2013-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658463摘要：多元胺(polyamines)是廣為存在於細胞內、結構中帶有多個正電荷的一級胺，包含腐胺(putrescine)、亞精胺(spermidine)、及精胺(spermine)等。多元胺能與核酸或蛋白質帶負電的區域(如 DNA、RNA 的核糖磷酸骨架；蛋白質之負電表面等)發生交互作用並影響其活性，對細胞的生長、分化及凋亡極為重要。由於過量的多元胺會導致細胞的變異與癌化，因此多元胺的胞內濃度受到嚴密的調控。人類鳥胺酸脫羧酶(ornithine decarboxylase; ODC)能將鳥胺酸分子之羧基移除而形成腐胺，此反應為多元胺合成途徑的起始與速率決定步驟，亦是多元胺生合成主要的調控點。當多元胺濃度升高時，存在於細胞質中的抗酶(antizyme; Az) mRNA 會藉由轉譯框架改變(translational frameshift)的調控機制開始產生全長的 Az 蛋白。Az 能藉由與 ODC 形成緊密的複合體有效抑制其酵素活性，更特別的是 Az-ODC 複合體會直接被 26S 蛋白酶體(26S proteasome)辨識；並在無須泛素化的情況下(ubiquitin-independent)造成 ODC 的降解，以降低多元胺的胞內濃度。目前關於 26S 蛋白酶體降解機制的結構研究皆集中於泛素相關的調控途徑(ubiquitin-dependent degradation pathways)，而對 Az 引發蛋白降解的結構機制則所知極為有限，本計畫的主要目標之一在於解析 Az-ODC 之蛋白複合體結構，並分析此複合體與26S蛋白酶體的交互作用，以深入瞭解Az辨識並導致其標的蛋白降解的分子機制。 除了上述的負向調控機制，抗酶抑制蛋白(antizyme inhibitor; AzI)亦參與胞內多元胺濃度的調節。當多元胺濃度不足時，細胞會增加 AzI 的表現量。由於 Az-AzI 複合體的穩定度高於 Az-ODC，因此 AzI 能抑制 Az 與 ODC 的結合並回復 ODC 的酵素功能，進而促進多元胺的生合成。有趣的是，AzI 與 ODC 雖為同源蛋白，且二者胺基酸序列高度相似，但 AzI 的降解卻受到泛素化(poly-ubiquitination)的調控，而 Az-AzI 複合體並不會被 26S 蛋白酶體所辨識，本計畫亦將針對 Az-AzI 複合體進行結構解析，以瞭解Az-ODC 和 Az-AzI 複合體的結構差異如何影響其與 26S 蛋白酶體之交互作用。 最近的結果顯示，除了 ODC 之外，許多細胞週期調控蛋白(如 cyclin D1、Aurora A kinase 等)也能透過與 Az 的結合，在無須泛素化的情況下藉由 26S 蛋白酶體進行降解。鑑於 Az 相關之蛋白降解途徑與日俱增的重要性，解析這些重要細胞週期調控蛋白與Az 形成的複合體結構亦為本計畫的研究重點。 目前本實驗室已建立 ODC、Az、AzI 等蛋白之表達與純化的標準流程，可製備大量Az-ODC 與 Az-AzI 蛋白複合體。經由結晶條件篩選，我們亦順利得到二者的蛋白單晶，並成功判定解析度約為 2.6 Å 的 Az-ODC 晶體結構。此結構揭示 Az 辨識並抑制 ODC活性的分子機制，後續的生化及結構分析將針對 Az-ODC 複合體與 26S 蛋白酶體之間的交互作用進行探討。而 Az-AzI 晶體之繞射解析度則可達 5.4 Å 左右，目前正致力於晶體品質的改良，以得到更佳的繞射數據。綜合以上敘述可知：本計畫的方向明確且已獲得豐富的初步結果，預期本研究應能順利進行，並可望為此重要的蛋白降解機制提供第一手的結構訊息。<br> Abstract: Ornithine decarboxylase (ODC) catalyzes the first and rate-limiting reaction in polyamine biosynthesis, which produces putrescine, spermidine, and spermine. By carrying multiple primary amine groups, the positively charged polyamines may regulate the functions of proteins and nucleic acids by interacting with acidic surface patches of protein and phosphoribosyl backbones of DNA and RNA. While suitable levels of polyamines are essential for cell growth and differentiation in eukaryotes, abnormal increase in cellular polyamine concentration is known to associate with many human malignancies, including cancers. Therefore, the ODC activity is tightly regulated during the cell cycle. Earlier studies revealed that the down-regulation of polyamine biosynthesis is achieved by degradation of ODC by the 26S proteasome. Interestingly, instead of being mediated by the well known ubiquitin-dependent tagging system, the down regulation of ODC activity is triggered by its direct association with antizyme (Az), a conserved small protein of 228 residues. Az binds to and inhibits the enzymatic activity of ODC. Moreover, Az promotes an unusual “ubiquitin-independent” proteasomal degradation of ODC. As structural information related to Az-mediated degradation pathway is currently limited, the main goals of this project include structural and biochemical characterization of how Az interacts with ODC and how the resulting Az-ODC heterodimer is recognized by the 26S proteasome. In contrast to Az function, the cellular ODC activity is positively regulated by the expression of antizyme inhibitor (AzI), a 50 kDa protein that restores cellular ODC function by disrupting the Az-ODC complex through its tight association with Az. While AzI shares over 50% similarity with ODC, the formation of Az-AzI complex does not target AzI for degradation. Instead, Az-binding suppressess the ubiquitin-dependent degradation of AzI. Therefore, it would be both interesting and important to know how Az binding may lead to distinct biological consequences. We will also attempt to determine the structure of Az-AzI complex. In addition to its role as a negative regulator for ODC, Az also down regulates the levels of cell cycle regulatory protein cyclin D1 as well as Aurora A kinase. Due to the increasing biological significance of Az-mediated protein degradation pathway, we intend to uncover the structural basis by which Az recognizes its target proteins by determining the crystal structures of the relevant protein complexes. One of the biggest hurdles in any crystallographic study is whether diffracting crystals of the target proteins or protein complexes can be obtained. To this end, we have reconstituted Az-ODC and Az-AzI heterodimers and produced crystals of both complexes. Moreover, the structure of Az-ODC has been successfully determined at 2.6 Å resolution, which explains clearly how the Az-mediated inhibition of ODC activity is achieved. Subsequent efforts will be initiated to address how Az-ODC is recognized by the 26S proteasome. The Az-AzI crystals only diffract X-ray to low resolution (~5.8 Å). We are now employing a variety of techniques, including surface entropy reduction, crystal dehydration, new cryo-protection protocol, to improve diffraction quality. Given our current progress, it is expected that we will be able to deliver new insights for the Az-mediated protein degradation pathway.多元胺鳥胺酸脫羧酶抗酶抗酶抑制蛋白26S 蛋白酶體晶體結構polyamineornithine decarboxylaseantizymeantizyme inhibitor26S proteasomecrystal structure