• Molecules and Cells
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• 제39권2호
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• pp.163-168
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• 2016

Caffeine has both positive and negative effects on physiological functions in a dose-dependent manner. C. elegans has been used as an animal model to investigate the effects of caffeine on development. Caffeine treatment at a high dose (30 mM) showed detrimental effects and caused early larval arrest. We performed a comparative proteomic analysis to investigate the mode of action of high-dose caffeine treatment in C. elegans and found that the stress response proteins, heat shock protein (HSP)-4 (endoplasmic reticulum [ER] chaperone), HSP-6 (mitochondrial chaperone), and HSP-16 (cytosolic chaperone), were induced and their expression was regulated at the transcriptional level. These findings suggest that high-dose caffeine intake causes a strong stress response and activates all three stress-response pathways in the worms, including the ER-, mitochondrial-, and cytosolic pathways. RNA interference of each hsp gene or in triple combination retarded growth. In addition, caffeine treatment stimulated a food-avoidance behavior (aversion phenotype), which was enhanced by RNAi depletion of the hsp-4 gene. Therefore, up-regulation of hsp genes after caffeine treatment appeared to be the major responses to alleviate stress and protect against developmental arrest.

• Animal cells and systems
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• 제7권3호
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• pp.271-277
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• 2003

Regulation of endoplasmic reticulum(ER) chaperone, ERp29, in traumatized rat spinal cord was investigated. Compared to the control, ERp29 expression was down-regulated at the lesion site 1 d after spinal cord injury. However, ERp29 expression gradually increased from 3 d after the injury and peaked remarkably after 7 d. Two ER chaperones (GRP94 and BiP) showed constantly strong expression levels 1 d after spinal cord injury while the expression levels of the other two (calnexin and PDI) were unchanged. In the case of ERp72, its expression level was increased 1 d after the injury and gradually decreased thereafter. This study suggests that ERp29 expression in the spinal cord after traumatic injury might be associated with the posttraumatic neural survival, playing a role as a molecular chaperone.

• 대한의생명과학회지
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• 제14권1호
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• pp.59-62
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• 2008

This experiment was performed to study the effect of TSH (thyroid-stimulating hormone) on the expression of endoplasmic reticulum (ER) chaperones in the rat thyrocytes FRTL-5 cells. Although the expressions of ER membrane kinases (ATF6, IRE1 and PERK) were specially enhanced under absence of TSH, no remarkable up- or down regulations of ER chaperones (BiP, CHOP and Calnexin) were detected by TSH. We firstly report here that TSH by dose up-regulated expression of ER membrane kinases in FRTL-5 culture thyrocytes.

• 한국동물학회지
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• 제38권2호
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• pp.167-176
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• 1995

Glucose-regulated proteins (grp's), srp94 3nd grp78/BiP, are a group of stress proteins which are highly synthesized in cells exposed to a variety of stressful agents including tunicamycin 3nd Ca2+ ionophore. Grp78/BiP is hon to function as a molecular chaperone which regulates the folding and assembly of secretory or membrane proteins, but the biological function of grp941 remains to be elucidated. In this study, we have examined the intracellular distribution of grV's and the function of srp94. Grp's are resident in the endoplasmic reticulum (ERI 3nd a specific sequence (Lys-Asp-Glu-Leu) at their C-terminus is known to be responsible for their retention within the ER. However, it has been unclear whether upon disturbance of cellular Caa+ homeostasis by the Ca2+ ionophore, grp94 is retained within the ER or secreted into the medium. In this study, we showed that in the presence of C3a+ ionophore, grp94 and gif78/BiP are present in the cells, mainly within the ER. We have also investigated whether grp94 might function as a molecular chaperone. Here we showed that in the immunoglobulin (Ig)-secreting hvbridom3 cells, grp94 transientlY interacts with fully glycosylated Is heavy chain, suggesting that grpg94 may be involved in facilitating the folding and assembly of Ig heavy chains.

