• Current Research on Agriculture and Life Sciences
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• 제17권
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• pp.59-63
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• 1999

벼의 엽록체 small HSP의 고온 스트레스 하에서의 기능을 밝히기 위하여 Oshsp21 cDNA를 pET 발현 vector에 도입하였다. 형질전환된 대장균 배양액에 IPTG를 첨가하여 단백질 발현을 유도시킨 다음 고온 stress 하에서의 생존율을 대조구와 비교하였다. 그 결과 대조균주의 경우 $50^{\circ}C$에서의 생존율이 크게 감소하였으나 Oshsp21이 발현된 대장균의 경우 70% 이상의 생존율을 나타내었다. 또한 대장균 단백질을 $55^{\circ}C$에서 30분간 열처리한 후, 대장균 단백질을 가용성과 비가용성 단백질로 분획한 다음, 각각의 비율을 조사한 결과, 대조균주의 경우 총 단백질의 약 60%가 비가용성 단백질로 변성되었으나, Oshsp21을 발현시킨 대장균의 경우 총 단백질의 약 35%만이 비가용성 단백질로 나타났다. 이러한 결과는 벼의 엽록체 small HSP는 세포내에서 분자 chaperone으로 기능하여 고온내성을 부여할 수 있음을 나타낸다.

• 한국자기공명학회논문지
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• 제15권2호
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• pp.137-145
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• 2011

Hsp33, a prokaryotic molecular chaperone, exerts holdase activity in response to oxidative stress. In this study, the stepwise conformational change of Hsp33 upon oxidation was monitored by NMR. In order to overcome its high molecular weight (33 kDa as a monomer and 66 kDa as a dimer), spectra were simplified using a selectively [$^{15}N$]His-labeled protein. All of the eight histidines were observed in the TROSY spectrum of the reduced Hsp33. Among them, three peaks showed dramatic resonance shifts dependent on the stepwise oxidation, indicating a remarkable conformational change. The results suggest that unfolding of the linker domain is associated with dimerization, but not entire region of the linker domain is unfolded.

• International Journal of Oral Biology
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• 제39권4호
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• pp.207-213
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• 2014

Antimicrobial actions of reactive oxygen/nitrogen species (ROS/RNS) derived from products of NADPH oxidase and inducible nitric oxide (NO) synthase in host phagocytes inactivate various bacterial macromolecules. To cope with these cytotoxic radicals, pathogenic bacteria have evolved to conserve systems necessary for detoxifying ROS/RNS and repairing damages caused by their actions. In response to these stresses, bacteria also induce expression of molecular chaperones to aid in ameliorating protein misfolding. In this study, we explored the function of a newly identified chaperone Spy, that is localized exclusively in the periplasm when bacteria exposed to conditions causing spheroplast formation, in the resistance of Salmonella Typhimurium to ROS/RNS. A spy deletion mutant was constructed in S. Typhimurium by a PCR-mediated method of one-step gene inactivation with ${\lambda}$ Red recombinase, and subjected to ROS/RNS stresses. The spy mutant Salmonella showed a modest decrease in growth rate in NO-producing cultures, and no detectable difference of growth rate in $H_2O_2$ containing cultures, compared with that of wild type Salmonella. Quantitative RT-PCR analysis showed that spy mRNA levels were similar regardless of both stresses, but were increased considerably in Salmonella mutants lacking the flavohemoglobin Hmp, which are incapable of NO detoxification, and lacking an alternative sigma factor RpoS, conferring hypersusceptibility to $H_2O_2$. Results demonstrate that Spy expression can be induced under extreme conditions of both stresses, and suggest that the protein may have supportive roles in maintaining proteostasis in the periplasm where various chaperones may act in concert with Spy, thereby protecting bacteria against toxicities of ROS/RNS.

