Ho Byoung Chae

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.

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.

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.

Heat‐induced chaperone activity of serine/threonine protein phosphatase 5 enhances thermotolerance in Arabidopsis thaliana

• This study reports that Arabidopsis thaliana protein serine/threonine phosphatase 5 (AtPP5) plays a pivotal role in heat stress resistance. A high-molecular-weight (HMW) form of AtPP5 was isolated from heat-treated A. thaliana suspension cells. AtPP5 performs multiple functions, acting as a protein phosphatase, foldase chaperone, and holdase chaperone. The enzymatic activities of this versatile protein are closely associated with its oligomeric status, ranging from low oligomeric protein species to HMW complexes. • The phosphatase and foldase chaperone functions of AtPP5 are associated primarily with the low-molecular-weight (LMW) form, whereas the HMW form exhibits holdase chaperone activity. Transgenic over-expression of AtPP5 conferred enhanced heat shock resistance to wild-type A. thaliana and a T-DNA insertion knock-out mutant was defective in acquired thermotolerance. A recombinant phosphatase mutant (H290N) showed markedly increased holdase chaperone activity. • In addition, enhanced thermotolerance was observed in transgenic plants over-expressing H290N, which suggests that the holdase chaperone activity of AtPP5 is primarily responsible for AtPP5-mediated thermotolerance. • Collectively, the results from this study provide the first evidence that AtPP5 performs multiple enzymatic activities that are mediated by conformational changes induced by heat-shock stress.

Hwanghaeicola aestuarii gen. nov., sp. nov., a moderately halophilic bacterium isolated from a tidal flat of the Yellow Sea.

A moderately halophilic Gram-staining-negative bacterium, designated strain Y26(T), was isolated from a tidal flat of Taean coast in South Korea. Cells were strictly aerobic, motile cocci with a single flagellum and showed catalase- and oxidase-positive reactions. Growth of strain Y26(T) was observed at 15-35 °C (optimum 25-30°C), pH 6.0-8.0 (optimum pH6.5-7.5) and with 1.5-6.0 % (w/v) NaCl (optimum 2.0-3.0 %). The predominant fatty acids were C(18 : 1)ω7c (66.2 %), C(16 : 0) (12.4 %) and C(10 : 0) 3-OH (5.0 %) and the G+C content of the genomic DNA was 61.0 mol%. Strain Y26(T) contained ubiquinone-10 (Q-10) as the major respiratory quinone. Comparative 16S rRNA gene sequence analysis showed that strain Y26(T) formed a distinct phyletic lineage from other genera within the Roseobacter clade of the class Alphaproteobacteria and was most closely related to members of the genera Maribius, Maritimibacter and Palleronia with 93.8-94.6 % sequence similarity. On the basis of chemotaxonomic data and molecular properties, strain Y26(T) represents a novel genus, Hwanghaeicola, within the family Rhodobacteraceae, for which the name Hwanghaeicola aestuarii gen. nov., sp. nov. is proposed. The type strain is Y26(T) (=KACC 13705(T) =DSM 22009(T)).

HY5, a positive regulator of light signaling, negatively controls the unfolded protein response in Arabidopsis

Significance In nature, plants are inevitably exposed to adverse conditions such as salinity, drought, and extreme temperatures. Recent reports suggest that environmental stresses critically affect the protein-folding capacity of endoplasmic reticula (ER), leading to ER stress. As the growth and development of plants significantly depend on light environment, the crosstalk between light signaling and ER stress response explained in current research can be a unique feature of plants. Our results suggest that light increases the ER stress sensitivity of plants and ELONGATED HYPOCOTYL 5, a positive regulator of light signaling, negatively regulates unfolded protein response gene expression in plant cells, which decreases the protein-folding capacity. The present study may form the basis for designing new strategies to increase stress tolerance of plants by tightly controlling light environment. Light influences essentially all aspects of plant growth and development. Integration of light signaling with different stress response results in improvement of plant survival rates in ever changing environmental conditions. Diverse environmental stresses affect the protein-folding capacity of the endoplasmic reticulum (ER), thus evoking ER stress in plants. Consequently, the unfolded protein response (UPR), in which a set of molecular chaperones is expressed, is initiated in the ER to alleviate this stress. Although its underlying molecular mechanism remains unknown, light is believed to be required for the ER stress response. In this study, we demonstrate that increasing light intensity elevates the ER stress sensitivity of plants. Moreover, mutation of the ELONGATED HYPOCOTYL 5 (HY5), a key component of light signaling, leads to tolerance to ER stress. This enhanced tolerance of hy5 plants can be attributed to higher expression of UPR genes. HY5 negatively regulates the UPR by competing with basic leucine zipper 28 (bZIP28) to bind to the G-box–like element present in the ER stress response element (ERSE). Furthermore, we found that HY5 undergoes 26S proteasome-mediated degradation under ER stress conditions. Conclusively, we propose a molecular mechanism of crosstalk between the UPR and light signaling, mediated by HY5, which positively mediates light signaling, but negatively regulates UPR gene expression.

