization (MALDI) signal of a peptide was enhanced 20-35-fold upon picolinamidination depending on the number of amino groups derivatized. The signal enhancement effect is much higher than that of acetamidination or guanidination previously reported. Improved protein identification by mass mapping of the derivatized peptides was demonstrated.

C-terminal de novo sequencing of peptides using oxazolone-based derivatization with bromine signature.

Due to almost identical chemical properties of C-terminal and side-chain carboxylic groups, selective C-terminal derivatization has been difficult. Although oxazolone-based C-terminal derivatization is the only selective C-terminal modification available, it has not been used widely because of its low derivatization efficiency. In this paper, an improved oxazolone chemistry for incorporation of Br signature to C-terminus is reported. MS/MS analysis of the brominated peptides led to a series of y ions with Br signature, facilitating de novo C-terminal sequencing.