2016-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/656463摘要：多元胺(polyamines)是一群結構中具有二個以上一級胺基(primary amine)的有機小分子，在生理條件下主要以多聚陽離子(polycation)的形式存在。正電態的多元胺能與核酸或蛋白質結構中帶負電的區域結合並影響其活性，是維繫生物細胞正常運作不可或缺的重要物質。目前已知細胞內多元胺濃度異常會影響細胞的生長並可能導致癌化，故細胞對多元胺的攝取(polyamine uptake)及其生合成途徑皆受到嚴密的調控。人類鳥胺酸脫羧酶(ornithine decarboxylase; ODC)負責催化多元胺生合成途徑的起始步驟，是此生合成途徑主要的調控點。當多元胺濃度升高時，抗酶亞型一(antizyme 1; Az1)之 mRNA 會藉由多元胺引發之轉譯框架改變(+1 translational frameshift)開始產生全長的 Az1 蛋白。Az1 不但能藉由與 ODC 形成緊密的複合體抑制其酵素活性，並可使 ODC在不須泛素化(ubiquitin-independent)的情況下直接經由 26S 蛋白酶體(26S proteasome)降解。此外、Az1 會與分佈於細胞表面的多元胺運輸蛋白(polyamine transporter)結合以抑制多元胺的攝取。實驗室目前執行中的國科會計畫即在探討 Az1 辨識並引發 ODC 降解的結構機制，此方面的研究進展順利，並已獲致數項關鍵性的成果(詳述於 C012-1)。 人類細胞中除了 Az1 之外、其他三種抗酶亞型(Az2、Az3、Az4)亦可抑制 ODC 與多元胺運輸蛋白的活性，對多元胺的胞內濃度進行負向調控。然而 Az2、Az3、Az4 並不會導致 ODC 降解，顯示具有組織分佈特異性的不同亞型各有其獨特的功能與重要性。相對於抗酶家族蛋白參與之負調控、抗酶抑制蛋白(antizyme inhibitor; AzIN)的表現則會提高多元胺的濃度。當細胞中多元胺濃度偏低時，AzIN 的表現量會顯著提升。由於AzIN-Az 複合體的穩定度高於 ODC-Az 複合體，因此 AzIN 能有效抑制 Az 與 ODC 的結合，藉以回復 ODC 的酵素功能並重新開啟多元胺的生合成途徑。最近的研究結果更指出 AzIN 的過度表現與多種癌症發生的關聯性極高，因此 AzIN-Az 複合體被視為癌症治療的新分子標靶。本計畫的主要目標之一即在於探討抗酶家族蛋白(Az1、Az2、Az3、Az4)與上述各類多元胺調控蛋白(ODC、AzIN、polyamine transporter)形成之複合體的結構與功能。此外、許多細胞週期調控蛋白(如 cyclin D1、Aurora A kinase 等)也能透過與 Az1 的結合，在無須泛素化的情況下藉由 26S 蛋白酶體降解，因此抗酶家族蛋白於調控細胞生長方面的重要性可謂與日俱增，本計畫亦將解析這些細胞週期調控蛋白與 Az1 形成的複合體結構。 為了瞭解抗酶家族蛋白辨識並誘發其標的蛋白降解之結構基礎，本實驗室已成功解析 ODC-Az1 之複合體晶體結構，並順利建構使用紅血球萃取液(reticulocyte lysate)分析 Az 誘發其標的蛋白降解的實驗技術，以及使用核磁共振光譜(NMR spectroscopy)分析 Az1 結合引起的蛋白構形改變。我們也已建立抗酶家族蛋白(Az1、Az2)與其標的蛋白(ODC、AzIN、Aurora kinase、cyclin D1)之表達與純化的標準流程，可用於製備 Az之相關蛋白複合體進行後續之結構分析。由於我們已順利取得到 ODC-Az1 之高品質晶體，因此未來將會使用「隨機微晶種誘導式結晶法」，以 ODC-Az1 晶體製備微晶種，輔助其他蛋白複合體的結晶。藉由 ODC-Az1 複合體之結構解析及後續的生化分析，我們也發現 ODC 之 C-端刪除突變蛋白在與 Az1 結合後可為 26S 蛋白酶體辨識但不會被降解，因此可應用於與 26S 蛋白酶體共結晶的實驗。綜合以上敘述可知：本研究計畫兼具基礎研究與醫學應用兩方面的重要性，且已獲得部分關鍵成果，預期將為抗酶家族蛋白參與的複雜調控機制提供豐富的結構資訊。<br> Abstract: Polyamines are multivalent organic polycations ubiquitously present in all organisms. Being positively charged at physiological pH, polyamines may interact with acidic surface patches of biomacromolecules to modulate their functions. While proper levels of polyamines are essential for cell growth and differentiation, abnormal increase in polyamine concentration is known to cause many types of human malignancies, including cancers. Therefore, the activities of cellular pathways involving polyamine biosynthesis and uptake are tightly regulated. Ornithine decarboxylase (ODC) catalyzes the first step in polyamine biosynthesis, and its enzymatic function is regulated by a delicate feedback mechanism. When cellular polyamine concentration increases, the translation of antizyme isoform 1 (Az1) mRNA is stimulated via a polyamine-induced +1 frameshift to produce full-length Az1 protein. Binding of Az1 to ODC not only inhibits ODC function, Az1 further channels ODC to 26S proteasome for degradation. Interestingly, the formation of ODC-Az1 heterodimer alone is sufficient for ODC to be