Inhibitor of Apoptosis (IAP)-like Protein Lacks a Baculovirus IAP Repeat (BIR) Domain and Attenuates Cell Death in Plant and Animal Systems

A novel Arabidopsis thaliana inhibitor of apoptosis was identified by sequence homology to other known inhibitor of apoptosis (IAP) proteins. Arabidopsis IAP-like protein (AtILP) contained a C-terminal RING finger domain but lacked a baculovirus IAP repeat (BIR) domain, which is essential for anti-apoptotic activity in other IAP family members. The expression of AtILP in HeLa cells conferred resistance against tumor necrosis factor (TNF)-α/ActD-induced apoptosis through the inactivation of caspase activity. In contrast to the C-terminal RING domain of AtILP, which did not inhibit the activity of caspase-3, the N-terminal region, despite displaying no homology to known BIR domains, potently inhibited the activity of caspase-3 in vitro and blocked TNF-α/ActD-induced apoptosis. The anti-apoptotic activity of the AtILP N-terminal domain observed in plants was reproduced in an animal system. Transgenic Arabidopsis lines overexpressing AtILP exhibited anti-apoptotic activity when challenged with the fungal toxin fumonisin B1, an agent that induces apoptosis-like cell death in plants. In AtIPL transgenic plants, suppression of cell death was accompanied by inhibition of caspase activation and DNA fragmentation. Overexpression of AtILP also attenuated effector protein-induced cell death and increased the growth of an avirulent bacterial pathogen. The current results demonstrated the existence of a novel plant IAP-like protein that prevents caspase activation in Arabidopsis and showed that a plant anti-apoptosis gene functions similarly in plant and animal systems.

Serine/threonine protein phosphatase 5 (PP5) interacts with substrate under heat stress conditions and forms protein complex in Arabidopsis.

Protein phosphatase 5 plays a pivotal role in signal transduction in animal and plant cells, and it was previously shown that Arabidopsis protein phosphatase 5 (AtPP5) performs multiple enzymatic activities that are mediated by conformational changes induced by heat shock stress. In addition, transgenic overexpression of AtPP5 gene conferred enhanced heat shock resistance compared with wild-type plant. However, the molecular mechanism underlying this enhanced heat shock tolerance through functional and conformational changes upon heat stress is not clear. In this report, AtPP5 was shown to preferentially interact with its substrate, MDH, under heat stress conditions. In addition, in co-IP analysis, AtPP5 was observed to form a complex with AtHsp90 in Arabidopsis. These results suggest that AtPP5 may enhance thermotolerance via forming multi-chaperone complexes under heat shock conditions in Arabidopsis. Finally, we show that AtPP5 is primarily localized in the cytoplasm of Arabidopsis.

The membrane-tethered NAC transcription factor, AtNTL7, contributes to ER-stress resistance in Arabidopsis

We screened for endoplasmic reticulum (ER) stress-resistant mutants among 25 mutants of the Arabidopsis NTL (NAC with Transmembrane motif 1-Like) family. We identified a novel mutant, SALK_044777, showing strong resistance to ER stress. RT-PCR and genomic DNA sequence analyses identified the mutant as atntl7, which harbors a T-DNA insertion in the fourth exon of AtNTL7. Two other atntl7-mutant alleles, in which T-DNA was inserted in the second exon and third intron of AtNTL7, respectively, showed ER-stress sensitive phenotypes, suggesting that SALK_044777 is a gain-of-function mutant. Arabidopsis plants overexpressing AtNTL7 showed strong ER-stress resistance. Our findings suggest that AtNTL7 fragment is cleaved from the ER membrane under ER stress and translocates into the nucleus to induce downstream ER-stress responsive genes.