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

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：13

、訪客IP：18.191.234.191

姓名

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)

相關論文

★ 結合奈米脂粒與抗體微陣列晶片的高通量快速檢測系統之發展並應用於婦女子宮頸炎病因之診斷與研究	★ 蛋白質 G 與具硫基反應性的釕複合物之生物接合作為螢光免疫試驗的通用試劑
★ 利用微陣列蛋白質晶片帥選GNRA tetraloop結合蛋白	★ 利用大腸桿菌蛋白質體晶片分析新生兒血液中的免疫球蛋白
★ 利用大腸桿菌蛋白質體晶片找出參與第一型線毛表現之細菌蛋白質	★ 利用人類蛋白質體微陣列晶片探究C型肝炎病毒非轉譯區與宿主之交互作用
★ 利用大腸桿菌蛋白體微陣列晶片系統性探討抗菌肽的胞內作用目標	★ 利用大腸桿菌蛋白質體晶片找出與2-氧基組胺酸交互作用之蛋白質
★ 發展微珠式96孔過濾盤競爭型免疫分析法偵測硫酸紫菌素	★ 異質性核醣核酸蛋白K (hnRNP K) 抑制成熟miRNA-122轉錄後調控機制之研究
★ 利用酵母菌蛋白質體晶片找出與前信使核糖核酸加工因子19泛素連接?經泛素化作用之受質	★ 腸道共生黴菌與酒精性肝病的相關性
★ 在大腸桿菌與酵母菌蛋白質體晶片中量化其蛋白質的濃度	★ 應用大腸桿菌與酵母菌蛋白質體晶片系統性分析抗菌肽及抗生素作用之目標蛋白質

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

[檢視]

[下載]

本電子論文使用權限為同意立即開放。
已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。
請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。

