人類鳥胺酸脫羧酶與抗酶複合體之結構研究

Structural study of human ornithine decarboxylase in complex with antizyme

周芳(Fang Chou)

指導教授 ： 詹迺立

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.02052

摘要

多元胺 (polyamines) 為結構中帶有多個胺基的脂肪族小分子，包含腐胺 (putrescine) 、亞精胺 (spermidine) 和精胺 (spermine)，此類帶正電的物質可和帶負電的DNA、RNA或蛋白質產生交互作用，參與細胞生長、分化與凋亡的過程。
人類鳥胺酸脫羧酶 (Ornithine decarboxylase, ODC；EC 4.1.1.17) 為依賴5’-磷酸吡哆醛 (pyridoxal 5’-phosphate, PLP) 輔酶之酵素，負責催化鳥胺酸 (ornithine) 之脫羧反應得到腐胺，此為胞內多元胺合成途徑之第一與速率決定的步驟，亦是多元胺合成的主要調控點。ODC結構包含兩個domains，N端為TIM-like α/β-barrel domain；C端為β-sheet domain。兩個ODC單體 (monomer) 以頭尾相連 (head-to-tail) 的方式組成雙聚體 (homodimer)，其活性中心之胺基酸Lys-69會以Schiff base方式和輔酶PLP結合，形成具有催化活性的酵素。
胞內多元胺的含量會直接影響多元胺的合成，當細胞內多元胺含量過高時，會使抗酶 (antizyme；AZ) 的mRNA藉由轉譯調控合成具有功能的全長AZ蛋白。AZ為ODC之負回饋調控因子，可和ODC單體結合形成異雙聚體 (heterodimer)。AZ與ODC結合後不但會使ODC失去酵素活性，還會使ODC C端發生構型變化，露出可被26S 蛋白酶體 (proteasome) 辨認的降解訊號，進行不依賴泛素的降解路徑 (ubiquitin-independent degradation pathway)，進而抑制多元胺的合成。除了上述的負調控機制外，細胞尚可藉由抗酶抑制因子 (Antizyme inhibitor, AZI) 正向調控多元胺的合成，AZI與ODC具有序列與結構上的同源性，能與AZ形成更穩定的複合體而釋放出ODC，使胞內ODC雙體的含量上升。
本篇論文的主要目的在於解析ODC-AZ蛋白複合體的晶體結構，以深入探討ODC與AZ交互作用的方式，進而解釋AZ如何促使ODC進行不依賴泛素的蛋白降解機制。我們已經成功置備ODC與多種AZ刪除突變形成之複合體，並順利得到複合體的晶體。目前最好的繞射數據其解析度約為3.2 Å，初步分析顯示此晶體屬於Primitive tetragonal 晶系，空間群為P41212，晶胞參數為 a=b=266.64 Å，c=52.37 Å，α=β=γ=90˚。未來希望能得到品質更高的晶體，以利後續結構的解析。

關鍵字

鳥胺酸脫羧酶抗酶

並列摘要

Polyamines, such as putrescine, spermidine and spermine, are abundant multivalent organic cations. These compounds can interact with negatively charged molecules like DNA and RNA to participate in many cellular processes, including chromatin condensation, maintenance of DNA structure, RNA processing, translation and protein activation. Polyamines are essential for normal cell growth and apoptosis, they also play crucial roles during cell differentiation and the development of many cancers.
Ornithine decarboxylase (ODC) is involved in the first and rate-limiting step of the polyamine biosynthesis pathway, catalyzing the decarboxylation of ornithine to produce putrescine, which is subsequently converted into spermindine and then to spermine. 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-barrel domain and a C-terminal β-sheet domain. The active form of ODC exists as a head-to-tail homodimer, and the active site Lys69 binds the PLP cofactor via a Schiff-base linkage. The cellular ODC level is tightly regulated by polyamine concentration via an antizyme (AZ) dependent protein degradation pathway. Accumulation of polyamines promotes translational frameshifting of AZ mRNA, allowing expression of the full-length 25 kDa AZ protein. AZ binds to and inactivates ODC by forming a tight non-covalent 1:1 complex. The ODC-AZ heterodimer formation induces conformation changes in the ODC C-terminal region (residues 425-461), which triggers its association and degradation by the 26S proteasome in an ubiquitin-independent fashion. Residues 117-140 of ODC are critical for its association with AZ.
In addition to the AZ-mediated negative regulation, the intracellular polyamine homeostasis is also regulated by antizyme inhibitor (AZI), an enzymatically inactive ODC homolog. Because AZI binds to AZ with high affinity, ODC can be released from ODC-AZ complex in the presence of AZI, leading to the restoration of ODC activity.
This study is aimed to understand how AZ recognizes ODC and how AZ binding promotes proteasomal degradation of ODC by determining the crystal structure of ODC-AZ complex. Using vapor-diffusion crystallization technique, we have successfully obtained crystals of many different forms of AZ truncation mutants in complex with both the full-length and a mutant ODC (ODCΔ299-310). A diffraction data set has been collected to 3.2 Å resolution. Preliminary diffraction analysis indicated that ODC-AZ complex belongs to space group P41212, with unit-cell parameters a=b=266.64 Å, c=52.37 Å, α=β=γ=90˚. We will continue to optimize the crystallization as well as the cryo-protection conditions to facilitate structural determination.

並列關鍵字

ornithine decarboxylase ； antizyme

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