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研究生:許智勇
研究生(外文):Chih-Yung Hsu
論文名稱:人類基因轉錄輔促因子及抗胰蛋白酵素C-5peptides二級結構之研究
論文名稱(外文):Studies of Secondary Structures of Human Positive Cofactor 4 and a1-antitrypsin C-5 peptides
指導教授:鄭 梅 芬
指導教授(外文):Mei-Fen Jeng
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所
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:2001
畢業學年度:90
語文別:中文
論文頁數:102
中文關鍵詞:人類基因轉錄輔促進因子抗胰蛋白酵素
外文關鍵詞:Human Positive Cofactor 4a1-antitrypsin C-5 peptides
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人類轉錄輔促因子4 (PC4) 二級結構之研究
在細胞合成蛋白質的過程中,必須以訊息RNA作為模板,才能合成正確的氨基酸序列以組成完整的蛋白質,因此在生物體蛋白質合成的調控中,訊息RNA (mRNA) 的合成是一個很重要的環節。
在轉錄作用進行的過程中,訊息RNA的合成需要一些轉錄因子(transcriptional factors) 等一起參與作用的進行。轉錄因子最主要的作用在於轉錄作用進行之初能夠協助RNA polymerase分子聚集到DNA啟動子的位置上,以利訊息RNA合成作用的進行。此外,轉錄輔促因子 (transcriptional cofactor) 也會參與作用的進行,它會結合在轉錄起始點上游一段已活化的DNA序列上,進而與轉錄因子作用以調控RNA polymerase分子與DNA啟動子的結合。
Positive cofactor 4 (PC4) 是一個由127個氨基酸所組成的轉錄輔促進因子,它能與TBP-associated factor (TAFs) 產生協同作用,調控轉錄活化因子與轉錄因子間的作用。非磷酸化的PC4具有促進轉錄的功能,而磷酸化型態的PC4則不具調控活性 (Ge et al. 1994)。
本研究以基因轉殖的方法產生PC4蛋白質,利用紅外線光譜儀(infrared spectrometer) 和環形兩色現象 (circular dichroism) 探討其二級結構的特性,進一步探討其因結構特性所產生的鍵結作用機制。由IR及CD光譜的研究結果發現, PC4蛋白質的二級結構以a-helix型態為主,並且存在少部分的b-sheet結構。利用此一結果,將有助於我們進一步探討PC4蛋白質因其本身二級結構的特性所形成的立體結構及其衍生的結構動力學特性。
抗胰蛋白酵素 (AAT) C-5 peptides二級結構之研究
許多前趨蛋白 (precursor proteins) 經蛋白質水解作用後所產生的氨基酸片段會聚集生成b-類澱粉纖維 (b-amyloid fibrils),目前已知氨基酸片段生成b-類澱粉纖維的現象與其本身二級結構的特性有極大的關係,因此我們合成許多氨基酸片段進行其二級結構特性的研究。
AAT (a1-antitrypsin) 屬於serpins (SERine Proteinase INhibitors) 的一種,其主要的作用是抑制肺臟中嗜中性白血球 (neutrophil) 所產生的彈性蛋白酵素 (elastase) 的活性。之前的研究發現當AAT水解後,其C端一段5個氨基酸序列 (FVFLM) 所組成的疏水性片段會有聚集的現象,產生與類澱粉蛋白沉澱 (amyloid deposit) 相似的沉澱物 (Sabina et al., 1995 )。為了進一步探討此片段中具有決定二級結構之關鍵性胺基酸的位置,以便於將來進行藥物設計方面的研究,我們合成了20個氨基酸片段,藉由置換特定氨基酸及位置,以了解不同氨基酸對於結構所造成的影響。
經由IR及CD光譜分析的結果得知,這20個胺基酸片段聚集時所產生的二級結構依其被置換胺基酸特性的不同而有所差異。大多數的胺基酸片段聚集時會形成不同比例的a-helix、b-sheet以及random coil的結構,但主要仍是以b-sheet的結構所佔的比例較高,其中以非極性脂肪族胺基酸取代的胺基酸片段所表現的b-sheet結構較為明顯。經由置換特定胺基酸的方法研究片段聚集時形成二級結構的特性,有助於了解特定胺基酸對於片段聚集時二級結構的影響,進一步可以運用於設計特定胺基酸片段蛋白質藥物研究。