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

參考文獻

1. Ganz T, "Defensins: antimicrobial peptides of innate immunity", Nat Rev Immunol, 3, 710-720, (2003)
2. Brogden KA, "Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?", Nat Rev Microbiol, 3, 238-250, (2005)
3. Cudic M, Otvos L, "Intracellular targets of antibacterial peptides", Current Drug Targets, 3, 101-106, (2002)
4. Hancock REW, Sahl HG, "Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies", Nature Biotechnology, 24, 1551-1557, (2006)
5. Yeaman MR, Yount NY, "Mechanisms of antimicrobial peptide action and resistance", Pharmacol Rev, 55, 27-55, (2003)
6. Park CB, Kim HS, Kim SC, "Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions", Biochem Biophys Res Commun, 244, 253-257, (1998)
7. Gudmundsson GH, Magnusson KP, Chowdhary BP, Johansson M, Andersson L, et al., "Structure of the gene for porcine peptide antibiotic PR-39, a cathelin gene family member: comparative mapping of the locus for the human peptide antibiotic FALL-39", Proc Natl Acad Sci U S A, 92, 7085-7089, (1995)
8. Jenssen H, Hamill P, Hancock RE, "Peptide antimicrobial agents", Clin Microbiol Rev, 19, 491-511, (2006)
9. Salomon RA, Farias RN, "Microcin 25, a novel antimicrobial peptide produced by Escherichia coli", J Bacteriol, 174, 7428-7435, (1992)
10. Wiedemann I, Breukink E, van Kraaij C, Kuipers OP, Bierbaum G, et al., "Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity", J Biol Chem, 276, 1772-1779, (2001)
11. Tomita M, Bellamy W, Takase M, Yamauchi K, Wakabayashi H, et al., "Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin", J Dairy Sci, 74, 4137-4142, (1991)
12. Bellamy W, Takase M, Wakabayashi H, Kawase K, Tomita M, "Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin", J Appl Bacteriol, 73, 472-479, (1992)
13. Vorland LH, Ulvatne H, Andersen J, Haukland HH, Rekdal O, et al., "Antibacterial effects of lactoferricin B", Scand J Infect Dis, 31, 179-184, (1999)
14. Hwang PM, Zhou N, Shan X, Arrowsmith CH, Vogel HJ, "Three-dimensional solution structure of lactoferricin B, an antimicrobial peptide derived from bovine lactoferrin", Biochemistry, 37, 4288-4298, (1998)
15. Podda E, Benincasa M, Pacor S, Micali F, Mattiuzzo M, et al., "Dual mode of action of Bac7, a proline-rich antibacterial peptide", Biochim Biophys Acta, 1760, 1732-1740, (2006)
16. Haukland HH, Ulvatne H, Sandvik K, Vorland LH, "The antimicrobial peptides lactoferricin B and magainin 2 cross over the bacterial cytoplasmic membrane and reside in the cytoplasm", Febs Letters, 508, 389-393, (2001)
17. Ulvatne H, Samuelsen O, Haukland HH, Kramer M, Vorland LH, "Lactoferricin B inhibits bacterial macromolecular synthesis in Escherichia coli and Bacillus subtilis", Fems Microbiology Letters, 237, 377-384, (2004)
18. Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A, et al., "Global analysis of protein activities using proteome chips", Science, 293, 2101-2105, (2001)
19. Chen CS, Korobkova E, Chen H, Zhu J, Jian X, et al., "A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli", Nature Methods, 5, 69-74, (2008)
20. Chen CS, Zhu H, "Protein microarrays", Biotechniques, 40, 423-+, (2006)
21. Chandra H, Reddy PJ, Srivastava S, "Protein microarrays and novel detection platforms", Expert Rev Proteomics, 8, 61-79, (2011)
22. Yang L, Guo S, Li Y, Zhou S, Tao S, "Protein microarrays for systems biology", Acta Biochim Biophys Sin (Shanghai), 43, 161-171, (2011)
23. Beranova-Giorgianni S, "Proteome analysis by two-dimensional gel electrophoresis and mass spectrometry: strengths and limitations", Trac-Trends in Analytical Chemistry, 22, 273-+, (2003)
24. Lueking A, Possling A, Huber O, Beveridge A, Horn M, et al., "A nonredundant human protein chip for antibody screening and serum profiling", Molecular & Cellular Proteomics, 2, 1342-1349, (2003)
25. Chen CS, Sullivan S, Anderson T, Tan AC, Alex PJ, et al., "Identification of Novel Serological Biomarkers for Inflammatory Bowel Disease Using Escherichia coli Proteome Chip", Molecular & Cellular Proteomics, 8, 1765-1776, (2009)
26. Thao S, Chen CS, Zhu H, Escalante-Semerena JC, "Nepsilon-lysine acetylation of a bacterial transcription factor inhibits Its DNA-binding activity", PLoS One, 5, e15123, (2010)
27. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, et al., "Gene ontology: tool for the unification of biology. The Gene Ontology Consortium", Nat Genet, 25, 25-29, (2000)
28. Bailey TL, Boden M, Buske FA, Frith M, Grant CE, et al., "MEME SUITE: tools for motif discovery and searching", Nucleic Acids Research, 37, W202-208, (2009)
