Heat Shock Protein 70 as a Molecular Chaperone for DNA Mismatch Repair Factors in Fish Embryos and Its Antagonism against Oxidative Stress

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Project title

Code/計畫編號

NSC102-2313-B019-017

Translated Name/計畫中文名

熱休克蛋白70對魚類胚胎DNA錯誤配對修補因子之分子監護與抗氧化壓力效應

Project Coordinator/計畫主持人

Todd Hsu

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=3104744

Year

2013

Start date/計畫起

01-08-2013

Expected Completion/計畫迄

01-07-2014

Bugetid/研究經費

1000千元

ResearchField/研究領域

生物技術(農)
漁業

"熱休克蛋白 70(Hsp70)為一種分子監護者，可將因緊迫狀態而變性之蛋白質摺疊回其原初構型，也可協助蛋白質由細胞質藉穿膜輸送至各胞器。DNA 錯誤配對修補作用 (MMR)校正因 DNA 複製或重組產生之錯誤配對，而真核細胞中單一鹼基錯誤配對與小型寡核酸環係由 MSH2 與 MSH6 蛋白質複合體進行辨識。本實驗室已發現將飼養在 28.5℃之斑馬魚胚胎移至 37℃30 分鐘後可激發大量 Hsp70 產生。最近研究顯示熱休克可提升斑馬魚胚胎中 MSH2/MSH6 於 RNA 及蛋白質層次之表現，但由過氧化氫、 paraquat 與鎘造成之氧化壓力卻明顯抑制 MSH 基因活性。因魚類胚胎對環境汙染之敏感度與 MMR 維持基因完整之重要性，本計畫將以斑馬魚與點帶石斑魚胚胎為對象，繼續探討由熱休克因子 1(HSF1)轉錄誘發之 Hsp70 對 MSH 之分子監護效應與 Hsp70 如何拮抗氧化壓力對 MSH 基因表現之抑制作用。研究上將以顯微注射反意寡核苷酸至胚胎內以抑制 HSF1/Hsp70 mRNA 轉譯，再藉全覆式原位雜交與定量 PCR 分析 HSF1/Hsp70 是否為調控 MSH 基因轉錄之因子。Hsp 70 與 MSH 分子於細胞核內之交互作用將藉雷射共軛焦顯微鏡與免疫沉澱進行分析，而一帶有雙約束限制序列之質體將用以檢測核蛋白中斷股指引 MMR 活性變化。除偵測 Hsp 70 與脂肪酸過氧化物間之關聯性外，反意寡核苷酸阻斷技術亦將用以探討氧化壓力是否藉激發缺氧因子與 Nrf2 轉錄因子等抑制 MSH 基因活性。 " "Heat shock protein 70 (HSP 70) is a molecular chaperone that assists the refolding of denatured proteins in stress organisms and it also facilitates the transmembrane transport of proteins into organelles. A drastic up-regulation of HSP 70 synthesis was found to appear in zebrafish (Danio rerio) embryos when embryos maintained at 28.5℃were heat stressed at 37 ℃for 30 min. Our recent studies on zebrafish embryos showed the ability of heat shock to stimulate the gene expression of MutS homolog 2/MSH6 that initiates DNA mismatch repair (MMR) of simple mispairs and small insertion-deletion loops via mismatch recognition. In contrast, oxidative stress caused by hydrogen peroxide, paraquat, or the heavy metal cadmium significantly down-regulated MSH gene activities. Heat shock response and oxidative stress may both be triggered in fish embryos due to the presence of chemical pollutants in the aquatic environment and changes in water body temperature. This research intends to explore if HSP 70 produced by heat shock factor 1 (HSF1)-dependent transcription acts as a chaperone for MSH2/MSH6 in zebrafish and grouper embryos and if HSP 70 protects against oxidative stress-induced gene down-regulation. To determine the chaperoning effect of HSP 70, blockage of HSF1/HSP 70 mRNA translation will be conducted by microinjecting morpholino-modified antisense oligonucleotides into embryos at 1-cell stage and the inhibition of HSF1/HSP 70-stimulated MSH gene expression will be analyzed by whole mount in situ hybridization and QPCR. The physical interactions between HSF1/HSP 70 and MSH2/MSH6 in the nuclei of fish embryos will be examined by confocal microscope and immunoprecipitation after protein crosslinking. A circular plasmid harboring a G-T mispair inserted into a double restriction recognition site will be employed as a repair substrate to test if heat shock stimulates MMR activity in the embryonic extracts. Evidences supporting the protection of HSP 70 against oxidative stress will include a negative correlation between HSP 70 production and oxidant-induced lipid peroxidtion plus the free radical-scavenging effect of HSP 70. Antisense technology combined with in situ hybridization and QPCR will be used to determine if HSF1/HSP 70 protect against hydrogen peroxide-induced MSH gene down-regulation by suppressing the transcription factor activities of NF-E2-related factor 2 and hypoxia-inducing factor 1. "

Keyword(s)

DNA 錯誤配對修補
熱休克蛋白 70
熱休克因子 1
氧化壓力
魚類
DNA mismatch repair
heat shock protein 70
heat shock factor 1
oxidative stress
fish

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Heat Shock Protein 70 as a Molecular Chaperone for DNA Mismatch Repair Factors in Fish Embryos and Its Antagonism against Oxidative Stress

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