• Proceedings of the Botanical Society of Korea Conference
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• 1999.04a
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• pp.18-18
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• 1999

We characterized a cDNA clone for a low molecular weight heat-shock protein (LMW HSP) from tobacco named TLHS-l. Nucleotide sequence determination of TLHS-1 identified an open reading frame for 159 amino acids. To the upstream of the open reading frame, a sequence of 124 nucleotides was determined. To the 3' downstream of the open reading frame, 212 nucleotides were identified which carried poly(A)-tail. Comparison of the open reading frame and hydropathy plot of TLHS-1 with the previously reported class I LMW HSPs showed high identity which classified TLHS-1 as a class I LMW HSP cDNA clone. We proposed that there are six consensus regions in class I LMW HSPs. RNA blot hybridization for TLHS-1 showed a typical expression pattern of heat-shock-inducible gene from three common tobacco cultivars. The open reading frame of TLHS-1 was overexpressed in Escherichia coli. TLHS-1 protein confers thermal protection of other proteins in vitro and in vivo. Thermal induced aggregation of citrate synthase was reduced by purified TLHS-1 protein, and thermal death rate at $50^{\circ}C$ was reduced in E. coli expressing TLHS-l. From these data, we can expect that TLHS-1 acts as a molecular chaperone.perone.

• Bulletin of the Korean Chemical Society
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• v.34 no.11
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• pp.3405-3409
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• 2013

The activation of heat shock factor 1 (HSF1) can be induced by the changes in environmental pH, but the mechanism of HSF1 activation by acidification is not completely understood. This paper reports that a low pH (pH~6.0) can trigger human HSF1 activation. Considering the involvement of the imidazole group of histidine residues under acid pH stress, an in vitro EMSA experiment, Trp-fluorescence spectroscopy, and protein structural analysis showed that the residue, His83, is the essential for pH-dependent human HSF1-activation. To determine the roles of His83 in the HSF1-mediated stress response affecting the cellular acid resistance, mouse embryo fibroblasts with normal wild-type or mutant mouse HSF1 expression were preconditioned by heating or pH stress. The results suggest that His83 is essential for HSF1 activation or the HSF1-mediated transcription of heat shock proteins, in protecting cells from acid pH stress.

• Korean Journal of Microbiology
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• v.38 no.4
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• pp.247-253
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• 2002

The cellular responses of TNT-degrading bacterium, Stenotrophomonas sp. OK-5 to explosive 2,4,6-trini-trotoluene (TNT) as an environmental contaminant were examined. Survival of the strain OK-5 with time in the presence of different concentrations of TNT under sublethal conditions was monitored, and viable counts paralleled the production of the stress shock proteins in this bacterium. Total cellular fatty acids analysis showed that strain OK-5 produced or disappeared several different kinds of lipids when grown on TNT media than when grown on TSA. Under scanning electron microscope, the cells treated with 0.5 mM TNT for 12 hrs showed irregular rod shapes with wrinkled surfaces. Analyses of SDS-PAGE and Western blot using anti-DnaK and anti-GroEL revealed that several stress shock proteins including 70 kDa DnaK and 60 kDa GroEL in strain OK-5 were newly synthesized at different TNT concentrations in exponentially growing cultures. 2-D PAGE of soluble protein fractions from the culture of OK-5 exposed to TNT demonstrated that approximately 300 spots were observed on the silver stained gel ranging from pH 3 to pH 10. Among them, 10 spots significantly induced and expressed in response to TNT were selected and analyzed. As the result of internal amino acid sequencing with ESI-Q TOF, two proteins, spot #1 and spot #10 were assigned the DnaK protein XF2340 of Xylella fastidiosa and stress-induced protein of Mesorhizobium loti, respectively.

• Microbiology and Biotechnology Letters
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• v.42 no.2
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• pp.190-193
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• 2014

CMP-Neu5Ac synthetase is a key enzyme for the synthesis of CMP-Neu5Ac, which is an essential precursor of sialylated glycoconjugates. For the soluble expression of the CMP-Neu5Ac synthetase gene (neuA) from Escherichia coli K1, various heat shock proteins were co-expressed in E. coli BL21 (DE3) Star. In order to do this, a pG-KJE8 plasmid, encoding genes for GroEL-ES and DnaK-DnaJ-GrpE, was co-transformed with neuA and was expressed at $20^{\circ}C$ by the addition of 0.01 mM IPTG and 0.005 mg/ml L-arabinose. The co-expression of a variety of heat shock proteins resulted in the remarkably improved production of soluble CMP-Neu5Ac synthetase in E. coli.

