以生物資訊分析與實驗驗證探討大腸桿菌蛋白質體晶片找出的乳鐵胜肽B胞內目標蛋白

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杜郁萱(Yu-Hsuan Tu) 查詢紙本館藏

畢業系所

系統生物與生物資訊研究所

論文名稱

以生物資訊分析與實驗驗證探討大腸桿菌蛋白質體晶片找出的乳鐵胜肽B胞內目標蛋白
(Validations and Bioinformatics Analyses ofLactoferricin B Intracellular Targets Identified by Escherichia coli Proteome Chips)

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摘要(中)

乳鐵胜肽B是一種廣受矚目的抗菌肽。許多研究指出乳鐵胜肽B可藉由影響細菌的胞內活動達到抑制細菌的效果，然而，卻不知道抗菌肽的胞內目標蛋白為何。因此，我們使用一種高通量方式─大腸桿菌蛋白質體晶片試圖找出乳鐵胜肽B的胞內目標蛋白。首先，將乳鐵胜肽B和大腸桿菌蛋白質體晶片進行交互作用，並且做正規化及基因本體分析。從基因本體分析結果發現，利用晶片找出的可能目標蛋白和代謝的過程是很有關係的。接著，我們利用螢光偏極化實驗來驗證乳鐵胜肽B和晶片實驗找出的可能目標蛋白之間的交互作用。從螢光偏極化實驗中，十六個蛋白質被找出並且透過大腸桿菌交互作用資料庫發現這些蛋白質大部分都和檸檬酸循環有關。另外，我們還使用基因剔除實驗為螢光偏極化的結果做進一步驗證。其結果顯示磷酸烯醇式丙酮酸羧化酶為乳鐵胜肽B的目標。因此，抑制細菌的機制之一可能和丙酮酸代謝有關係。所以，我們利用丙酮酸實驗以活體證實乳鐵胜肽B和大腸桿菌中丙酮酸含量的關係。從結果可以發現當大腸桿菌在有乳鐵胜肽B的環境生長時，有異於正常情況的丙酮酸含量；也就是說，乳鐵胜肽B造成大腸桿菌體內有丙酮酸堆積的現象。此研究成功利用大腸桿菌蛋白質體晶片找出乳鐵胜肽B於細菌內部的目標蛋白，並藉由結合實驗驗證及生物資訊的方法找出其抑制細菌的可能機制。

摘要(英)

Lactoferricin B (LfcinB) is a well-known antimicrobial peptide (AMP). Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this AMP have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO) analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we
validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP) assays. Sixteen proteins were identified, and an E. coli
interaction database (EcID) analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA) cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets and the possible mechanism of LfcinB using an E. coli proteome chip approach with the combination of validations and
bioinformatics analyses.

關鍵字(中)

★ 抗菌肽
★ 蛋白質體晶片
★ 乳鐵胜肽B
★ 胞內目標蛋白

關鍵字(英)

★ antimicrobial peptides
★ intracellular targets
★ lactoferricin B
★ proteome chips

論文目次

摘要 i
ABSTRACT iii
誌謝 iv
Table of Contents v
List of Figures vii
List of Tables ix
I INTRODUCTION 1
I. 1. Immune System 1
I. 2. Antimicrobial Peptides 1
I. 2. 1. Different modes of inhibiting activities 2
I. 3. Lactoferricin B 2
I. 3. 1. Source and structure of Lactoferricin B 2
I. 3. 2. The inhibiting activities of Lactoferricin B 2
I. 4. Proteome Chips 3
I. 4. 1. Applications of proteome chips 3
I. 5. Bioinformatics Analyses 4
I. 5. 1. Gene Ontology 4
I. 5. 2. MEME and Pfam 4
I. 5. 3. E. coli interaction database 4
I. 6. Study Outline 5
II MATERIALS AND METHODS 5
II. 1. Fabrication of an E. coli proteome chip 5
II. 2. The influence of biotinylated LfcinB, LfcinB and biotin on E. coli growth 6
II. 3. E. coli proteome chip assays with LfcinB 7
II. 4. Bioinformatics analyses of the chip assay results 7
II. 5. Fluorescence polarization assays 8
II. 6. Functional interaction analysis 8
II. 7. Knockout assays 9
II. 8. Pyruvate assays 9
II. 9. Overexpression assays 10
III RESULTS 10
III. 1. E. coli proteome chip assays 10
III. 2. Fluorescence polarization assays 12
III. 3. Motifs and domains 13
III. 4. Functional interaction analysis 13
III. 5. Knockout assays 14
III. 6. Pyruvate assays 15
III. 7. Overexpression assays 16
IV DISCUSSION 17
IV. 1. A rapid, reliable and high-throughput tool to study antimicrobial peptides 17
IV. 2. Limitations of knockout assays 17
IV. 3. The EcID analysis 18
IV. 4. LfcinB and pyruvate accumulation 19
V CONCLUSION 20
VI REFERENCES 20
FIGURES 25
TABLES 47
APPENDIX: PLoS ONE publication reprint 53

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指導教授

陳健生(Chien-Sheng Chen)

審核日期

2012-1-10

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