• BMB Reports
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• 제44권12호
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• pp.816-820
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• 2011

There is a broad range of different small heat shock proteins (sHSPs) that have diverse structural and functional characteristics. To better understand the functional role of mitochondrial sHSP, NtHSP24.6 was expressed in Escherichia coli with a hexahistidine tag and purified. The protein was analyzed by non-denaturing PAGE, chemical cross-linking and size exclusion chromatography and the $H_6NtHSP24.6$ protein was found to form a dimer in solution. The in vitro functional analysis of $H_6NtHSP24.6$ using firefly luciferase and citrate synthase demonstrated that this protein displays typical molecular chaperone activity. When cell lysates of E. coli were heated after the addition of $H_6NtHSP24.6$, a broad range of proteins from 10 to 160 kD in size remained in the soluble state. These results suggest that NtHSP24.6 forms a dimer and can function as a molecular chaperone to protect a diverse range of proteins from thermal aggregation.

• Molecules and Cells
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• 제19권3호
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• pp.328-333
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• 2005

Small heat shock proteins (sHSPs) are widely distributed, and their function and diversity of structure have been much studied in the field of molecular chaperones. In plants, which frequently have to cope with hostile environments, sHSPs are much more abundant and diverse than in other forms of life. In response to high temperature stress, sHSPs of more than twenty kinds can make up more than 1% of soluble plant proteins. We isolated a genomic clone, NtHSP18.3, from Nicotiana tabacum that encodes the complete open reading frame of a cytosolic class I small heat shock protein. To investigate the function of NtHSP18.3 in vitro, it was overproduced in Escherichia coli and purified. The purified NtHSP18.3 had typical molecular chaperone activity as it protected citrate synthase and luciferase from high temperature-induced aggregation. When E. coli celluar proteins were incubated with NtHSP18.3, a large proportion of the proteins remained soluble at temperatures as high as $70^{\circ}C$. Native gel analysis suggested that NtHSP18.3 is a dodecameric oligomer as the form present and showing molecular chaperone activity at the condition tested. Binding of bis-ANS to the oligomers of NtHSP18.3 indicated that exposure of their hydrophobic surfaces increased as the temperature was raised. Taken together, our data suggested that NtHSP18.3 is a molecular chaperone that functions as a dodecameric complex and possibly in a temperature-induced manner.

• Journal of Microbiology and Biotechnology
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• 제31권4호
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• pp.570-583
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• 2021

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100℃), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90℃ for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

• International Journal of Oral Biology
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• 제45권4호
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• pp.190-196
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• 2020

Several factors, including genetic and environmental insults, impede protein folding and secretion in the endoplasmic reticulum (ER). Accumulation of unfolded or mis-folded protein in the ER manifests as ER stress. To cope with this morbid condition of the ER, recent data has suggested that the intracellular event of an unfolded protein response plays a critical role in managing the secretory load and maintaining proteostasis in the ER. Tauroursodeoxycholic acid (TUDCA) is a chemical chaperone and hydrophilic bile acid that is known to inhibit apoptosis by attenuating ER stress. Numerous studies have revealed that TUDCA affects hepatic diseases, obesity, and inflammatory illnesses. Recently, molecular regulation of ER stress in tooth development, especially during the secretory stage, has been studied. Therefore, in this study, we examined the developmental role of ER stress regulation in tooth morphogenesis using in vitro organ cultivation methods with a chemical chaperone treatment, TUDCA. Altered cellular events including proliferation, apoptosis, and dentinogenesis were examined using immunostaining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. In addition, altered localization patterns of the formation of hard tissue matrices related to molecules, including amelogenin and nestin, were examined to assess their morphological changes. Based on our findings, modulating the role of the chemical chaperone TUDCA in tooth morphogenesis, especially through the modulation of cellular proliferation and apoptosis, could be applied as a supporting data for tooth regeneration for future studies.