• Molecules and Cells
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• v.21 no.3
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• pp.381-388
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• 2006

Heat shock proteins (Hsp) 70 are a ubiquitous family of molecular chaperones involved in many cellular processes. A yeast strain, ssa1/2, with two functionally redundant cytosolic Hsp70s (SSA1 and SSA2) deleted shows thermotolerance comparable to mildly heatshocked wild type yeast, as well as increased protein synthesis and ubiquitin-proteasome protein degradation. Since mRNA abundance does not always correlate well with protein expression levels it is essential to study proteins directly. We used a gel-based approach to identify stress-responsive proteins in the ssa1/2 mutant and identified 43 differentially expressed spots. These were trypsin-digested and analyzed by nano electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). A total of 22 non-redundant proteins were identified, 11 of which were confirmed by N-terminal sequencing. Nine proteins, most of which were up-regulated (2-fold or more) in the ssa1/2 mutant, proved to be stress-inducible proteins such as molecular chaperones and anti-oxidant proteins, or proteins related to carbohydrate metabolism. Interestingly, a translational factor Hyp2p up-regulated in the mutant was also found to be highly phosphorylated. These results indicate that the cytosolic Hsp70s, Ssa1p and Ssa2p, regulate an abundance of proteins mainly involved in stress responses and protein synthesis.

• Bulletin of the Korean Chemical Society
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• v.33 no.8
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• pp.2508-2512
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• 2012

Cold shock proteins (CSPs) are a family of proteins induced at low temperatures. CSPs bind to single-stranded nucleic acids through the ribonucleoprotein 1 and 2 (RNP 1 and 2) binding motifs. CSPs play an essential role in cold adaptation by regulating transcription and translation via molecular chaperones. The solution nuclear magnetic resonance (NMR) or X-ray crystal structures of several CSPs from various microorganisms have been determined, but structural characteristics of psychrophilic CSPs have not been studied. Therefore, we optimized the purification process to obtain highly pure Lm-Csp and determined the three-dimensional structure model of Lm-Csp by comparative homology modeling using MODELLER on the basis of the solution NMR structure of Bs-CspB. Lm-Csp consists of a ${\beta}$-barrel structure, which includes antiparallel ${\beta}$ strands (G4-N10, F15-I18, V26-H29, A46-D50, and P58-Q64). The template protein, Bs-CspB, shares a similar ${\beta}$ sheet structure and an identical chain fold to Lm-Csp. However, the sheets in Lm-Csp were much shorter than those of Bs-CspB. The Lm-Csp side chains, E2 and R20 form a salt bridge, thus, stabilizing the Lm-Csp structure. To evaluate the contribution of this ionic interaction as well as that of the hydrophobic patch on protein stability, we investigated the secondary structures of wild type and mutant protein (W8, F15, and R20) of Lm-Csp using circular dichroism (CD) spectroscopy. The results showed that solvent-exposed aromatic side chains as well as residues participating in ionic interactions are very important for structural stability. Further studies on the three-dimensional structure and dynamics of Lm-Csp using NMR spectroscopy are required.

• Korean Journal of Microbiology
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• v.34 no.1_2
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• pp.43-50
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• 1998

Induction and purification of the GroEL homolog from Streptococcus pneumolliae were characterized. The stress conditions were determined by temperature, ethanol, NaCI, $H_2O_2$ methyl methanesulfonate, and ethyl methanesulfonate. And stress induced proteins were analyzed using [$^{35}S$]-methionine labeling method. Heat shock induced the synthesis of a set of about 3 heat shock proteins (hsps) (65, 73, and 84-kDa). Of those 3 hsps, a 65 kDa protein, hsp65, was purified by DEAE-Sephacel and ATP-agarose column chromatography, and used for antibody preparation. Immunoblot analysis employing antisera raised against pneumococcus hsp65 demonstrated cross-reactivity with a 60 kDa protein in Eschericha coli. Also cross reaction of the purified p65 with anti-Escherichia coli GroEL monoclonal antibody demonstrated that pneumococcal hsp65 is the GroEL homolog.

