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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/69408


    Title: 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;Identification of 2-oxohistidine interacting proteins using E. coli proteome chips
    Authors: 林均穆;Lin,Jun-mu
    Contributors: 系統生物與生物資訊研究所
    Keywords: 大腸桿菌蛋白質體晶片;二氧基組胺酸;胜肽與蛋白質交互作用;轉譯後修飾;氧化;人類S100A1蛋白質;E. coli proteome chip;2-oxohistidine;Peptide-protein interaction;Post-translational modification;Oxidation;Human S100A1 protein
    Date: 2016-01-21
    Issue Date: 2016-03-17 20:35:35 (UTC+8)
    Publisher: 國立中央大學
    Abstract: 在細胞呼吸作用下,活性氧化物不斷地產生,而細胞內的蛋白質也同時遭受到活性氧化物的破壞。由於組胺酸在其強金屬螯合的能力下,組胺酸容易受到銅離子、鐵離子以及過氧化氫的金屬催化氧化作用,造成其容易被氧化成2-氧基組胺酸。我們假設生物會演化出抗氧化能力來對抗這種2-氧基組胺酸的產生,因此,在細胞內會有蛋白質可以辨認這樣的氧化官能基(2-氧基組胺酸)。我們利用兩條人造且含有2-氧基組胺酸的肽鏈,以及含有大於五千種蛋白質的大腸桿菌蛋白質體晶片,我們篩選出十個會與2-氧基組胺酸產生交互作用的蛋白質,並且利用偏極化螢光試驗和第三條含有2-氧基組胺酸的肽鏈驗證其交互作用的能力,最後我們測量了各個蛋白質與2-氧基組胺酸之間的親和力。我們利用生物資訊的方式在這十個蛋白質內找出一段共有結合序列,此序列也被發現在人類蛋白質S100A1上,而S100A1也利用上述的方式成功的驗證其具有與2-氧基組胺酸產生交互作用的能力,而這一發現說明了辨認2-氧基組胺酸這樣的修飾在演化上是具有共通性的。我們結合了人造肽鏈與蛋白質體晶片,建立一個有效且高敏感度的研究平台,可提供轉譯後修飾更進一步的研究,即使是小到只有一個氧基的轉譯後修飾。;Cellular proteins are constantly damaged by reactive oxygen species generated by cellular respiration. Due to its metal-chelating property, histidine residues are easily oxidized in the presence of Cu/Fe ions and H2O2 via metal-catalyzed oxidation, usually converted to 2-oxohistidine. We hypothesize that cells may have evolved antioxidant defenses against the generation of 2-oxohistidine residues on proteins, and therefore there would be cellular proteins which specifically interact with this oxidized side chain. Using two chemically synthesized peptide probes containing 2-oxohistidine, high-throughput interactome screening was conducted using the E. coli K12 proteome microarray containing >5000 proteins. The 10 interacting proteins were successfully validated using fluorescence polarization assay, a third peptide probe of different sequence, as well as binding constant measurements. A consensus binding motif was identified among these 10 bacterial interacting proteins based on bioinformatic prediction, which also appeared to be present on human S100A1 protein. The preferential binding of S100A1 with 2-oxohistidine over histidine was successfully validated using all three peptide probes, suggesting that the capacity to recognize 2-oxohistidine modification may be evolutionarily conserved from bacteria to humans. The combination of chemically engineered peptide probes with proteome microarrays proves to be an efficient discovery platform for protein interactomes of unusual post-translational modifications, sensitive enough to detect even the insertion of a single oxygen atom in this case.
    Appears in Collections:[系統生物與生物資訊研究所] 博碩士論文

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