29. Bailey TL, Elkan C, "Fitting a mixture model by expectation maximization to discover motifs in biopolymers", Proc Int Conf Intell Syst Mol Biol, 2, 28-36, (1994)
30. Finn RD, Mistry J, Tate J, Coggill P, Heger A, et al., "The Pfam protein families database", Nucleic Acids Research, 38, D211-222, (2010)
31. Andres Leon E, Ezkurdia I, Garcia B, Valencia A, Juan D, "EcID. A database for the inference of functional interactions in E. coli", Nucleic Acids Res, 37, D629-635, (2009)
32. Saka K, Tadenuma M, Nakade S, Tanaka N, Sugawara H, et al., "A complete set of Escherichia coli open reading frames in mobile plasmids facilitating genetic studies", DNA Res, 12, 63-68, (2005)
33. Zhu X, Gerstein M, Snyder M, "ProCAT: a data analysis approach for protein microarrays", Genome Biol, 7, R110, (2006)
34. Maere S, Heymans K, Kuiper M, "BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks", Bioinformatics, 21, 3448-3449, (2005)
35. Cline MS, Smoot M, Cerami E, Kuchinsky A, Landys N, et al., "Integration of biological networks and gene expression data using Cytoscape", Nature Protocols, 2, 2366-2382, (2007)
36. Canelas AB, ten Pierick A, Ras C, Seifar RM, van Dam JC, et al., "Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics", Anal Chem, 81, 7379-7389, (2009)
37. Lundblad JR, Laurance M, Goodman RH, "Fluorescence polarization analysis of protein-DNA and protein-protein interactions", Mol Endocrinol, 10, 607-612, (1996)
38. Parker GJ, Law TL, Lenoch FJ, Bolger RE, "Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase/phosphatase assays", J Biomol Screen, 5, 77-88, (2000)
39. Allen M, Reeves J, Mellor G, "High throughput fluorescence polarization: a homogeneous alternative to radioligand binding for cell surface receptors", J Biomol Screen, 5, 63-69, (2000)
40. Moerke NJ (2009) Fluorescence Polarization (FP) Assays for Monitoring Peptide-Protein or Nucleic Acid-Protein Binding: John Wiley & Sons, Inc.
41. Jameson DM, Seifried SE, "Quantification of protein-protein interactions using fluorescence polarization", Methods-a Companion to Methods in Enzymology, 19, 222-233, (1999)
42. Zhu J, Shimizu K, "Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for D-lactate production under microaerobic condition", Metab Eng, 7, 104-115, (2005)
43. Phue JN, Noronha SB, Bhattacharyya R, Wolfe AJ, Shiloach J, "Glucose metabolism at high density growth of E. coli B and E. coli K: Differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and northern blot analyses (vol 90, pg 805, 2005)", Biotechnology and Bioengineering, 91, 649-649, (2005)
44. Webb M, "Pyruvate accumulation in growth-inhibited cultures of Aerobacter aerogenes", Biochem J, 106, 375-380, (1968)
45. Knight SM, Umezawa N, Lee HS, Gellman SH, Kay BK, "A fluorescence polarization assay for the identification of inhibitors of the p53-DM2 protein-protein interaction", Analytical Biochemistry, 300, 230-236, (2002)
46. Jordan A, Pontis E, Atta M, Krook M, Gibert I, et al., "A second class I ribonucleotide reductase in Enterobacteriaceae: characterization of the Salmonella typhimurium enzyme", Proc Natl Acad Sci U S A, 91, 12892-12896, (1994)
47. Boal AK, Cotruvo JA, Jr., Stubbe J, Rosenzweig AC, "Structural basis for activation of class Ib ribonucleotide reductase", Science, 329, 1526-1530, (2010)
48. Kolberg M, Strand KR, Graff P, Andersson KK, "Structure, function, and mechanism of ribonucleotide reductases", Biochimica Et Biophysica Acta, 1699, 1-34, (2004)
49. Nordlund P, Sjoberg BM, Eklund H, "Three-dimensional structure of the free radical protein of ribonucleotide reductase", Nature, 345, 593-598, (1990)
50. Zhu J, Shimizu K, "The effect of pfl gene knockout on the metabolism for optically pure D-lactate production by Escherichia coli", Appl Microbiol Biotechnol, 64, 367-375, (2004)
51. Ulvatne H, Samuelsen O, Haukland HH, Kramer M, Vorland LH, "Lactoferricin B inhibits bacterial macromolecular synthesis in Escherichia coli and Bacillus subtilis", FEMS Microbiol Lett, 237, 377-384, (2004)
52. Bailey TL, "Discovering novel sequence motifs with MEME", Curr Protoc Bioinformatics, Chapter 2, Unit 2 4, (2002)
53. Andres Leon E, Ezkurdia I, Garcia B, Valencia A, Juan D, "EcID. A database for the inference of functional interactions in E. coli", Nucleic Acids Research, 37, D629-635, (2009)
54. Chuang Y-C (2010) Identification of intracellular target proteins of lactoferricin B using Escherichia coli K12 proteome chips: National Taiwan Ocean University.

指導教授

陳健生(Chien-Sheng Chen)

審核日期

2012-1-10

推文