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標題: A Study on the Regulatory Mechanisms of Protein-Protein Interactions among Human Ornithine Decarboxylase, Antizyme and Antizyme Inhibitor, and Their Biological Significanc
作者: 洪慧芝
關鍵字: 基礎研究;ornithine decarboxylase;生物科學類;人類鳥胺酸脫羧?;蛋白質-蛋白質交互作用;定位突變;分析型超高速離心;protein-protein interaction;site-directedmutagenesis;analytical ultracentrifugation
在細胞週期過程中,鳥胺酸脫羧酶 (ODC) 的濃度是被嚴密地調控的。活化的鳥胺酸脫羧酶和人類許多種類之惡性腫瘤有著高度相關性。抗酶 (antizyme) 可直接與鳥胺酸脫羧酶結合,抑制其活性,並且使鳥胺酸脫羧酶不需與泛素結合就能被26S 蛋白酶體降解。另一方面,過度產生的抗酶也和抑制許多種類的癌細胞生長有關。因此,抗酶也許具有抑制腫瘤形成的功能。抗酶抑制蛋白 (antizymeinhibitor) 可使所有抗酶家族的成員失去活性,進而使鳥胺酸脫羧酶回復其活性且不受降解。由上述,推測在腫瘤形成的過程中,抗酶是負向的調控因子,而抗酶抑制蛋白是正向的調控因子。在此三年的計畫中,我們首先將目標放在釐清鳥胺酸脫羧酶和抗酶抑制蛋白間,其四級結構的形成及酵素活性上決定差異性之決定因子。第二,我們將研究人類鳥胺酸脫羧酶,人類抗酶抑制蛋白及錐蟲鳥胺酸脫羧酶,對於抗酶具有不同結合能力之決定因素。第三,我們將致力於尋找抗酶的二級結構片段,可以有效抑制鳥胺酸脫羧酶酵素活性或是阻斷此酶形成二聚體。我們將利用分析型超高速離心技術,分析這些蛋白質的四級結構,以及利用X-ray 繞射,分析這些蛋白質複合物的結晶結構。此外,在本計畫中,定點突變、酵素動力學及生物物理學之方法也會參與解決這些問題。這些結果在發展鳥胺酸脫羧酶之抑制劑用於癌症治療上將賦予價值,進而開啟一條在癌症治療之康莊大道。

Levels of the enzyme ornithine decarboxylase (ODC) are tightly regulated during thecell cycle. Elevated ODC activity is associated with many forms of humanmalignancies. Antizyme (AZ) directly binds to ODC, inhibits ODC activity andpromotes the ubiquitin-independent degradation of ODC by the 26S proteasome. Incontrast, the overproduction of AZ has been shown to correlate with cell growthinhibition in a variety of different cell types. Therefore, AZ may function as a tumorsuppressor. Antizyme inhibitor (AZI) inactivates all members of the AZ family,reactivates ODC and prevents the proteolytic degradation of ODC. Overall, theseobservations suggest a role for AZ as a negative modulator, and AZI as a positivemodulator, of tumor progression. In this three-year proposal, we first aim to identifythe underlying mechanisms that direct the differential quaternary structure formationand enzyme activity of ODC and AZI. Secondly, we will investigate the essentialfactors that control the AZ-binding affinity of human ODC, human AZI andtrypanosomal ODC. Thirdly, we aim to identify the essential secondary structureelements of AZ that can effectively inhibit the activity or disrupt the dimeric structureof ODC. The quaternary structure of these proteins will be examined usinganalytical ultracentrifugation and the structures of these protein complexes will bedetermined by X-ray crystallography. In addition, site-directed mutagenesis, kineticanalysis and biophysical methods will be used to address the questions in thisproposal. These results will be valuable in the development of an ODC inhibitor forcancer therapy, and may have a significant impact in opening this pathway fortherapeutic innovation.
其他識別: NSC98-2113-M005-009
Appears in Collections:生命科學系所

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