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研究生:高孝元
研究生(外文):Kao Hsiao Yuan
論文名稱:新奇蛋白BC1上相似YTH區域之功能分析
論文名稱(外文):Functional analysis of YTH-like domain of novel protein BC1
指導教授:李玉梅李玉梅引用關係
指導教授(外文):Yu-May, Lee Ph.D
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:53
中文關鍵詞:遺傳性漸進式肌緊張力不足GTP cyclohydrolase IBC1ATP-結合ATP水解
外文關鍵詞:Hereditary progressive DystoniaGTP cyclohydrolase IBC1ATP-bindingATPase
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GTP cyclohydrolase I (GCH)的突變會導致遺傳性漸進式肌緊張力不足(Hereditary progressive Dystonia, HPD)或對多巴反應肌緊張力不足。在tetrahydrobiopterin (BH4)生合成的過程中,GCH是第一個作用的酵素,也是速率決定步驟酵素。GTP cyclohydrolase I (GCH)的突變會降低BH4及其下游tyrosine hydrolase的含量,導致多巴量的不足而影響身體正常功能的運作。即使DRD病人中含有正常對偶基因,但其腦部的GCH活性卻只有正常人的20%,表示DRD的致病機制為顯性抑制作用(dominant negative effect, DN)。
在建立DN的HeLa細胞株的同時,我們很意外的篩選出非顯性抑制(Non-DN)的細胞株,利用PCR selected cDNA subtraction技術,許多已知基因如熱休克蛋白(heat shock protein) 70以及新奇基因如BC1均在non-DN細胞中大量的表現。經過蛋白資料庫做序列比對,發現在BC1的C端,也就是胺基酸437-526的區域與YT521b之YTH保守區域具有非常高的相似度。
我們發現純化後的BC1上YTH相似區域的重組蛋白具有與ATP結合和水解ATP的能力,其比活性為在45°C每毫克YTH蛋白在每小時可以從1毫摩爾之ATP釋放0.52 奈摩爾的磷酸根,但BC1不具有傳統保守ATP結合區域,因此要證明BC1為一新奇ATP水解蛋白則需更進一步的探討。
Hereditary progressive dystonia (HPD) or Dopa-repsonsive dystonia (DRD) is a heredity form of dystonia caused by mutation in GTP cyclohydrolase I (GCH) gene, which is the first and rate limiting enzyme of tetrahydrobiopterin (BH4) synthesis. GCH mutations cause a reduction in BH4 and consequently decrease tyrosine hydrolase(TH) content, which diminished dopamine production that leads to symptoms of dopamine deficiency. GCH activity of DRD patients in brain is only 20% of normal value regardless of the presence of active allele suggesting a dominant negative (DN) effect.
While establishing a HeLa cell line showing DN effect of GCH mutation, we accidentally screened a line of HeLa that did not have the DN phenomenon. Through PCR selected cDNA subtraction method, we found many well studied genes such as Hsp70 along with novel gene BC1 (named after Institute of Biological Chemistry) are distinctly and highly expressed in non-DN cells. Blast search of the non-redundant NCBI database with the complete BC1 amino acid sequence detected a conserve YTH-like domain located at the C-terminal stretch (437-526aa) of BC1.
In this study, we discovered that purified recombinant YTH domain of BC1 possesses ATP binding ability and a maximum specific ATPase activity of 0.52 nmole Pi released per min per mg at 45°C. Since BC1 doesn’t contain traditional ATP binding domain such as Walker A, Walker B, P loop motif, more evidence are required to prove BC1 as a novel ATPase.
Chapter 1: Introduction
1.1 Hereditary progressive dystonia
1.2 GTP cyclohydrolase I (GCH)
1.3 Discovery of BC1
1.4 Preliminary data of BC1
Chapter 2: Material and Methods
2.1 Cloning
2.1.1 PCR amplification
2.1.2 Gel extraction
2.1.3 Enzyme digestion
2.1.4 Ligation
2.1.5 Transformation
2.1.6 Plasmid Purification
2.1.7 Restriction analysis
2.1.8 Sequence analysis
2.2 Expression
2.2.1 YTH-like domain (YTH) of BC1
2.2.2 Heat shock cognate protein70, Hsc70
2.2.3 Glutathione-S transferase, GST
2.3 Purification
2.3.1 YTH
2.3.1.1 Ni-NTA chromatography under native condition
2.3.1.2 Ni-NTA chromatography under denaturing condition
2.3.1.3 GST-affinity chromatography
2.3.1.4 Gel filtration chromatography
2.3.2 Hsc70
2.3.2.1 Anion exchange chromatography
2.3.2.2 ATP-agarose chromatography
2.3.3 GST
2.3.3.1 GST-affinity chromatography
2.4 Refolding
2.5 Biophysical analysis
2.5.1 CD spectra
2.5.2 1D NMR
2.5.3 Crystal screen
2.6 Cell culture and transfection
2.7 Functional analysis of YTH-like domain
2.7.1 ATPase assay
2.7.2 Chaperon-like assay
2.7.2.1 DTT induced insulin aggregation assay
2.7.2.2 Alcohol dehydrogenase thermostabilty assay
2.7.3 Gelatin zymography
2.7.4 ATP-agarose affinity chromatography
2.7.5 GST-pull down
2.8 Polyclonal antibody generation
2.9 SDS-PAGE and Western blot
Chapter 3: Results
Chapter 4: Discussion
Chapter 5: Conclusion
Chapter 6: Figures and Tables
Chapter 7: Reference
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