Abstract
摘要:維持細胞胞器的恆定可經由合成與分解速率的調整來達成。過去的研究已對調控胞器新生成的機制建立基本的認識,而對於胞器分解的調節,則僅有相對較少的著墨,但此部分自從出芽酵母菌的細胞自噬(autophagy)分子機制開始被解析後,目前已逐漸受到重視。由酵母菌中所確認的細胞自噬基因已接近30個,其中許多在包括人類的多細胞生物中具有同源基因存在,而這些基因的功能則被發現與神經退化、肌肉萎縮、腫瘤、乃至於老化有關。因此,引發細胞自噬及其後續調控的分子機制之研究,為近年來相當熱烈的一個領域。本計畫將以三年時間來研究活化細胞自噬功能的分子機制。酵母菌的細胞自噬受生長環境中養分缺乏的訊息而活化,我們發現伴隨而來的是細胞自噬蛋白Atg9磷酸化(phosphorylation)程度的增強。Atg9為一膜蛋白,在正常細胞內會出現於正在形成細胞自噬體(autophagosome)的構造上,功能則被推測為協調其他細胞自噬蛋白,以便促成細胞自噬體的合成。我們的初步實驗則發現,無法被磷酸化的突變Atg9,不但在細胞內的分佈受影響,也無法正常的控制細胞自噬功能的執行。因此,在活化細胞自噬過程中,磷酸化Atg9很可能是一個重要的調控機制,我們計畫尋找Atg9受磷酸化的位置,並進一步確認細胞自噬活化與Atg9蛋白間的關聯。
Abstract: Cells maintain the organelle homeostasis by modulation both synthetic and degradative activities. In the past, signals and mechanisms that stimulate organelle biogenesis have attracted many research efforts, whereas the mechanisms to regulate organelle degradation were much ignored. This situation, however, has changed in recent years. Studies of the major organelle degradation pathway, autophagy, have been rejuvenated due to the identification of its regulatory proteins in the budding yeast Saccharomyces cerevisiae. Many of the autophagy proteins found in yeast have metazoan orthologues and their roles are being examined in growing number of organisms. In mammalian cells, superfluous and dysfunctional organelles are selectively degraded by autophagy. Abnormal autophagy activities are associated with many human diseases, such as neurodegenerative diseases, muscular dystrophy, and cancers. In C. elegans, aging process is found accelerated by compromised autophagy functions. All these phenomena point to the possibility of manipulating autophagy for medical applications. To reach this purpose, knowledge on the regulatory mechanisms of autophagy induction in molecular details is critical. We propose to study autophagy induction in yeast. Despite of close to 30 yeast autophagy proteins isolated, how autophagy is induced by starvation signals is not clear. The available information suggests that the Atg1 kinase complex regulates autophagy execution by switching vesicle formation machinery for autophagosome assembly. The kinase activity of Atg1 is stimulated by starvation-induced association with the Atg13-Atg17 complex. The role of Atg1’s kinase activity in autophagy induction, however, is still in debate, and its substrates remain to be identified. We have found that Atg9, as an integral membrane protein required for autophagosome formation, was hyper-phosphorylated concomitantly with autophagy induction. The presences of Atg1 and Atg13 are required for this modification. N-terminal truncated Atg9 proteins lose starvation-induced hyper-phosphorylation and mislocalize in vivo. Taken together, because Atg9 is proposed to recruit other autophagy proteins to and coordinate their functions at the vesicle formation site, we suspect that Atg9 hyper-phosphorylation is responsible for autophagy induction. We propose to map the phosphorylation sites of Atg9 and study their involvement in autophagy control. We will also find more Atg9 interaction proteins to evaluate the molecular mechanisms of autophagy induction and how autophagy affects cell survival.
Keyword(s)
細胞自噬
磷酸化
蛋白質傳遞
腫瘤
老化
細胞死亡
autophagy
Atg9
phosphorylation
vacuole
protein targeting
membrane trafficking
cancer
aging
programmed cell death
Saccharomyces cerevisiae