2007-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/714519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 大多是接受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、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。其主要功能在於凝血、瘍口癒口、調控細胞自戕死亡、神導傳導物質釋放、訊息傳導及細胞間質形成等。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，因此我們認為轉穀胺醯氨酶及其受<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. TGs are abundant enzymes which are involved in a number of different physiologic processes, e.g., plasma transglutaminase factor XIII stabilizes the fibrin clot during hemostasis, and keratinocyte and epidermal transglutaminase contribute to the formation of the cornified envelope in skin. Several physiological roles for tissue transglutaminases have been demonstrated, such as wound healing, fibrosis, apoptosis, and matrix formation. 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. To this end, we have identified 29 potential transglutaminase substrates from mouse liver extract and over 100 potential substrates from mouse testis extract by substrate purification and tandem Mass Spectrometry analysis. 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 cellular proteins with functions related to cellular response to stress. More importantly, many of the tTG substrates have not been reported. In light of the fact that oxidative stress or UV irradiation elevates in situ tTG activity, we seek to study the role of tTG in HepG2 cells and other cell lines in response to oxidative stress, heat shock and ER stress. In addition, we wish to study the interactions between heat shock and ER stress. More specifically, we will determine whether our tTG substrates identified in vitro are genuine substrates in cells in response to oxidative stress, heat shock and ER stress and further to determine the consequences of tTG activation; (in)activation of substrates, alteration of protein-protein interactions between tTG substrates and partners and alteration of subcellular localization of tTG substrates following transmidation. Furthermore, we will examine whether tTG is involved in the formation of stress-induce RNA granules.轉穀胺醯氨酶蛋白質體熱休克壓力反應蛋白質轉譯transglutaminaseheat shockstress responseprotein translationTissue type Transglutaminase於細胞壓力反應所扮演之角色:生化與細胞學之研究