[논문]붕어와 마우스의 간세포 배양에서 열 스트레스에 의해 유도되는 heat shock factor1 (HSF1)의 비교

김소선; 소재형; 박장수

doi:10.5352/jls.2016.26.12.1360

붕어와 마우스의 간세포 배양에서 열 스트레스에 의해 유도되는 heat shock factor1 (HSF1)의 비교
Comparison of Thermal Stress Induced Heat Shock Factor 1 (HSF1) in Goldfish and Mouse Hepatocyte Cultures 원문보기

생명과학회지 = Journal of life science, v.26 no.12 = no.200, 2016년, pp.1360 - 1366

김소선 (부산대학교 화학과) , 소재형 (부산대학교 화학과) , 박장수 (부산대학교 화학과)

초록
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Heat shock proteins (HSPs)은 다양한 생리학적인 또는 환경적 스트레스에 응답하여 유도된다. 그러나 HSPs의 전사 활성은 heat shock factors (HSFs)에 의해 조절 된다. 현재 연구에서는 붕어와 마우스의 간세포 배양에서 열 스트레스에 의한 heat shock factor 1 (HSF1)의 패턴 차이와 heat shock protein 70 (HSP70)의 발현을 면역분석법을 이용하여 조사하였다. 붕어의 간세포는 $33^{\circ}C$에서 trimer를 이루지만 마우스의 간세포는 $42^{\circ}C$에서 trimer를 이루었다. 이 연구는 붕어와 마우스의 HSF1은 열 스트레스로부터 다른 온도에서 반응을 한다는 것을 보여준다. 또한 재조합 단백질을 이용하여 붕어와 인간의 HSF1의 온도에 따른 활성 조건을 CD spectroscopy와 면역분석을 이용하여 조사하였다. 이러한 결과들은 인간과 마우스 HSF1과 붕어의 HSF1은 온도에 의한 활성 변화를 보이지만 그들의 최적 활성 온도는 다르다는 것을 알 수 있다.

Abstract ▼ AI-Helper

Heat shock proteins (HSPs) are induced in response to various physiological or environmental stressors. However, the transcriptional activation of HSPs is regulated by a family of heat shock factors (HSFs). Fish models provide an ideal system for examining the biochemical and molecular mechanisms of adaptation to various temperatures and water environments. In this study, we examined the pattern differentials of heat shock factor 1 (HSF1) and expression of heat shock protein 70 (HSP70) in response to thermal stress in goldfish and mouse hepatocyte cultures by immune-blot analysis. Goldfish HSF1 (gfHSF1) changed from a monomer to a trimer at $33^{\circ}C$ and showed slightly at $37^{\circ}C$, whereas mouse HSF1 (mHSF1) did so at $42^{\circ}C$. This experiment showed similar results to a previous study, indicating that gfHSF1 and mHSF1 play different temperature in the stress response. We also examined the activation conditions of the purified recombinant proteins in human HSF1 (hmHSF1) and gfHSF1 using CD spectroscopy and immune-blot analysis. The purified recombinant HSF1s were treated from $25^{\circ}C$ to $42^{\circ}C$. Structural changes were observed in hmHSF1 and gfHSF1 according to the heat-treatment conditions. These results revealed that both mammal HSF1 (human and mouse HSF1) and fish HSF1 exhibited temperature-dependent changes; however, their optimal activation temperatures differed.

주제어

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문제 정의

The cells responded to unexpected adverse environmental changes by processes referred to as thermal or heavy metal responses, which were accompanied by a rapid increase in the synthesis of stress proteins [9]. This study aimed to understand the changes and roles of HSF1 in hepatocytes. Upon stress, HSF1 is the key transcriptional activator of chaperones, cochaperones and ubiquitin, and coordinates the expression of many transcriptional and translational regulators, signaling molecules and mitotic determinants [18, 31, 32].
This species has been used as a model to evaluate environmental contamination [13]. This study was designed to show the trimerization of HSF1s (mHSF and gfHSF1) exposed to thermal stress. The HSF1s were investigated to analyze their protein synthesis pattern in response to thermal stress from hepatocyte cultures.

제안 방법

The CD spectra were used to examine the changes in the conformation of the proteins. The CD scans were performed on a Jasco J-715 spectropolarimeter in the far-UV range at 25℃, 33℃ and 37℃ in the case of gfHSF1 and at 25℃, 37℃ and 42℃ in the case of hmHSF1. A cell with a 0.

대상 데이터

Goldfish (Carassius auratus), approximately 10-15 cm in length and 20 g in weight, were purchased from a local market and maintained in dechlorinated freshwater tanks. The fish were killed by a sharp blow to the head followed by severance of the spinal cord, and were dissected on ice.
After exposure, the harvested cells were extracted using RIPA buffer (Sigma) for western blot analysis. The Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell electroblotted SDS-PAGE samples, including the 42℃ and 25℃ treated cell pellet, 33℃ and 37℃ treated cell pellet and control cell pellets in transfer buffer (15.6 mM Tris-HCl, 120 mM glycine, pH 8.3) to the nitrocellulose membrane, were examined. After being blocked with 3% bovine serum albumin (BSA) in Tris-Buffered saline (TBS), the polyclonal antibodies of antigoldfish HSP70, HSF1 and β-actin, which were prepared in this laboratory [4, 12], a rat monoclonal antibody HSF1 Ab-1, anti-HSP70 and β-actin were purchased from Thermo scientific and used for specific binding.

성능/효과

In conclusion, the heat shock factor1s (gfHSF1 and mHSF1) from hepatocyte cultures showed similar patterns in vitro, in that there was a difference in their activation temperatures. This study confirmed that their optimal temperatures forming trimer were different by immunoblot analysis and circular dichroism analysis.
The biochemical characterization of HSF1 and the mechanism of HSF1-mediated transcription have been extensively studied and are well understood [16, 27]. The results of the present study revealed the different optimal temperature activation of two HSF1s (mHSF1 and gfHSF1) under heat stress conditions in cultured hepatocytes. GfHSF1 was activated at 33℃, whereas mHSF1 was activated at 42℃.
2D). This study confirmed that fish and mouse have different activated temperature by thermal stress. Circular dichroism (CD) can screen the changes in the conformation of biopolymers and has previously been used to analyze the effects of heat stress on hmHSF1 and gfHSF1 [12].
In conclusion, the heat shock factor1s (gfHSF1 and mHSF1) from hepatocyte cultures showed similar patterns in vitro, in that there was a difference in their activation temperatures. This study confirmed that their optimal temperatures forming trimer were different by immunoblot analysis and circular dichroism analysis.

후속연구

It would seem important to study the different structural changes of fish HSF1and mammal HSF1. These functional differences will need to be examined in a future study to determine the reasons for them.

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