• Archives of Pharmacal Research
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• 제26권12호
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• pp.1047-1054
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• 2003

Molecular chaperones have a crucial role in the folding of nascent polypeptides in endoplasmic reticulum. Some of them are known to be sensitive to the modification by electrophilic metabolites of organic pro-toxicants. In order to identify chaperone proteins sensitive to alkyators, ER extract was subjected to alkylation by 4-acetamido-4 -maleimidyl-stilbene-2,2 -disulfonate (AMS), and subsequent SDS-PAGE analyses. Protein spots, with molecular mass of 160, 100, 57 and 36 kDa, were found to be sensitive to AMS alkylation, and one abundant chaperon protein was identified to be protein disulfide isomerase (PDI) in comparison with the purified PDI. To see the reactivity of PDI with cysteine alkylators, the reduced form ($PDI_{red}$) of PDI was incubated with various alkylators containing Michael acceptor structure for 30 min at $38^{\circ}C$ at pH 6.3, and the remaining activity was determined by the insulin reduction assay. Iodoacetamide or N-ethylmaleimide at 0.1 mM remarkably inactivated $PDI_{red}$ with N-ethylmaleimide being more potent than iodoacetamide. A partial inactivation of $PDI_{oxid}$ was expressed by iodoacetamide, but not N-ethylmaleimide (NEM) at pH 6.3. Of Michael acceptor compounds tested, 1,4-benzoquinone ($IC_{50}, 15 \mu$ M) was the most potent, followed by 4-hydroxy-2-nonenal and 1,4-naphthoquinone. In contrast, 1,2-naphthoquinone, devoid of a remarkable inactivation action, was effective to cause the oxidative conversion of $PDI_{red}$ to $PDI_{oxid}$. Thus, the action of Michael acceptor compounds differed greatly depending on their structure. Based on these, it is proposed that POI, one of chaperone proteins in ER, could be susceptible to endogenous or xenobiotic Michael acceptor compounds in vivo system.

• Biomolecules & Therapeutics
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• 제20권3호
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• pp.346-351
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• 2012

Cells show various stress signs when they are challenged with severe physiological problems. Majority of such cellular stresses are conveyed to endoplasmic reticulum (ER) and unfolded protein response (UPR) serves as typical defense mechanism against ER stress. This study investigated an interaction between ER stress agents using macropage cell line Raw 264.7. When activated by lipopolysaccharide (LPS), the cell lines showed typical indicators of ER stress. Along with molecular chaperones, the activation process leads to the production of additional inflammatory mediators. Following activation, the macrophage cell line was further treated with TUN and characterized in terms of chaperone expression and cytokine secretion. When treated with TUN, the activated macrophage cell leads to increased secretion of IL-6 although expression of ER stress markers, GRP94 and GRP78 increased. The secretion of cytokines continued until the addition of BFA which inhibits protein targeting from ER to Golgi. However, secretion of cytokines was ceased upon dual treatments with BFA and TG. This result strongly implies that cells may differently deal with various polypeptides depending on the urgency in cellular function under ER stress. Considering IL-6 is one of the most important signal molecules in macrophage, the molecule might be able to circumvent ER stress and UPR to reach its targeting site.

• 생명과학회지
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• 제22권8호
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• pp.1132-1136
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• 2012

많은 종류의 세포스트레스는 unfolded protein response (UPR)관련인자의 유전자발현을 조절한다. 본 연구결과 부동스트레스(immobilization stress)는 세포의 소포체스트레스(ER stress)와 관련된 유전자발현의 변화를 유도한다; Heart, spleen, thymus, kidney, testis에서는 유전자발현 변화가 없었지만 adrenal gland, liver, lung에서는 유의할만한 상승변화가 있었다. 그러나 muscle에서는 다른 것들과 대조적으로 발현이 감소되었다. 이 결과는 부동스트레스도 다른 종류의 세포스트레스와 같이 세포수준에서 UPR을 조절할 수 있다는 최초의 보고이다.

