Characterization of Serine Protease 23 and its potential roles in cancer invasion

絲氨酸蛋白酶23的功能定性以及其在癌症入侵中可能的角色探討

林有志(Yu-Chi Lin)

指導教授 ： 莊永仁

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2008年)

摘要

Cell migration and extracellular matrix degradation are important properties of the cancer cells. Several proteases are known to participate in these processes. In order to understand the molecular mechanisms underlying these processes, we identified a novel tumor-related protease, serine protease 23 (PRSS23/SPUVE), by SAGE database mining. The current findings have shown that the gene expression of PRSS23 is significantly up-regulated in various cancers such as breast, cervical and thyroid. Furthermore, the protein sequence analysis has predicted that PRSS23 is consisted of a hydrophobic signal peptide (hSP), a stem region which is annotated as N domain, and a trypsin-like proteolytic (P) domain.
To characterize PRSS23, we first analyzed the function of each domain by expressing various truncated forms of PRSS23 protein. The Western blot showed that hSP was essential for full-length PRSS23 cleavage at a site within N domain. The confocal imaging showed that the cleaved PRSS23, P domain, could be transported from the cytoplasm into the nucleus. Moreover, we used GST-pull down assay to identify the interaction molecules of PRSS23. The assay revealed that the N domain was critical for PRSS23 to interact with β-actin, which may account for its transient stay in the cytoplasm. To investigate the functional roles of PRSS23, we knockdowned PRSS23 expression in the high invasive breast (MD-MB-21) and lung cancer (CL1-5) cell lines. The PRSS23 knockdown MDA-MB-231 cells were found to exhibit enhanced cell migration ability. Interestingly, the RNA expression of MMP9 and MT1-MMP (MMP14) were also increased in the PRSS23 knockdown MDA-MB-231 cells. Moreover, The PRSS23 knockdown CL1-5 cells could enhance their cell invasiveness. Based on these observations, my results supported that PRSS23 may play an important role in regulating cell mobility in cancer.

關鍵字

絲氨酸蛋白酶23 ； 癌症轉移 ； 癌症入侵

並列摘要

細胞移動與胞外間質分解是癌細胞的重要特性。現今已經知道有許多蛋白酶參與此過程。在我們先前的研究中，利用比較不同血管內皮層細胞基因表達系列分析資料庫發現一可能與癌症相關的新穎蛋白酶-絲氨酸蛋白酶23。相關文獻指出絲氨酸蛋白酶23在乳癌、子宮頸癌與甲狀腺癌等癌症中被高度表現。此外，分析絲氨酸蛋白酶23的氨基酸序列後，發現其具有一疏水性胜肽訊號、一個莖柄功能域稱之為N功能域，以及一個胰蛋白酶相似功能域稱之為P功能域。文獻中對於此蛋白酶功能的瞭解至今仍不清楚。
□畯怑漸□ル悛竁F個別功能域的方式來探討絲氨酸蛋白酶23的功能。由西方點墨吸漬結果指出，疏水性胜肽訊號對於截切絲氨酸蛋白酶23的全長前趨物是必須的。被截切的氨基酸序列可能位於N功能域間。由共軛焦影像分析指出被截切後的絲氨酸蛋白酶23會從細胞質內被運送到細胞核中。我們亦利用GST沈降試驗來確定與絲氨酸蛋白酶23有交互作用的分子，結果顯示絲氨酸蛋白酶23中的N功能域與肌動蛋白分子間具有交互作用。因此，我們認為全長的絲氨酸蛋白酶23會藉由與肌動蛋白分子間的作用，停留在細胞質中。為了進一步地研究絲氨酸蛋白酶23的功能，我們利用核酸干擾的方式來默化MDA-MB-231與CL1-5兩癌細胞株中的絲氨酸蛋白酶23的表現。結果顯示默化絲氨酸蛋白酶23後能增進MDA-MB-231的細胞移動，同時默化後亦提升基質金屬蛋白酶9與基質金屬蛋白酶14的基因表現。此外，默化絲氨酸蛋白酶23後的CL1-5亦能增加細胞的轉移。由上述的結果顯示絲氨酸蛋白酶23對於癌症細胞的移動特性扮演一重要的調控角色。

並列關鍵字

PRSS23 ； metastasis ； invasion

參考文獻

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