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研究生： 唐蒂琪
Duong Ngoc Kieu Thi
論文名稱： 酵母菌組蛋白的物種專一性生物素化
Species-specific biotinylation of yeast histones
指導教授： 王健家
Wang Chien-Chia
口試委員:
學位類別： 碩士
Master
系所名稱： 生醫理工學院 - 生命科學系
Department of Life Science
論文出版年： 2018
畢業學年度： 106
語文別： 英文
論文頁數： 52
中文關鍵詞： 組蛋白 、H2B 、生物素蛋白連接酶 、白色念珠菌 、生物素化
外文關鍵詞： Histone, H2B, biotin protein ligase, Candida albicans, biotinylation
相關次數： 點閱：15  下載：0 
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    • x中文摘要

    生物素化是 種必需的維生素與蛋白質的特定賴氨酸殘基共價結合的生物素化作用，由酵母中的生物素蛋白連接酶（BPL1）介導。生物素化是一種罕見的翻譯後修飾，通常發生在位於廣泛分化物種中羧化酶內共有序列（A / V）MKM中的特定賴氨酸殘基上。雖然釀酒酵母的Arc1p缺少共有序列（A / V）MKM，但它在其SSKD處被生物素化。更有趣的是，儘管白色念珠菌（CaH2B）的組蛋白蛋白缺乏（A / V）MKM和SSKD，它們可以在體內生物素化，而釀酒酵母（ScH2B）的組蛋白不能。從Western blot檢測HRP-Streptavidin，我們的結果表明，CaH2B的生物素化似乎是溫度依賴性的;將生長溫度從20℃升高至37℃顯著增加其生物素化水平。基於外源生物素補充劑（〜0.2μg/ L至200μg/ L）的增強，組蛋白的生物素化水平也增加。儘管測試了10種BPL1（ScBPL1，CaBPL1，SpBPL1，PsBPL1，VpBPL1，KlBPL1，LeBPL1，PgBPL1，TpBPL1 ，DhBPL1）可以成功替代ScBPL1敲除菌株，但是它們都不能在釀酒酵母體內對天然組蛋白或CaH2B轉化體進行生物素化。然而，體外生物素化測定結果表明，CaBPL1和ScBPL1都能直接將生物素附著到組蛋白H2B上。這些結果表明釀酒酵母可能含有它們自己的機制來抑制生物素蛋白連接酶在體內對蛋白質組蛋白的生物素化。
    關鍵詞：組蛋白，H2B，生物素蛋白連接酶，白色念珠菌，生物素化

    Abstract

    Biotinylation, which is covalent binding of a biotin - an essential vitamin to a specific lysine residue of a protein, is mediated by biotin protein ligase (BPL1) in yeast. Biotinylation is a rare post-translational modification and normally occurs in a specific lysine residue positioned in the consensus sequence (A/V)MKM within carboxylases in widely divergent species. While Arc1p of Saccharomyces cerevisiae lacks the consensus sequence (A/V)MKM, it is biotinylated at its SSKD. More interestingly, even though the histone proteins of Candida albicans (CaH2B) lack both (A/V)MKM and SSKD, they can be biotinylated in vivo while histone proteins of S. cerevisiae (ScH2B) cannot. Judging from Western blot probed with HRP-Streptavidin, our results showed that the biotinylation of CaH2B appears to be temperature-dependent; rising the growth temperature from 20oC to 37oC dramatically increased its biotinylation level. Biotinylation level of histones also increased based on the enhancement of exogenous biotin supplement (~0.2 µg/L to 200 μg/L). Despite ten of BPL1s tested (S. cerevisiae BPL1 INVSc1, C. albicans BPL1 SC 5314, S. pombe BPL1 972, and all wild-type of P. stipitis BPL1, V. polyspora BPL1, K. lactis BPL1, L. elongisporus BPL1, P. guilliermondii BPL1, T. phaffii BPL1, D. hansennii BPL1) can successfully substitute ScBPL1 knockout strain, none of them can biotinylate the native histone protein or CaH2B transformants in S. cerevisiae. However, result from in vitro biotinylation assay showed that both of the CaBPL1 and ScBPL1 could attach biotin into the histone protein H2B directly. These results suggesting that S. cerevisiae might contain their own mechanism to inhibit biotinylation of protein histone by biotin protein ligase in vivo.

    Keywords: Histone, H2B, biotin protein ligase, Candida albicans, biotinylation

    Table of Contents 中文摘要 i Abstract ii Acknowledgement iii Table of Contents iv List of figures vi List of Tables vii Abbreviations viii I. Introduction 1 1. Post-translational modification 1 2. Biotin 1 3. Biotin protein ligase 2 4. Targets of biotinylation 3 5. Biological function of histone biotinylation from various species 5 II. Materials and methods 8 1. Strains and culture media 8 2. Plasmid constructions 8 3. Complementation assays for cytoplasmic function and in vivo biotinylation assays 9 4. Western blot with HRP-anti-His6 Ab, HRP-anti mouse IgG-anti H2B, Streptavidin-HRP 10 5. Purification of His6-tagged Proteins 11 6. In vitro biotinylation assays 13 III. Results 14 1. Only C. albicans histones are biotinylated among 10 yeast species tested 14 2. Biotinylation of histone in C. albicans is biotin concentration dependent 14 3. Biotinylation of histone in C. albicans is temperature dependent 15 4. Rescue of ScBPL1 knockout strain by BPL1s of other yeasts in vivo 15 5. Co-transformation of CaH2B and yeast BPL1s into a ScBPL1 knockout strain 16 6. Purification of BPL1 and H2B 17 7. In vitro biotinylation of H2B by BPL1 18 IV. Discussion 19 FIGURES 23 TABLES 32 REFERENCES 33 Appendixes 36 Appendix 1. Schematic structure of the three classes biotin protein ligase 36 Appendix 2. Sequence alignment of the biotin domains of biotin carboxylase from diverse organisms. 36 Appendix 3. Biotinylation of Arc1p. 37 Appendix 4. Sequence of C. albicans Histone H2B. The sites of biotinylation are in bold 38 Appendix 5. Sequence of C. albicans Histone H4. The sites of biotinylation are in bold 38 Appendix 6. Plasmid list 39

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