Kyun Oh Lee

A Chinese cabbage cDNA with high sequence identity to phospholipid hydroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase

A cDNA, PHCC-TPx , specifying a protein highly homologous to known phospholipid hydroperoxide glutathione peroxidases was isolated from a Chinese cabbage cDNA library. PHCC-TPx encodes a preprotein of 232 amino acids containing a putative N-terminal chloroplast targeting sequence and three conserved Cys residues (Cys107, Cys136, and Cys155). The mature form of enzyme without the signal peptide was expressed in Escherichia coli, and the recombinant protein was found to utilize thioredoxin (Trx) but not GSH as an electron donor. In the presence of a Trx system, the protein efficiently reduces H2O2 and organic hydroperoxides. Complementation analysis shows that overexpression of the PHCC-TPx restores resistance to oxidative stress in yeast mutants lacking GSH but fails to complement mutant lacking Trx, suggesting that the reducing agent of PHCC-TPx in vivo is not GSH but is Trx. Mutational analysis of the three Cys residues individually replaced with Ser shows that Cys107 is the primary attacking site by peroxide, and oxidized Cys107 reacts with Cys155-SH to make an intramolecular disulfide bond, which is reduced eventually by Trx. Tryptic peptide analysis by matrix-assisted laser desorption and ionization time of flight mass spectrometry shows that Cys155 can form a disulfide bond with either Cys107 or Cys136.

Phosphorylation and concomitant structural changes in human 2-Cys peroxiredoxin isotype I differentially regulate its peroxidase and molecular chaperone functions

The H2O2‐catabolizing peroxidase activity of human peroxiredoxin I (hPrxI) was previously shown to be regulated by phosphorylation of Thr90. Here, we show that hPrxI forms multiple oligomers with distinct secondary structures. HPrxI is a dual function protein, since it can behave either as a peroxidase or as a molecular chaperone. The effects of phosphorylation of hPrxI on its protein structure and dual functions were determined using site‐directed mutagenesis, in which the phosphorylation site was substituted with aspartate to mimic the phosphorylated status of the protein (T90D‐hPrxI). Phosphorylation of the protein induces significant changes in its protein structure from low molecular weight (MW) protein species to high MW protein complexes as well as its dual functions. In contrast to the wild type (WT)‐ and T90A‐hPrxI, the T90D‐hPrxI exhibited a markedly reduced peroxidase activity, but showed about sixfold higher chaperone activity than WT‐hPrxI.

Rice 1Cys-peroxiredoxin over-expressed in transgenic tobacco does not maintain dormancy but enhances antioxidant activity

Possible functions that have been proposed for the plant 1Cys‐peroxiredoxin, include activity as a dormancy regulator and as an antioxidant. The transcript level of ice ys‐ eroxi edo in (R1C‐Prx) rapidly decreased after imbibition of rice seeds, but the protein was detected for 15 days after imbibition. To investigate the function of this protein, we generated transgenic tobacco plants constitutively expressing the R1C‐Prx gene. The transgenic R1C‐Prx plants showed a germination frequency similar to control plants. However, the transgenic lines exhibited higher resistance against oxidative stress, suggesting that antioxidant activity may be its primary function.

Heat-shock dependent oligomeric status alters the function of a plant-specific thioredoxin-like protein, AtTDX

We found that Arabidopsis AtTDX, a heat-stable and plant-specific thioredoxin (Trx)-like protein, exhibits multiple functions, acting as a disulfide reductase, foldase chaperone, and holdase chaperone. The activity of AtTDX, which contains 3 tetratricopeptide repeat (TPR) domains and a Trx motif, depends on its oligomeric status. The disulfide reductase and foldase chaperone functions predominate when AtTDX occurs in the low molecular weight (LMW) form, whereas the holdase chaperone function predominates in the high molecular weight (HMW) complexes. Because deletion of the TPR domains results in a significant enhancement of AtTDX disulfide reductase activity and complete loss of the holdase chaperone function, our data suggest that the TPR domains of AtTDX block the active site of Trx and play a critical role in promoting the holdase chaperone function. The oligomerization status of AtTDX is reversibly regulated by heat shock, which causes a transition from LMW to HMW complexes with concomitant functional switching from a disulfide reductase and foldase chaperone to a holdase chaperone. Overexpression of AtTDX in Arabidopsis conferred enhanced heat shock resistance to plants, primarily via its holdase chaperone activity.

