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標題: Caspase-14抗細胞凋亡特性之分子機制
Molecular analysis of the anti-apoptotic property of caspase-14
作者: 洪敏發
Hung, Min-Fa
關鍵字: apoptosis
protein interaction
出版社: 生物醫學研究所
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摘要: 半胱胺酸蛋白酶14 (caspase-14)隸屬於caspase家族一員,然而caspase-14並不歸屬於目前已知三類caspase分類,其胺基酸序列與第一類發炎相關caspase較相似,在細胞內功能又不直接參與第二與第三類caspase促細胞凋亡訊號路徑。先前有關caspase-14的研究大多與其表現於上皮細胞角質化過程為主,在此我們探討caspase-14是否參與細胞凋亡路徑。利用自製的caspase-14抗體,我們發現在肺癌細胞株中大量表現全長型態的caspase-14。本實驗室以酵母菌雙雜交系統 (yeast two hybrid system)發現細胞凋亡誘導因子 (apoptosis inducing factor, AIF)能與caspase-14交互作用,因此本研究進一步以GST pull-down試驗與免疫沉澱法 (immunoprecipitation, IP)測試兩者是否有交互作用。結果顯示caspase-14確實能與AIF有交互作用,並且是經由caspase-14大次單元與AIF之氧化還原結合區域所達成。為了解caspase-14與AIF如何交互作用,我們在caspase-14大次單元內疏水性胺基酸設計點突變為親水性胺基酸,結果顯示casapse-14第21、22與25個胺基酸點突變會影響其與AIF交互作用的能力。先前我們已證實AIF能與hHR23A有交互作用而促進細胞凋亡能力,然而以GST pull-down試驗顯示caspase-14並不與hHR23A有直接交互作用,故在此我們推測caspase-14與hHR23A對於AIF作用位置與功能可能不相同。另外,caspase-14若以牛小腸鹼性去磷酸酶 (calf intestinal alkaline phosphatase, CIP)處理後則會使其蛋白表現量降低,暗指磷酸化具有維持caspase-14蛋白穩定性的功能。而帶螢光之GFP-caspase-14顯示其主要存在於細胞質中,推測caspase-14在此扮演攔截AIF進入細胞核的角色。總結上述結果,我們推測caspase-14在caspase不相關的細胞凋亡過程中扮演抑制AIF進入細胞核的角色。
Caspase-14 is a member of caspase family base on the amino acid sequence. Caspase-14 is most similar to the type I pro-inflammatory caspase on amino acid sequence. Besides, caspase-14 doesn't participate in type II, III caspase-dependent apoptosis. Most of the caspase-14 studies showed its correlation with keratinocyte differentiation. Here we show that caspase-14 plays a role as an anti-apoptotic factor in apoptosis pathway. Previously, by an yeast two hybrid method we demonstrated that apoptosis inducing factor (AIF) interacted with caspase-14. Furthermore, using a GST-pull down system and an immunoprecipitation (IP) we confirmed they did interact with each other. The interaction was through caspase-14 large subunit and the NADH/FAD binding domain of AIF. Using caspase-14 point-mutation by turning hydrophobic amino acid into hydrophilic, our results showed that amino acids 21, 22 and 25 mutations affected the interaction between caspase-14 and AIF. Because AIF interacts with human homologue of yeast Rad23 protein A (hHR23A) to execute apoptosis function. Therefore, we test whether caspase-14 interacts with hHR23A as well. No direct interaction was observed. Caspase-14 was mainly located in the cytoplasm by GFP-caspase-14 transfection. Overall, our data show that caspase-14 intercepts AIF from mitochondria during apoptosis as an anti-apoptotic protein.
其他識別: U0005-1808201012564000
Appears in Collections:生物醫學研究所



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