跳到主要內容

臺灣博碩士論文加值系統

(44.220.62.183) 您好!臺灣時間:2024/02/27 20:28
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:鄭大山
研究生(外文):Cheng, Da-Shan
論文名稱:利用酵母菌雜交系統配合隨機突變和點突變研究NADPHoxidasep67phoxC端SH3B區域
論文名稱(外文):Genetic Determination of Boundary Sequence of NADPH Oxidase p67 phox C-Terminal SH3 Domain Using the Yeast Two-Hybrid System
指導教授:洪義人洪義人引用關係
指導教授(外文):Yi-Ren Hong
學位類別:碩士
校院名稱:高雄醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:66
中文關鍵詞:酵母菌雜交系統隨機及點突變
外文關鍵詞:SH3區域NADPH氧化酵素Yeast two-hybrid systemNADPH oxidasep67phoxSH3 domainRandom mutagensissite-directed mutagenesis
相關次數:
  • 被引用被引用:0
  • 點閱點閱:131
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
【摘要】人類免疫系統的巨噬細胞﹑多核性白血球及嗜中性白血球細胞等
會利用NADPH oxidase系統大量產生superoxide (O2_)來防禦外來侵害人
體的病原微生物。NADPH oxidase系統中,二個細胞內次單位因子p47phox
和p67phox各含有二個相當專一性且和Src homology 3區域(SH3 domain)
類似的同源區域和一個可能與SH3區域專一結合的多脯氨酸區域(proline-
rich region)。在本研究中,我們利用酵母菌雜交系統並結合隨機及點突
變方法來探討p67phox C端的SH3B區域與p47phox多脯氨酸區域的結合情形
,針對p67phox C端的SH3B區域之末端序列P55KVF58V59E60作各種不同的
突變,利用酵母菌雜交系統之功能性分析其結合的情形,期能找到專一性
SH3區域之真正界限(boundary sequence),並能對NADPH oxidase中SH3區
域更進一步的了解。結果顯示當p67phox C端的SH3B區域之P55被置換成
Ile﹑Gly﹑Val﹑Trp﹑Asn﹑Glu﹑His和Phe或中止信號均完全阻斷與p47
phox C端的多脯氨酸區域相結合的能力,顯示其為一高度保留性區域,且
具穩定整個SH3區域結構的角色。在F58位置經改變為同性質之Tyr,並不
影響其結合能力,但置換為中止信號,則完全喪失其結合能力。更進一步
進行置換V59和E60為中止信號,結果顯示E60為中止信號一點也不影響結
合能力,但V59若為中止信號,則完全喪失其結合能力。綜合這些結果可
知,V59位置係p67phox C端SH3B區域之界限序列。另一方面;在大腸桿菌
中大量表現融合蛋白時,我們可發現當序列停止在F58(V59stop)或V59(
E60stop)﹐二者所表現之融合蛋白對於蛋白脢的感受度有所差異。而在in
vitro蛋白連結實驗中﹐此二種融合蛋白均可結合至GST-p47phox融合蛋白
。從X-ray結晶和核磁共振的結構揭示SH3區域包含五條β-strands﹐形成
二組反平行之β-sheets。而由分子模擬顯示,V59所在的位置同時也是提
供結構中第五條b-strand所必需存在的序列。我們也對此V59作各種不同
氨基酸之突變取代,結果發現置換成Phe﹑Ser﹑Gly及Ile不致影響其結合
能力,但置換成Glu﹑Asn及Leu則會影響其結合能力。令人訝異的是,此
位置不能被置換為Leu (NADPH oxidase中p67phox 和p47phox所含之另三
個SH3區域在此相對位置均為Leu)。上述結果建議V59序列之存在不只扮演
了穩定SH3區域結構的角色,也可能參與了NADPH oxidase在信號傳遞中專
一性(specificity)的決定。

AbstractSuperoxide and its reactive byproducts are generated in
large amounts as defense mechanism by neutrophils against
pathogenic microbes. Superoxide is produced by a membrane-bound
multi-subunit NADPH oxidase that transfers electrons from NADPH
to molecular oxygen. In this study, we are investigating the p67
phox C-terminal SH3B domain which binds to p47phox proline-rich
region using the yeast two-hybrid system combine with random
mutagenesis and site-directed mutagenesis to identify mutations
of boundary sequence in p67phox C-terminal SH3B domain. The
amino acid P55 has been changed to Ile, Gly, Val, Trp, Asn, Glu,
His and Phe or produce stop codon, resulting in the binding to
p47phox proline-rich region activity was completely abolished.
Changing F58 to Tyr has no effect of this binding, while change
to stop codon completely abolished the activity. Furthermore,
the amino acid V59 is replaced by stop codon, the activity was
also completely abolished. The amino acid E60 is replaced by
stop codon shown no effect of binding. Taken together, the
resultsOverexpressing of the V59stop and E60stop two mutant of
p67phox C-terminal SH3B domain in the bacterial pET expressing
system. The product of the E60stop fusion protein is showing
unstable compare to the product of V59stop fusion protein. This
suggests that this two fusion protein may exist structure
difference in their conformation. In vitro binding assays show
that both two forms have binding activity of target GST-p47
differ in the yeast two-hybrid system. This suggests that in the
bacterial expression sThe mutation of V59 to Phe﹑Ser﹑Gly or
Ile did not effect the ability of the p67phox C-terminal SH3B
domain to bind p47phox , which shown this residue may not a
conserved residue . However, replaced by Leu or Glu result in
loss of in interaction ability. We speculate that this residue
may play its specificity for protein-protein interactions in
phagocyte NADPH oxidase (since four SH3 domains existing in p67
phox and p47phox of NADPH oxidase, only p67phox C-terminal SH3
domain containing V59, the other three

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top