人類第二亞型抗酶與鳥胺酸脫羧酶複合體之結構研究

Abstract

多胺(polyamine)是一群帶有正電荷的多價有機小分子，主要包含了腐胺、亞精胺與精胺。由於帶有正電荷的特性，其能夠與帶負電荷的DNA、RNA或是蛋白表面帶負電的區域產生交互作用，進而影響細胞的存活與凋亡。過去的研究也發現過量的多胺與細胞的癌化有關，因此，細胞內的多胺含量必須受到嚴密的調控。 鳥胺酸脫羧酶(ornithine decarboxylase, ODC)是5’-磷酸吡哆醛(pyridoxal 5’-phosphate, PLP)依賴型酵素，可將鳥胺酸(ornithine)催化為腐胺(putrescine)，此催化作用不僅是多胺生合成途徑的第一個步驟，更是整個多胺生合成的速率決定步驟與主要的調控點。當細胞內的多胺含量過高時，會促進抗酶蛋白(antizyme, Az)的轉譯作用，Az能與ODC形成異質二聚體(heterodimer)，而阻止ODC形成具有催化活性的同質二聚體(homodimer)，Az與ODC所形成的複合體會被26S蛋白酶體(26S proteasome)辨認，並在不需泛素(ubiquitin)參與的情況下造成ODC的降解，另外，Az也能阻斷細胞對外來多胺的攝取，因此，Az是多胺生合成的負調控因子。人類的Az有三種亞型，第一亞型(Az1)與第二亞型(Az2)在組織中的分布很相似，但在in vitro的實驗中卻發現僅有Az1具備讓ODC降解的能力，而Az2的功能或許是用來暫時性地抑制ODC的活性。本研究希望藉由比較兩者在與ODC形成複合體的結構差異，從而釐清兩者在功能上的區別。 本研究著重於解析人類ODC-Az2蛋白複合體之晶體結構，以便與實驗室已有的ODC-Az1蛋白複合體結構進行比較。首先，建構一系列Az2重組蛋白與ODC的表達質體，以利兩者於大腸桿菌(Escherichia coli, E. coli)進行共同表現，接著利用固定性金屬離子親和層析法和分子篩層析法進行複合體的純化，最後以蒸氣擴散法進行晶體的培養。目前已獲得相當純度的蛋白複合體，並針對蛋白複合體在層析圖所展現的特性進行初步的分析，但尚未獲得適合晶體生成的條件。未來將試著搭配不同截切長度的Az2與ODC片段進行蛋白質複合體的結晶實驗。

Polyamines are polyvalent organic cations found in all eukaryotes and most bacteria. Because of their cationic nature, polyamines can interact with acidic patches on protein surfaces and negatively charged DNA and RNA to alter their functions. Therefore, polyamines are known to regulate cell growth, survival, and proliferation, and abnormal polyamine levels may induce tumorigenesis. To control the cellular levels of polyamines, their biosynthesis is tightly regulated. Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to form putrescine as the first and step in polyamine biosynthesis. When polyamine level is high enough, the translation of antizyme (Az) is stimulated to suppress ODC function and polyamine uptake via the formation of an ODC-Az heterodimer. Moreover, Az-binding allows ODC to be recognized and degraded by the 26S proteasome in an ubiquitin-independent manner. In human, four Az isoforms have been identified to date. Among them, isoform 1 (Az1) and 2 (Az2) exhibit similar tissue distribution. Previous studies demonstrated that only Az1 can efficiently promote ODC degradation in vitro, suggesting that Az2 might act as a reservoir for transient suppression of ODC function. To understand how the ODC-Az1 and ODC-Az2 complexes diverge in function, our laboratory has determined the crystal structure of a truncated Az1 in complex with ODC. The aim of my thesis research is to obtain the crystal structure of the ODC-Az2 complex. To this end, I first constructed plasmids for expressing different truncations of human Az2 with either N-terminal or C-terminal His tag. Then, ODC and different forms of Az2 were co-expressed in E. coli BL21 (DE3) cells. Immobilized metal affinity chromatography was used to capture the ODC-Az2 complexes. Gel filtration column was used next to remove excess Az2 in the sample. Using vapor diffusion crystallization technique, a preliminary condition for growing crystals of the ODC-Az2 was identified. However, subsequent efforts for reproducing these crystals were not successful. A retrospective examination of the purification profiles revealed the ODC-Az2 complex eluted in two peaks, indicating that crystallization may be hampered by structural heterogeneity of the protein sample. Cross-linking experiments suggested that the complex may undergo fast equilibrium between the dimeirc and tetrameric forms and that pooling protein samples of two peaks for crystallization was feasible. Moreover, a new crystallization strategy termed random microseed matrix seeding (rMMS) was performed to facilitate crystallization by using micro-crystals of the ODC-Az195-228 complex. Although a few micro-crystals were observed in some screening conditions, they likely correspond unbroken crystals of the ODC-Az195-228 complex rather than new crystals of the ODC-Az257-189 complex. Additional crystallization trials will be performed using different constructs of Az2 and ODC.