Heat-Shock and Redox-Dependent Functional Switching of an h-Type Arabidopsis Thioredoxin from a Disulfide Reductase to a Molecular Chaperone

A large number of thioredoxins (Trxs), small redox proteins, have been identified from all living organisms. However, many of the physiological roles played by these proteins remain to be elucidated. We isolated a high Mr (HMW) form of h-type Trx from the heat-treated cytosolic extracts of Arabidopsis (Arabidopsis thaliana) suspension cells and designated it as AtTrx-h3. Using bacterially expressed recombinant AtTrx-h3, we find that it forms various protein structures ranging from low and oligomeric protein species to HMW complexes. And the AtTrx-h3 performs dual functions, acting as a disulfide reductase and as a molecular chaperone, which are closely associated with its molecular structures. The disulfide reductase function is observed predominantly in the low Mr forms, whereas the chaperone function predominates in the HMW complexes. The multimeric structures of AtTrx-h3 are regulated not only by heat shock but also by redox status. Two active cysteine residues in AtTrx-h3 are required for disulfide reductase activity, but not for chaperone function. AtTrx-h3 confers enhanced heat-shock tolerance in Arabidopsis, primarily through its chaperone function.

Regulation of seed germination and seedling growth by an Arabidopsis phytocystatin isoform, AtCYS6

Phytocystatins are cysteine proteinase inhibitors in plants that are implicated in the endogenous regulation of protein turnover and defense mechanisms against insects and pathogens. A cDNA encoding a phytocystatin called AtCYS6 (Arabidopsis thaliana phytocystatin6) has been isolated. We show that AtCYS6 is highly expressed in dry seeds and seedlings and that it also accumulates in flowers. The persistence of AtCYS6 protein expression in seedlings was promoted by abscisic acid (ABA), a seed germination and post-germination inhibitory phytohormone. This finding was made in transgenic plants bearing an AtCYS6 promoter–β-glucuronidase (GUS) reporter construct, where we found that expression from the AtCYS6 promoter persisted after ABA treatment but was reduced under control conditions and by gibberellin4+7 (GA4+7) treatment during the germination and post-germinative periods. In addition, constitutive over-expression of AtCYS6 retarded germination and seedling growth, whereas these were enhanced in an AtCYS6 knock-out mutant (cys6-2). Additionally, cysteine proteinase activities stored in seeds were inhibited by AtCYS6 in transgenic Arabidopsis. From these data, we propose that AtCYS6 expression is enhanced by the germination inhibitory phytohormone ABA and that it participates in the control of germination rate and seedling growth by inhibiting the activity of stored cysteine proteinases.

Abnormal Chloroplast Development and Growth Inhibition in Rice Thioredoxin m Knock-Down Plants

Plant cells contain several thioredoxin isoforms that are characterized by subcellular localization and substrate specificity. Here, we describe the functional characterization of a rice (Oryza sativa) thioredoxin m isoform (Ostrxm) using a reverse genetics technique. Ostrxm showed green tissue-specific and light-responsive mRNA expression. Ostrxm was localized in chloroplasts of rice mesophyll cells, and the recombinant protein showed dithiothreitol-dependent insulin β-chain reduction activity in vitro. RNA interference (RNAi) of Ostrxm resulted in rice plants with developmental defects, including semidwarfism, pale-green leaves, abnormal chloroplast structure, and reduced carotenoid and chlorophyll content. Ostrxm RNAi plants showed remarkably decreased Fv/Fm values under high irradiance conditions (1,000 μmol m−2 s−1) with delayed recovery. Two-dimensional electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight analysis showed that the levels of several chloroplast proteins critical for photosynthesis and biogenesis were significantly decreased in Ostrxm RNAi plants. Furthermore, 2-Cys peroxiredoxin, a known target of thioredoxin, was present in oxidized forms, and hydrogen peroxide levels were increased in Ostrxm RNAi plants. The pleiotropic effects of Ostrxm RNAi suggest that Ostrxm plays an important role in the redox regulation of chloroplast target proteins involved in diverse physiological functions.

