人類類哺乳類Ste20激酵素3 (Mst3)在細胞凋亡中之角色與作用機轉研究

Abstract

本計畫的主要目標為瞭解人類Ste20 蛋白激酵素3 (Mst3)在一些妊娠期病變中在胎 盤上的生理功能，以及其在壓力下引發細胞凋亡作用中的分子作用機轉。在先前的研 究中，我們已証實大量表現Mst3 在細胞中，會導致許多類細胞株的死亡。近期，我們 也發現Mst3 可同時存在於細胞質及粒線體中。進一步研究發現，Mst3 存在於粒線體 的膜間隙(intermembrane space)中，在此一區域，Mst3 會與存於此處的凋亡促進因子 (apoptosis-inducing factor; AIF)及核酸內切酵素G (Endonuclease G; EndoG)結合成為一 促凋亡的蛋白複合體。因此，這一三年計畫希望研續先前的研究規畫，探討Mst3 在形 成Mst3/AIF/EndoG 蛋白複合體上的角色。另外我們也將研究Mst3 是如何接受凋亡信 號而進行調控AIF 及EndoG 的活性。此外，我們也將找出在Mst3 上會與AIF 及EndoG 結合的區域，並研究Mst3 會與AIF 及EndoG 結合成為促凋亡的蛋白複合體的目的。 這些實驗所獲得的結果，可讓我們更加瞭解Mst3 是如何藉著AIF 及EndoG 來調控細 胞凋亡的作用。在近期的研究中，我們發現Mst3 可利用與caspase 關聯 (caspase-dependent)及非與caspase 關聯(caspase-independent)的訊息傳遞路徑，來參與由 氧化逆境所引發的人類胎盤上的凋亡現象。有趣的是，在發生妊娠毒血症的婦女身上 取得的胎盤組織切片，可測得Mst3 的大量表現。妊娠毒血症是一懷孕期常見病變，是 婦女與胎兒在懷孕期間罹病及致死的主要成因之一。雖然在發生妊娠毒血症的胎盤組 織中可發現Mst3，但它在妊娠毒血症的胎盤中的角色並不清楚。因此，我們希望首先 以免疫病理染色技術及活組織培養技術來釐清Mst3 在發生妊娠毒血症的胎盤中之角 色。接下來，我們將以一人類胎盤基質細胞株作為模型，來進一步研究Mst3 在缺氧情 況下(hypoxia)所引發胎盤基質細胞凋亡的分子作用機轉。希望這些實驗所得的結果可 至少部份解開Mst3 在婦女妊娠毒血症中的角色與分子作用機轉。

The main objective of this project is to understand the physiological function of mammalian Ste20-like protein kinases 3 (Mst3) in placenta during pregnancy disorder and its molecular mechanism in the stress-induced apoptosis. In previous studies, we have demonstrated that overexpression of Mst3 could induce apoptosis in several cell lines. Recently, we found that endogenous Mst3 may be present in both mitochondria and cytoplasm. In mitochondria, Mst3 resides in the intermembrane space and associates with pro-apoptotic proteins, including apoptosis-inducing factor (AIF) and endonuclease G (EndoG), forming a pro-apoptotic complex. Therefore, in this three-year proposal, we want to extend this study to further investigate the role of Mst3 in the formation of Mst3/AIF/EndoG complex and in regulating the activity of AIF and EndoG in response to apoptotic signals. Furthermore, the mapping of the binding domain in Mst3 for AIF and/or EndoG will be performed. The effect of Mst3 without this domain will be studied. The results of these studies may provide evidences explaining how Mst3 mediates apoptosis via AIF and EndoG. Recently, Mst3 was found to mediate oxidative stress-induced placental apoptosis through both the caspase-dependent and -independent pathways. Interestingly, the expression of Mst3 was also found in the placenta from woman complicated with preeclampsia. Preeclampsia, a disorder of pregnancy, is the leading cause of maternal and fetal morbidity and mortality. However, the role of Mst3 in preeclampsia is unclear so far. Hence, immunohistochemical study and explant culture will be performed first to try to elucidate the role of Mst3 in placenta with preeclampsia. Subsequently, a human trophoblast cell line will be used as a model to further investigate the molecular mechanism Mst3 in the hypoxia-induce trophoblast apoptosis. The results from these studies may reveal, at least partially, the role and molecular mechanism of Mst3 in preeclampsia.