Studies of the secondary structures of Human positive cofactor 4 (PC4)
In the process of protein synthesis in cells, mRNA must be used as template to translate intact proteins in correct sequence. Therefore, synthesis of mRNA is an important process in the regulation of protein synthesis in organisms. In the process of transcription, synthesis of mRNAs need some transcriptional factors participate in the reaction. The major role of transcriptional factors is to help RNA polymerase bind to the promoter of DNA template in the beginning of transcription. Otherwise, transcriptional cofactors also participate in the reaction by binding on the upstream of promoter in an activated DNA template, and then regulate the binding reaction between RNA polymerase and the promoter of DNA template.
Positive cofactor 4 (PC4) is a transcriptional cofactor composed of 127 amino acids. PC4 can make a synergistic effect with TBP-associated factors (TAFs) and regulate the interaction between transcription-activating factors and transcriptional factors。Unphosphorylated PC4 is the functional form in positive-regulated transcription, and phosphorylated PC4 can not regulate transcription (Ge et al. 1994).
In this study, PC4 gene was cloned into pET-21a vector and expressed in E coli BL21 strain. The secondary structures of PC4 were monitored by infrared (IR) and circular dichroism (CD) spectroscopy, and furthermore to confer the binding mechanism affected by the characteristics of secondary structures of PC4。The results have shown that the predominant secondary structure of PC4 is a-helix, and there are still little parts of PC4 in b-sheet structure. Based on the results of this study, we can further confer the factors that three dimensional structure of PC4 mediated by its secondary structures.
Studies of the secondary structures of a1-antitrypsin (AAT) C-5 peptides
Fragments from various proteolytically degraded precursor proteins can form b-amyloid fibrils. The phenomenon of peptides’ aggregation in the form of b-amyloid fibrils may be related to their secondary structures. Therefore, we synthesized many peptides to study the characteristics of their secondary structures.
a1-antitrypsin (AAT), the predominant serine proteinase inhibitor (serpin) in human serum, is an inhibitor of serine proteases in general but its most important target enzyme is neutrophil elastase. The hydrophobic C-5 peptide in hydrolyzed C-terminal peptide compos of 5 amino acids (FVFLM), aggregates and forms deposit similar with amyloid deposit. Therefore, we have synthesized 20 wild-type and constant C-5 peptides which the amino acid were replaced with a particular amino acid at the third position, in order to study the effects of different amino acid on the formation of secondary structures of the peptides. The information can be used further for structure-based drug design.
The results of IR and CD spectroscopy have shown the secondary structures of these twenty peptides are different because of the characteristics of the replaced amino acid. Most peptides aggregated and formed a-helical structure, b-sheet and random coil at different ratios. The main secondary structure is b-sheet, especially for those peptides having nonpolar, aliphatic amino acid. From the results obtained, we can understand more about the factors of different characteristics of amino acid determining the formation of secondary structure.

一、背景介紹
1. 人類轉錄輔促因子4 (human positive cofactor 4)………………...1
1.1原核生物的轉錄作用…………………………………………...1
1.2真核生物的轉錄作用…………………………………………...1
1.3 PC4的組成與作用機制………………………………………...6
1.4 PC4的磷酸化作用……………………………………………...8
2. 抗胰蛋白酵素 (a1-antitrypsin, AAT) 之概述………………………9
2.1 Serine protease inhibitors簡介………………………………….9
2.2抗胰蛋白酵素的生理作用機制……………………………….11
2.3抗胰蛋白酵素的基因多樣性………………………………….12
2.4抗胰蛋白酵素與類澱粉病變的關係……………………….…15
二、研究方法
1. Fourier transform infrared (FT-IR) spectroscopy原理及介紹..…..17
2. Circular dichroism (CD) spectroscopy原理及介紹…………...….18
三、試驗步驟
1. PC4 DNA轉殖實驗……………………………………………….20
1.1 pET-21a轉殖載體系統………………………………………..20
1.2 Primer設計…………………………………………………….21
1.3 PCR產物純化…………………………………………………21
1.4 Plasmid純化…………………………………………………...23
1.5限切酵素反應 (digestion)…………………………………….24
1.6 DNA接合反應………………………………………………...25
1.7形質轉換 (transformation)……………………………………26
1.8含PC4基因之菌種選殖……………………………………….27
2. PC4蛋白質表現…………………………………………………...28
2.1 PC4蛋白質於不同生長期之表現量………………………….28
3. PC4蛋白質的純化………………………………………………...29
3.1誘導蛋白質表現及菌種細胞打破之實驗………………….…29
3.2 Heparine sepharose column的純化…………………………...30
3.3 Cellulose phosphate column的純化…………………………..32
4. PC4蛋白質IR及CD光譜實驗…………………………………..34
4.1 Fourier transform infrared spectroscopy………………………34
4.2 Circular dichroism spectroscopy………………………………35
5. AAT peptide試驗………………………………………………….36
5.1 FVDLM peptide之純化……………………………………….36
5.2 Fourier transform infrared spectroscopy………………………37
5.3 Circular dichroism spectroscopy………………………………37
四、結果與討論
1. PC4二級結構之探討……………………………………………...38
1.1 IR圖譜分析……………………………………………………38
1.2 CD圖譜分析…………………………………………………..42
2. AAT C-5 胜二級結構之探討…………………………………..43
2.1 IR圖譜分析……………………………………………………43
2.1-1 Nonpolar, aliphatic amino acids 取代之C-5胜系列…..43
2.1-2 Aromatic amino acids 取代之C-5胜系列…………….44
2.1-3 Polar, uncharged amino acids 取代之C-5胜系列……..45
2.1-4 Positively charged amino acids 取代之C-5胜系列…...46
2.1-5 Negatively charged amino acids 取代之C-5胜系列….47
2.2 CD圖譜分析…………………………………………………..49
3. 討論
3.1 PC4蛋白質…………………………………………………….50
3.2 AAT C-5 peptides……………………………………………...51
五、文獻參考…………………………………………………………...82

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