• Journal of Genetic Medicine
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• 제20권1호
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• pp.6-14
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• 2023

Fabry disease (FD), a rare X-linked lysosomal storage disorder, is caused by mutations in the α-galactosidase A gene gene encoding α-galactosidase A (α-Gal A). The functional deficiency of α-Gal A results in progressive accumulation of neutral glycosphingolipids, causing multi-organ damages including cardiac, renal, cerebrovascular systems. The current treatment is comprised of enzyme replacement therapy (ERT), oral pharmacological chaperone therapy and adjunctive supportive therapy. ERT has been introduced 20 years ago, changing the outcome of FD patients with proven effectiveness. However, FD patients have many unmet needs. ERT needs a life-long intravenous therapy, inefficient bio-distribution, and generation of anti-drug antibodies. Migalastat, a pharmacological chaperone, augmenting α-Gal A enzyme activity only in patients with mutations amenable to the therapy, is now available for clinical practice. Furthermore, these therapies should be initiated before the organ damage becomes irreversible. Development of novel drugs aim at improving the clinical effectiveness and convenience of therapy. Clinical trial of next generation ERT is underway. Polyethylene glycolylated enzyme has a longer half-life and potentially reduced antigenicity, compared with standard preparations with longer dosing interval. Moss-derived enzyme has a higher affinity for mannose receptors, and seems to have more efficient access to podocytes of kidney which is relatively resistant to reach by conventional ERT. Substrate reduction therapy is currently under clinical trial. Gene therapy has now been started in several clinical trials using in vivo and ex vivo technologies. Early results are emerging. Other strategic approaches at preclinical research level are stem cell-based therapy with genome editing and systemic mRNA therapy.

• 생명과학회지
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• 제29권4호
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• pp.492-498
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• 2019

기존 연구를 통하여 토양에서 분리한 Acinetobacter schindleri DYL129로부터 3개의 lipase 유전자(lipAD1, lipAD2와 lipAD3)들과 1개의 chaperone (lipBD) 유전자를 보고하였다. 본 연구에서는 각 유전자들의 발현을 위해서 pET32a(+)와 pGEX-6P-1 벡터에 클로닝하여 각각을 pETLAD1-3와 pETLBD 또는 pGEXLAD1-3와 pGEXLB로 명명하였으며, 단백질의 발현량은 pET 시스템을 사용할 때 1.5 배 정도 향상됨을 확인하였다. LipAD1과 LipAD2는 inclusion body 형태로 발현이 되었으며, LipAD3과 LipBD는 soluble type으로도 발현되었다. Inclusion body 형태의 LipAD1과 LipAD2는 고농도의 우레아를 처리하여 refolding 시켰다. LipAD1은 C4와 C2를, LipAD2는 C2와 C14를 그리고 lipAD3은 C2, C4와 C14를 기질로 잘 이용하는 것을 확인하였다. 그리고 모든 효소들은 $50^{\circ}C$에서 최적 활성을 나타내었다.

• 대한유전성대사질환학회지
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• 제13권1호
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• pp.37-42
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• 2013

Gaucher disease is a lysosomal storage disease caused by deficiency of glucocerebrosidase (GBA). This condition is characterized by accumulation of glucocerebrosidase in liver, spleen, lung, skeletal system, and central nervous system. Gaucher disease is the prototype of disease in which efficacy of enzyme replacement therapy has been established. However, because recombinant enzyme is not able to enter the central nervous system, its efficacy is limited to the non-neurological manifestations of Gaucher disease. Importantly, approximately a half of Korean patients with Gaucher disease suffer from neurological manifestations. In addition, Korean Gaucher disease patients exhibit distinct mutation spectrum from those in other populations. Common mutations in Korean patients with Gaucher disease are also associated with neurological phenotype. Therefore, therapeutic strategies tailored to Korean patients were necessary. Interestingly, a chemical chaperone, ambroxol, has been known to increase residual enzymatic activities of the select mutant GBAs encoded by mutations prevalent in Korean patients. One promising aspect of this drug is that it can cross blood-brain barrier, and enhance the enzyme activity in the brain. In vitro study suggested this chemical chaperone as one of new therapeutic agents in Gaucher disease, and a well-designed human trial is required to confirm its efficacy.