Thioredoxin Reductase Type C (NTRC) Orchestrates Enhanced Thermotolerance to Arabidopsis by Its Redox-Dependent Holdase Chaperone Function

Genevestigator analysis has indicated heat shock induction of transcripts for NADPH-thioredoxin reductase, type C (NTRC) in the light. Here we show overexpression of NTRC in Arabidopsis (NTRC°(E)) resulting in enhanced tolerance to heat shock, whereas NTRC knockout mutant plants (ntrc1) exhibit a temperature sensitive phenotype. To investigate the underlying mechanism of this phenotype, we analyzed the proteins biochemical properties and protein structure. NTRC assembles into homopolymeric structures of varying complexity with functions as a disulfide reductase, a foldase chaperone, and as a holdase chaperone. The multiple functions of NTRC are closely correlated with protein structure. Complexes of higher molecular weight (HMW) showed stronger activity as a holdase chaperone, while low molecular weight (LMW) species exhibited weaker holdase chaperone activity but stronger disulfide reductase and foldase chaperone activities. Heat shock converted LMW proteins into HMW complexes. Mutations of the two active site Cys residues of NTRC into Ser (C217/454S-NTRC) led to a complete inactivation of its disulfide reductase and foldase chaperone functions, but conferred only a slight decrease in its holdase chaperone function. The overexpression of the mutated C217/454S-NTRC provided Arabidopsis with a similar degree of thermotolerance compared with that of NTRC°(E) plants. However, after prolonged incubation under heat shock, NTRC°(E) plants tolerated the stress to a higher degree than C217/454S-NTRC°(E) plants. The results suggest that the heat shock-mediated holdase chaperone function of NTRC is responsible for the increased thermotolerance of Arabidopsis and the activity is significantly supported by NADPH.

GSH-dependent peroxidase activity of the rice (Oryza sativa) glutaredoxin, a thioltransferase

Glutaredoxin (Grx) is a 12-kDa thioltransferase that reduces disulfide bonds of other proteins and maintains the redox potential of cells. In addition to its oxidoreductase activity, we report here that a rice Grx (OsGrx) can also function as a GSH-dependent peroxidase. Because of this antioxidant activity, OsGrx protects glutamine synthetase from oxidative damage. Individually replacing the conserved Cys residues in OsGrx with Ser shows that Cys(23), but not Cys(26), is essential for the thioltransferase and GSH-dependent peroxidase activities. Kinetic characterization of OsGrx reveals that the maximal catalytic efficiency (V(max)/K(m)) is obtained with cumene hydroperoxide rather than H(2)O(2) or t-butyl hydroperoxide.

The unfolded protein response in plants: a fundamental adaptive cellular response to internal and external stresses.

In eukaryotic cells, proteins that enter the secretory pathway are translated on membrane-bound ribosomes and translocated into the endoplasmic reticulum (ER), where they are subjected to chaperone-assisted folding, post-translational modification and assembly. During the evolution of the eukaryotic cell, a homeostatic mechanism was developed to maintain the functions of the ER in the face of various internal and external stresses. The most severe stresses imposed on eukaryotic cells can induce ER stress that can overwhelm the processing capacity of the ER, leading to the accumulation of unfolded proteins in the ER lumen. To cope with this accumulation of unfolded proteins, the unfolded protein response (UPR) is activated to alter transcriptional programs through inositol-requiring enzyme 1 (IRE1) and bZIP17/28 in plants. In addition to transcriptional induction of UPR genes, quality control (QC), translational attenuation, ER-associated degradation (ERAD) and ER stress-induced apoptosis are also conserved as fundamental adaptive cellular responses to ER stress in plants. This article is part of a Special Issue entitled: Translational Plant Proteomics.