李玉梅臺灣大學：生化科學研究所高孝元Hsiao-Yuan, KaoKaoHsiao-Yuan2007-11-262018-07-062007-11-262018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/52814GTP 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生成，並導致多巴量的不足而影響身體正常弁鄋犒B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，釵h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 responsive for the first and rate limiting enzyme of tetrahydrobiopterin (BH4) synthesis. GCH mutations cause a reduction in BH4 and consequently in low TH tyrosine hydrolase, 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 effect. While establishing a HeLa cell lines 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 appears to be functional. YTH 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 10 1.1 Hereditary progressive dystonia 10 1.2 Cause of HPD 10 1.3 Discovery of BC1 11 1.4 Hsp70 11 1.5 Preliminary data of BC1 12 Chapter 2: Material and Methods 14 2.1 Cloning 14 2.1.1 PCR amplification 14 2.1.2 Gel extraction 15 2.1.3 Restriction Enzyme digestion 15 2.1.4 Ligation 15 2.1.5 Transformation 15 2.1.6 Plasmid Purification 16 2.1.7 Restriction enzyme analysis 16 2.1.8 Sequence analysis 17 2.2 Expression of recombinant protein 17 2.2.1 YTH-like domain 17 2.2.2 Hsc70 18 2.2.3 GST 18 2.3 Purification of recombinant protein 18 2.3.1 YTH 18 2.3.1.1 Native Nickel-NTA affinity chromatography 18 2.3.1.2 Denaturing Nickel-NTA affinity chromatography 19 2.3.1.3 GST-affinity chromatography 19 2.3.1.4 Gel filtration chromatography 19 2.3.2 Hsc70 20 2.3.2.1 Anion exchange chromatography 20 2.3.2.2 ATP-agarose chromatography 20 2.3.3 GST 20 2.3.3.1 GST-affinity chromatography 20 2.4 Protein Refolding 21 2.5 Biophysical analysis 21 2.5.1 Circular dichroism (CD) spectroscopy 21 2.5.2 CD-pro analysis 21 2.5.3 One-dimensional NMR analysis 21 2.5.4 Crystal screen 22 2.6 Cell culture and transfection 22 2.7 Functional analysis 22 2.7.1 ATPase assay 22 2.7.2 Chaperon-like assay 23 2.7.2.1 DTT induced insulin aggregation assay 23 2.7.2.2 Alcohol dehydrogenase thermostabilty assay 23 2.7.3 ATP-agarose affinity chromatography 23 2.7.4 GST-pull down 24 2.7.5 Gelatin zymography 24 2.8 Generation of polyclonal antibody 24 2.9 SDS-PAGE 25 2.10 Western blot 25 2.11 Immunocytochemistry 25 Chapter 3: Results 27 3.1 Gene cloning 27 3.2 Protein expression 27 3.3 Purification of Hisx10 tagged GST-YTH 28 3.4 ATPase assay 29 3.5 ATP-agarose affinity chromatography 29 3.6 GST-pull down 30 3.7 Chaperon-like activity 30 3.8 Zymography 30 3.9 Denaturing Ni-NTA purification and refolding of YTH 31 3.10 CD spectra 31 3.11 Characteristics of rabbit anti-YTH polyclonal antibody 31 Chapter 4: Discussion 33 Chapter 5: Conclusion 37 Chapter 6: Reference 38834039 bytesapplication/pdfen-USATP-結合BC1GTP cyclohydrolase IATP水解遺傳性漸進式肌緊張力不足Hereditary progressive DystoniaGTP cyclohydrolasotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52814/1/ntu-93-R91242013-1.pdf