• Biomedical Science Letters
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• v.12 no.4
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• pp.419-425
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• 2006

Phospholipase $C-{\gamma}1\;(PLC-{\gamma}1)$ is an important signaling molecule for cell proliferation and differentiation. $PLC-{\gamma}1$ contains two pleckstrin homology (PH) domains, which are responsible for protein-protein interaction and protein-lipid interaction. $PLC-{\gamma}1$ also has two Src homology (SH)2 domains and a SH3 domain, which are responsible for protein- protein interaction. To identity proteins that specifically binds to PH domain of $PLC-{\gamma}1$, we prepared and incubated the glutathione S-transferase(GST)-fused PH domains of $PLC-{\gamma}1$ with COS7 cell lysate. We found that 90 kDa protein specifically binds to PH domain of $PLC-{\gamma}1$. By matrix-assisted laser desorption ionization time of flight-mass spectrometry, the 90 kDa protein revealed to be heat shock protein (Hsp) $90{\beta}$. Hsp $90{\beta}$ is a molecular chaperone that stabilizes and facilitates the folding of proteins that are involved in cell signaling, including receptors for steroids hormones and a variety of protein kinases. To know whether Hsp $90{\beta}$ affects on $PLC-{\gamma}1$ activity, we performed $PIP_2$ hydrolyzing activity of $PLC-{\gamma}1$ in the presence of purified Hsp $90{\beta}$ in vitro. Our results show that the Hsp $90{\beta}$ dose-dependently inhibits the enzymatic activity of $PLC-{\gamma}1$ and further suggest that Hsp $90{\beta}$ regulates cell growth and differentiation via regulation of $PLC-{\gamma}1$ activity.

• Journal of Plant Biotechnology
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• v.44 no.3
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• pp.287-295
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• 2017

Water temperature is one of the major factors that impacts the growth and life cycle of Pyropia tenera, one of the most valuable and cultivated marine red algae belonging to Bangiales (Rhodophytes). We analyzed transcriptome from gametophyte of P. tenera under normal and high temperature conditions, and identified four small heat shock proteins (sHSPs). They have no significant amino acid sequence homology with known proteins in public databases except PhsHSP22 from Pyropia haitanensis. PtsHSP19.3 gene responded to high temperature but slightly or not to desiccation, freezing or high salt condition. When the PtsHSP19.3 gene was overexpressed in Chlamydomonas reinhardtii, transformed Chlamydomonas lines revealed much higher growth rate than that of control cells under heat stress condition. Transformed cells also grew well in those of the control cell onto the medium containing high salt or $H_2O_2$. When the PtsHSP19.3 was fused to GFP and introduced into tobacco protoplast, fluorescence was detected at several spots. Results indicate that PtsHSP19.3 may form super-molecular assembles and be involved in tolerance to heat stress.

• Molecules and Cells
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• v.46 no.6
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• pp.374-386
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• 2023

Thermal stress induces dynamic changes in nuclear proteins and relevant physiology as a part of the heat shock response (HSR). However, how the nuclear HSR is fine-tuned for cellular homeostasis remains elusive. Here, we show that mitochondrial activity plays an important role in nuclear proteostasis and genome stability through two distinct HSR pathways. Mitochondrial ribosomal protein (MRP) depletion enhanced the nucleolar granule formation of HSP70 and ubiquitin during HSR while facilitating the recovery of damaged nuclear proteins and impaired nucleocytoplasmic transport. Treatment of the mitochondrial proton gradient uncoupler masked MRP-depletion effects, implicating oxidative phosphorylation in these nuclear HSRs. On the other hand, MRP depletion and a reactive oxygen species (ROS) scavenger non-additively decreased mitochondrial ROS generation during HSR, thereby protecting the nuclear genome from DNA damage. These results suggest that suboptimal mitochondrial activity sustains nuclear homeostasis under cellular stress, providing plausible evidence for optimal endosymbiotic evolution via mitochondria-to-nuclear communication.