以抗酶抑制人類鳥胺酸脫羧酶活性暨誘發其經由蛋白酶體降解之結構機轉

Abstract

多元胺(polyamines) 為結構中帶有許多正電荷的一級胺，包含了亞精胺(spermidine)、精胺酸(spermine)和腐胺(putrescine)等，此類物質可和DNA、RNA或蛋白質表面帶負電的區域交互作用，參與蛋白質活性調控、核酸代謝、維持各種核酸結構的穩定，因此多元胺在細胞生長與分化的過程皆扮演重要角色，由於過高濃度的多元胺會導致細胞癌化，因此在細胞內多元胺的含量受到嚴密控制。 人類鳥胺酸脫羧酶(ODC)為5’ -磷酸吡哆醛 (pyridoxal 5’ -phosphate，PLP) 依賴型酵素，負責將鳥胺酸 (ornithine)催化生成腐胺 (putrescine)，此反應為多元胺合成途徑中之速率決定步驟，亦是多元胺合成主要之調控點，ODC結構包含兩個domains，N端為TIM-like α/β-barrel domain; C端為β-sheet domain，其活性中心之胺基酸Lys-69 會以Schiff base方式與輔酶PLP結合。兩個單體(monomer)會以頭尾相連(head to tail)的方式形成同質雙聚體(homodimer)，形成具有催化活性之酵素，ODC酵素活性的高低與胞內多元胺濃度息息相關，故受到嚴密調節。 當細胞內多元胺濃度過高時，會藉由mRNA轉譯調控合成具有功能的全長抗酶蛋白(Antizyme ; Az)，Az為ODC負回饋調控因子，能夠與ODC單體(monomer)結合形成異質雙聚體(heterodimer)，Az與ODC結合後，ODC的酵素活性會受到Az抑制並,且ODC會發生構型變化，使其能被26S蛋白酶體(26Sproteasome)所辨識，進行獨特的不依賴泛素的降解路徑(ubiquitin-independent degradation pathway)，而Az則仍以聚泛素化(poly-ubiquitination)方式進行降解。除了上述的負回饋機制外，細胞內可藉由抗酶抑制因子(Antizyme inhibitor; AzIN) 正向調控多元胺的合成，AzIN 在序列與結構上與ODC具有同源性，但缺乏ODC脫羧作用之酵素活性，其利用與Az形成更穩定之複合體而釋出ODC，使ODC免於和Az形成異雙聚合體，因而提高胞內ODC雙聚體含量。 本研究的主要目的在於解析ODC-Az蛋白質結構，以深入分析Az如何與ODC交互作用並抑制其酵素活性，並試圖探討Az如何促使26S蛋白酶體進行獨特的、不依賴泛素的降解路徑。我們成功製備ODC與Az之N端刪除突變形成之複合體，並順利獲得此複合體之晶體結構。發現Az結合ODC與ODC形成具酵素活性蛋白同質雙聚體的交互作用介面具有重疊性，這可以解釋為何ODC同質雙聚體的生成會因Az出現而受到抑制。此外，當Az-ODC形成異質雙聚體時，ODC-Az會曝露出一個推測可為26S蛋白酶體所辨識的區域，而此一由ODC-Az形成之連續表面能有效驅使26S蛋白酶體辨識並降解ODC。透過核磁共振(Nuclear magnetic resonance, NMR)實驗發現Az結合至ODC並未導致ODC C端(殘基424-461)區域發生構型變化。因此推論ODC C端區域的主要功能在於做為26S蛋白酶體降解ODC之起始片段，在26S蛋白酶體與ODC-Az複合體結合後、此區域可媒介26S蛋白酶體開始降解ODC。我們亦利用同源結構模擬來探討抗酶亞型Az2、Az3(antizyme isoforms; Az2, Az3)與ODC形成之複合體的性質，發現降解辨識相關之連續表面與ODC-Az具有明顯差異。此外，我們亦解出AzIN-Az蛋白質結構，此結構解釋了AzIN如何有效的抑制ODC與Az結合，進而恢復細胞內多胺的濃度。綜合以上所述，透過解析ODC-Az及AzIN-Az此二蛋白質複合體的晶體結構，我們提供了一個結構及機制上的新觀點來解釋Az如何抑制ODC的活性並促進其降解以維持細胞內多胺的恆定。

Polyamines, including spermidine, spermine, and putrescine, are positively charged small organic cations. By interacting with negatively charged nucleic acids and acidic surface patches of proteins, these compounds participate in a large number of cellular processes, ranging from functional modulations of proteins to nucleic acid metabolism and packaging. Therefore, polyamines are essential for cell growth and differentiation, whereas aberrant cellular polyamine level has been implicated in neoplastic transformation. Human L-Ornithine decarboxylase (ODC；EC 4.1.1.17) catalyzes the first and rate-limiting step in the polyamine biosynthetic pathway, catalyzing the decarboxylation of ornithine to putrescine. Human ODC is a 53 kDa pyridoxal 5’-phosphate (PLP)-dependent enzyme consists of 461 amino acids. The ODC monomer consists of two domains: an N-terminal TIM-like α/β domain and a C-terminal β-sheet domain. The active form of ODC is a head to tail homodimer, and the active site residue Lys69 forms a Schiff-base linkage with PLP. It’s not surprisingly the enzymatic activity of ODC and cellular level is subjected to a tight regulation. As polyamine accumulates, ODC activity is inhibited and targeted for proteasomal degradation by interacting with antizyme (Az), a 26.5 kDa intracellular polyamine-inducible protein that binds ODC to form a non-covalent 1:1 complex. Notably, the Az-mediated degradation via the 26S proteasome bypasses the common requirement of poly-ubiquitination. In addition to the Az-mediated negative regulation, the intracellular polyamine homeostasis is also regulated by antizyme inhibitor (AzIN), which displays extensive homology to ODC but lacks decarboxylating activity. Because AzI binds Az with higher affinity than ODC binds to Az, ODC can be released form ODC-Az complex in the presence of AzI, it may save ODC from Az-mediated proteasomal degradation. To decipher how Az recognizes and inhibits ODC, and how Az-binding promotes proteasomal proteolysis of ODC, we have obtained the crystal structure of N-terminal truncated Az in complex with ODC or AzIN. The substantial overlap between the Az-binding surface and the homodimerization interface of ODC readily explains why the formation of a catalytically active ODC dimer is blocked in the presence of Az. Moreover, the assembly of Az-ODC heterodimer results in the juxtaposition of the proposed proteasome-targeting regions of both ODC and Az. The formation of this extensive and exposed surface likely allows the Az-ODC to be efficiently recognized by the 26S proteasome. In addition, NMR spectroscopic analysis reveals that Az-binding affects neither the structure nor dynamics of the ODC C-terminal region, therefore, rather than serving as a proteasome-targeting element, this region likely mediates ODC degradation after the Az-ODC is captured by the proteasome. Our findings also address the functional similarity and uniqueness of different Az isoforms. Finally, the AzIN-Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Take together, our findings provide new structural and mechanistic insights into how the unusual ubiquitin-independent degradation of ODC by the 26S proteasome is achieved.