recognized by proteasome, and its subsequent degradation is entirely “ubiquitin-independent”. Moreover, Az decreases polyamine uptake by shutting down cell surface polyamine transporter. Our on-going NSC grant is aimed at elucidating the structural basis by which Az1 mediates the degradation of ODC, and we have achieved major breakthrough during the past year (please refer to C012-1 for details). Besides Az1, human genome encodes three additional Az isoforms (Az2, Az3, Az4) that may also down-regulate polyamine biosynthesis and uptake. Despite their comparable affinity toward ODC, only Az1 is capable of promoting ODC degradation, suggesting that each isoform possesses distinct cellular functions. In contrast to the inhibitory roles exhibited by Az isoforms, the cellular polamine level is positively regulated by antizyme inhibitor protein (AzIN), which associates tightly with Az to sequester its function. A recently published study revealed a strong correlation between AzIN expression and tumorigenesis, making AzIN-Az complex a legitimate new target for anticancer therapy. This research proposal is aimed to provide a better understanding on the regulatory mechanism of cellular polyamine homeostasis by characterizing the structures of Az family members (Az1-4) in complexes with their target proteins (ODC, AzIN, polyamine transporters). In addition, Az1 also involves in cell cycle regulation by mediating the “ubiquitin-independent proteasomal degradation” of cyclin D1 and Aurora A kinase. Given the increasing biological significance of Az family, we will also perform structural analyses on the cyclin D1-Az1 and Aurora A kinase-Az1 complexes to uncover the structural basis by which Az1 recognizes its target proteins. To understand the structural basis of Az-mediated recognition and degradation of its target proteins, we have successfully determined the crystal structure of ODC-Az1 complex and characterized the Az1-induced ODC conformational changes by NMR. In addition, a reticulocyte lysate-based assay has been implanted to allow in vitro examination of Az-mediated protein degradation. We have also established the expression and purification protocols for Az family members (Az1, Az2) and their target proteins (ODC, AzIN, cyclin D1, Aurora A kinase), which will be used to prepare the relevant protein complexes for structural analyses. Since ODC-Az1 crystals can now be routinely prepared in my lab, the newly developed “random microseeding technique” can be readily employed by using microcrystals of ODC-Az1 to facilitate the crystallization of other protein complexes. Given our current progress, it is expected that the majority of the proposed specific aims can be achieved successfully.多元胺鳥胺酸脫羧酶抗酶家族蛋白抗酶抑制蛋白26S 蛋白酶體細 胞週期調控蛋白隨機微晶種誘導式結晶晶體結構解析polyamineornithine decarboxylaseantizyme familyantizyme inhibitor26S proteasomecell-cycle regulationrandom microseedingcrystal structure