• 생명과학회지
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• 제17권6호통권86호
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• pp.847-858
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• 2007

ER-Golgi 분비 경로를 통해서 정확한 구조를 가지면서 post-translational modification 과정을 거친 재조합 단백질의 발현을 최대화하는 것은 ER stress반응에 대한 연구의 중요한 계기가 된다. 세포가 스트레스를 받지 않는 상태라도 ER stress signaling은 재조합 단백질의 생산량을 제한하고 품질을 떨어뜨리는 여러 가지 조건을 만들게 된다. ER stress signaling을 막는 여러 가지 방법들이 제시되고 있으며 표 2는 이러한 방법들 중 일부를 나타내고 있다. 일반적으로는 pro-survival 경로에 관련되어 있는 인자를 촉진하고 pro-apoptosis에 관련되어 있는 인자를 억제하는 것들이다. 그러나 ER stress 반응은 매우 복잡하고 적응과 사멸 기작(adaptation and elimination mechanism)의 중간 역할을 하기 때문에 ER stress에 관련된 주요 인자를 산업적으로 응용하기 위해선 이들의 기능에 대해 보다 깊은 연구가 이루어져야 한다. 현재까지 재조합단백질의 생산량을 최대한으로 높이는 방법은 ER stress 반응이 생기지 않도록 fed-batch process를 개선하고 세포 사멸 기작을 조절하며 단백질의 glycosylation 처리를 하는 것이다.

• Biomolecules & Therapeutics
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• 제15권4호
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• pp.240-244
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• 2007

Cigarette smoking causes serious health problems in humans, especially if smoking habits are established during their adolescence. Nicotine is known to mutate DNA and interfere with apoptosis. Apoptosis is considered as a potent defense mechanism against cellular damaging agents. This study aims to investigate the effect of nicotine on the progression of apoptosis induced under ER stress conditions using four different established cell lines: HEK293, 3T3-L1, C2C12, and HepG2. When treated with nicotine, the progression of apoptosis was notably inhibited in the four cell lines according to the assays of caspase-3 activation and DNA fragmentation. In ER-stressed cells, nicotine appears to inhibit the progression of apoptosis in a concentration-dependent manner. When cells were treated with nicotine prior to ER stress, GRP94 level significantly increased compared to other ER stress markers of PDI and GRP78. This observation suggests that the inhibitory effect of nicotine may results from up-regulation of GRP94, an anti-apoptotic chaperone, under nicotine treatment. Taken together, the present study strongly implies that nicotine may inhibit apoptosis, caused by prolonged ER stress, based on promotion of GRP94 expression.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권3호
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• pp.192-198
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• 2003

BiP, immunoglobulin binding protein, is an ER homologue of Hsp70. However, unlit other Hsp70 proteins, regulatory protein(s) for BiP has not been identified. Here, we demo strafed the presence of potential regulatory proteins for BiP using a pull -down assay. Since BiP can bind any unfolded protein, only the ATPase domain of BiP was used for the pull -down assay in order to minimize nonspecific binding. The ATPase domain was cloned to produce recombinant protein, which was then conjugated to CNBr-activated agarose. The structural conformation and ATP hydrolysis activity of the recombinant ATPase domain were similar to those of the native protein, light proteins from metabolically labeled mouse plasmacytoma cells specifically bound to the recombinant ATPase protein. The binding of these proteins was inhibited by excess amounts of free ATPase protein, and was dependent on the presence of ATP. These proteins were eluted by ADP. Of these proteins, Grp170 and BiP where identified. while the other were not identified as known ER proteins, from Western blot analyses. The presence of the ATPase-binding proteins for BiP was first demonstrated in this study, and our data suggest similar regulatory machinery for BiP may exist in the ER, as found in prokaryotes and other cellular compartments.