2008-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/678481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 可能接受另一蛋白質上Lys 的ε-amino group 攻擊形成Isopeptide bond 而酵素離開。此Thioester 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) 或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。其主要功能在於腫瘤形成及轉移、凝血、瘍口癒口、調控細胞自戕死亡、神經傳導物質釋放、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，活化小鼠(>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。在超過一百個鑑定受質中，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。本計畫擬以小鼠睪丸為研究對象，調查tTG受質經polyamine conjugation 造成之影響。假設polyamine conjugation 造成之影響有：酵素之活化或去活化、蛋白質交互作用能力改變、及細胞分佈改變，計對上述可能影響我們設計實驗來逐一探討。實驗項目包括: 建立研究polyamine-conjugation之方法學, 自小鼠睪丸純化polyamine-binding proteins , 檢查in vitro polyamine-conjugation對特定酵素功能之影響,建立研究tTG及polyamine-conjugation於睪丸功能之動物模式; 一是在小鼠青春期前給予tTG抑制劑(Cystamine),觀察其對小鼠睪丸發育之影響, 另一是於小鼠青春期前後施以化療藥物Dorxorubicin造成對睪丸的氧化壓力,並探討tTG及polyamine-conjugation在此的作用.<br> Abstract: 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 over 70 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 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 and 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 analytical methods for purification and identification of polyamine-conjugated proteins, 2) conduct large-scale purification and identification of polyamine-conjugated proteins in mouse testis and 3) investigate the effects of polyamine-conjugation on the tTG substrates. Further, we will use two animal models to examine the role of tTG catalyzed polyamine-conjugation in spermatogenesis ; 1) normal male mice with pre-pubertal treatment of tTG inhibitor, cystamine and 2) pre-pubertal and mature mice treated with dorxorubicin to induce arrest of spermatogenesis.多胺精胺睪丸發育轉穀胺醯氨酶transglutaminasespermine conjugationtestis developmentpolyamine睪丸發育過程轉穀胺醯氨酶摧化之polyamine修飾所扮演的角色