2009-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/653249摘要：轉穀胺醯氨酶摧化之反應基本上是以酵素Cys 的thiol group 去攻打Gln 上的carbonyl group，NH3離開，形成Thioester bond。而此Thioester bond可接受Lys 的ε-amino group 攻擊形成Isopeptide bond 而酵素離開。此Isopeptide bond 亦可接受另一個一級氨、水分子或者醇類的攻擊。因此此酵素作用的反應為Transadmidation 造成蛋白質之聚合、Amine incorporation 造成polyamines共價結合上蛋白質(Polyamine conjugation)、Esterification 造成Gln 轉換成酸酯、Deamidation 造成Gln 轉換成Glu、及Isopeptide cleavage 造成聚合蛋白質的分開。轉穀胺醯氨酶之功能必需透過受質之鑑定以及Transamidation 造成之影響オ得以瞭解。本實驗室以新穎方法來純化及鑑定轉穀胺醯氨酶之受質，小鼠(>10 周齡)之肝臟及睪丸其轉穀胺醯氨酶酵素活性，並純化出受質，通過質譜分析鑑定受質的身分，最後以免疫轉漬法以確認受質之真正身分。在超過一白個鑑定受質中，大概屬下列性質蛋白；細胞骨架及其調節蛋白、Chaperones 及Co-chaperones、內質網蛋白、細胞Detoxification用蛋白、蛋白質轉譯調節蛋白等，大多是與細胞壓カ反應有關之蛋白。更重要的是其中多個受質屬新的發現。由於轉穀胺醯氨酶本身是氧化壓力所活化的酵素，而且多數受質屬壓力反應有關之蛋白。本計畫擬以培養細胞加以誘發氧化壓力、內質網壓力、熱休克，造成轉穀胺醯氨酶的活化，藉此調查受質Transamidation 造成之影響。根據這些結果 本計畫擬建立方法學尋求一、純化Spermine-conjugated proteins，二、利用質譜分析鑑定Spermine modification sites，三、利用蛋白質體學方法純化及鑑定小鼠睪丸中Spermine-binding proteins之種類，四、利用蛋白質體學方法純化及鑑定小鼠睪丸中Spermine-conjugated proteins之種類。 <br> Abstract: Transglutaminases (TG) are Ca2+-dependent enzymes which catalyze a post-translational modification of proteins. The enzyme reaction leads to the formation of an isopeptide bond either within or between polypeptide chains. The γ-glutamyl-ε-lysine crosslinks are formed between the γ-carboxamide group of peptide-bound glutamine residues and the ε-amino group of peptide-bound lysine residues. Polyamines can replace lysine residue in the transamidation reaction in vitro and in vivo. In order to understand the physiological functions of tissue transglutaminase (tTG or TG2), one needs to identify the acyl donor and the acyl acceptor substrates in the transamidation reaction. Five groups of substrates were disclosed; cytoskeleton proteins, proteins involved in ER stress response, molecular chaperones, proteins involved in redox regulation and proteins involved in stress-induced translation arrest. Most of the tTG substrates are intracellular proteins with functions related to cellular response to stress. Importantly, many of the tTG substrates have not been reported. The polyamines, putrescine, spermidine and spermine are present in most living cells and are required for normal cell function, cellular growth and differentiation. Ornithine decarboxylase (ODC) catalyzes the initial step in the biosynthesis of polyamines. ODC activities are associated with different states of spermatogenesis and play an important role during late meiosis and early spermiogenesis, but action of polyamines on the proliferating spermatogonia has not been investigated. Parts of the polyamine function are mediated by tTG since polyamines can act as the acyl acceptor substrates in the transamidation reaction. Therefore, we wish to ask whether and which effects of tTG in mouse testis are mediated by tTG catalyzed polyamine-conjugation. What proteins are polyamine-conjugated in mouse testis? How polyamine conjugation affects the function of tTG substrates? Thus, we will 1) establish novel methods for identification of polyamine-conjugated proteins and polyamine modification sites by enrichment of polyamine-conjugated proteins and de novo sequencing by Mass Spectrometry, and 2) conduct large-scale purification and identification of polyamine-conjugated proteins and polyamine-binding proteins in mouse testis.轉穀胺醯氨酶多胺多胺修飾小鼠睪丸Transglutaminasepolyaminespolyamine modificationmouse testis睪丸發育過程轉穀胺醯氨酶摧化之polyamine修飾所扮演的角色