Toshio Kojima

Molecular characterization of a novel soybean gene encoding a neutral PR-5 protein induced by high-salt stress

In this study, we characterized a novel soybean gene encoding a neutral PR-5 protein and compared it to two acidic isoforms of soybean PR-5 protein. This gene, designated as Glycine max osmotin-like protein, b isoform (GmOLPb, accession no. AB370233), encoded a putative protein having the greatest similarity to chickpea PR-5b (89% identity). Unlike the two acidic PR-5, GmOLPa and P21, the protein had a C-terminal elongation responsible for possible vacuolar targeting and after maturation showed a calculated molecular mass of 21.9kDa with pI 6.0. The 3D models, predicted by the homology modeling, contained four alpha-helixes and 16 beta-strands and formed three characteristic domains. The two acidic PR-5 proteins also showed a 3D structure very similar to GmOLPb, although the electrostatic potential on molecular surface of each PR-5 was significantly different. In the study of the gene expression under conditions of high-salt stress, GmOLPb was highly induced in the leaves of the soybean, particularly in the lower part of a leaf. The expression started at 2h after initiation of the stress and was highly induced between 18-72h. Gene expression of P21e (protein homologous to P21) was transiently induced by high-salt stress, but took place earlier than the gene expressions of GmOLPa and GmOLPb. Such differential expression was observed also under investigation with methyl jasmonate and salicylic acid. These results suggested that each soybean PR-5 might play a distinctive role in the defensive system protecting the soybean plant against high-salt stress, particularly in the leaves of the soybean.

Cloning of two cysteine proteinase genes, CysP1 and CysP2, from soybean cotyledons by cDNA representational difference analysis.

By cDNA representational difference analysis (cDNA RDA) and rapid amplification of cDNA ends (RACE), we isolated two cDNAs, CysP1 and CysP2, from the cotyledons of growing soybean (Glycine max (L.) Merr.) seedlings. CysP1 cDNA is 1265 bp in size with a 1089-bp open reading frame (ORF), and CysP2 cDNA is 1270 bp in size with a 1089-bp ORF. Either CysP1 or CysP2 encodes a cysteine proteinase (CPR) with a C-terminal KDEL motif. The similarities between CysP1 and CysP2 are 93.5% in nucleotide sequences and 93.6% in deduced amino acid sequences. Furthermore, we determined the nucleotide sequences of CysP1 genomic DNA (1846 bp) and CysP2 genomic DNA (1831 bp). Both consisted of four exons and three introns. RNA-blot analysis revealed that both CysP1 and CysP2 were expressed from 6 days after germination (DAG) to 13 or 14 DAG in the cotyledons of growing seedlings and did so in a short period (9-12 DAG) in rejuvenated cotyledons. The transcripts of CysP1 and CysP2 were also detected in the root, flower and pod of soybean plants. Their physiological roles in the cotyledons of growing seedlings are discussed.

Transcriptional regulation of peptidylarginine deiminase expression in human keratinocytes

Peptidylarginine deiminase (PAD, EC 3.5.3.15) enzyme catalyzes the conversion of arginine residues to citrulline residues in the presence of calcium ion, which is an elaborate post-translational modification on the target protein. Recently, five isoforms have been identified in mammals. Among them, three isoforms (type I, II, III) are expressed in the human epidermis, and involved in several skin physiological and pathological processes. In the past few years, several researches concerning the transcriptional regulation of three human PADI type genes (PADI1, PADI2 and PADI3) in the epidermis have been carried out. In this review, we describe an overview of the current outcomes about these studies with their significance. It is anticipated that these investigations will provide novel therapeutic and prophylactic targets for future approaches to the treatment or prevention of severe psoriasis and bullous congenital ichthyosiform erythroderma.

NF-Y and Sp1/Sp3 are involved in the transcriptional regulation of the peptidylarginine deiminase type III gene (PADI3) in human keratinocytes

Human peptidylarginine deiminase type III gene (PADI3) encodes a crucial post-translational modification enzyme that converts protein-bound arginine residues into citrulline residues. Its expression is restricted to a few cell types, including keratinocytes in the granular layer of the epidermis and in the inner root sheath of hair follicles. In these cells, the enzyme is involved in terminal processing of intermediate filament-binding proteins such as filaggrin and trichohyalin. To study the molecular mechanisms that control the expression of PADI3 in human keratinocytes at the transcriptional level, we characterized its promoter region using human keratinocytes transfected with variously deleted fragments of the 5-upstream region of PADI3 coupled to the luciferase gene. We found that as few as 129 bp upstream from the transcription initiation site were sufficient to direct transcription of the reporter gene. Electrophoretic mobility-shift and chromatin immunoprecipitation assays revealed that NF-Y (nuclear factor Y) and Sp1/Sp3 (specificity protein 1/3) bind to this region in vitro and in vivo. Moreover, mutation of the Sp1- or NF-Y-binding motif markedly reduced PADI3 promoter activity. Furthermore, Sp1 or NF-YA (NF-Y subunit) small interfering RNAs effectively diminished PADI3 expression in keratinocytes cultured in both low- and high-calcium medium. These data indicate that PADI3 expression is driven by Sp1/Sp3 and NF-Y binding to the promoter region.

Molecular Characterization of a Novel Armadillo Repeat-Like Protein Gene Differentially Induced by High-Salt Stress and Dehydration from the Model Legume Lotus Japonicus

Armadillo (ARM) repeat proteins have tandem repeats of a degenerate sequence motif required for protein–protein interaction and are distributed widely in eukaryotes. In this study, we isolated and characterized a novel ARM repeat-like protein gene from the model legume Lotus japonicus that is differentially induced by abiotic stress. The gene, LjTDF-5, encodes a hypothetical protein of 369 aa with a protein signature ARM-type fold. Three-dimensional protein structure was predicted to consist of 23 α-helices and no β-sheets by homology modeling. The LjTDF-5-homologous genes were distributed broadly in the plant kingdom and the C-terminal region, around 60 amino acids in length, was highly conserved in all of the homologs examined although any known functional domains or protein signatures in this region were not detected in silico analyses. Subcellular localization assays revealed that the sGFP-fused LjTDF-5 protein localized to the nuclei of onion epidermis cells, despite the protein not containing a typical nuclear localization signal. In quantitative real-time RT-PCR, the expression of LjTDF-5 was highly induced by 100xa0mM NaCl in the roots and by dehydration in the shoot, but not by abscisic acid (ABA, 10xa0μM). These results suggest that the ARM repeat-like protein LjTDF-5 functions in or around the nucleus in response to high-salt stress and dehydration in L. japonicus.