- 學位論文
探討在parthanatos過程中細胞能量適應性之改變
Investigating Cellular Energy Adaptation during Parthanatos
摘要
Parthanatos是一種PARP1相關之有計畫性細胞壞死死亡機制，且被發現在許多病理情況下扮演一定的重要性。這種細胞死亡會需要消耗ATP，主要是由於PARP1對目標蛋白進行PARylation時會需要大量NAD+。近年來有愈來愈多的證據指出額外添加一些生物能量物質可以影響細胞對parthanatos的敏感度，然而目前兩者之間的關係還尚未明瞭。在本篇研究中，我們發現Atg5缺乏之老鼠纖維母細胞對MNNG所引起之parthanatos相當敏感，且伴隨著ATP回復上的受損。接著，我們檢驗MAPKs和活性含氧物種（ROS）對parthanatos過程中細胞能量適應的貢獻。意外的是，我們發現到PD98059經由一個與MKK抑制無關之機制促進胞內ATP回復，來達到對MNNG處理之老鼠纖維母細胞的保護作用。此外，我們也指出AMPK在parthanatos的重要性。有趣的是，我們發現細胞自噬（autophagy）對於MNNG所誘發之能量適應是非必需的，儘管AMPK是一個為人所知的細胞自噬啟動者。相對地，我們也檢測在parthanatos過程中SIRT1與PARP1的活性平衡，因為這兩者會互相競爭NAD+為活化基質。由於文獻指出AMPK可以經由SIRT1來增加粒線體DNA合成與粒線體呼吸，於此我們也發現MNNG處理會增加老鼠纖維母細胞的粒線體DNA含量。的確，我們也證明粒線體活性在對抗MNNG毒性是必需的。更重要的是，我們發現Atg5缺乏之老鼠纖維母細胞有相對較差的粒腺體呼吸能力，且這或許能解是Atg5缺乏之老鼠纖維母細胞對MNNG之敏感度。最後，由於轉化後之細胞（癌細胞）的能量代謝模式與正常細胞大相逕庭，我們也強調HeLa（一種子宮頸上皮癌細胞）對於MNNG藥物有相較於正常細胞不同的反應。概括而言，這篇研究提供了在parthanatos過程中一個更明確的細胞能量適應情況，也同時暗示一個Atg5在粒線體呼吸的功能來對抗parthanatos。
關鍵字
能量並列摘要
Parthanatic cell death (parthanatos) is a PARP1-dependent programmed necrotic demise with its role highlighted in several pathologic conditions. This type of cell death is characteristic of ATP consumption due to the massive NAD+ depletion by PARP1-dependent poly(ADP-ribosyl)ation on target proteins. Accumulative evidence has suggested that exogenous administration of bioenergetic fuels could modulate the severity of parthanatos. However, how the cellular bioenergetics engages in this process still remains unclear. Here, we first showed that Atg5-/- MEFs were susceptible to MNNG-induced parthanatic cell death with insufficiency for ATP recovery. Next, we examined the contribution of MAPKs and ROS to cellular bioenergetics in MNNG-treated MEFs. Unexpectedly, we found that PD98059 could strongly protect MNNG-treated MEFs from death via facilitating energy restoration independently of MEK1/2 inhibition. Moreover, we further highlighted an essential role of AMPK in parthanatos. Intriguingly, we demonstrated that autophagy is not obligatory in MNNG-elicited energy adaptation during parthanatos, despite the well-known role of AMPK in autophagy induction. By contrast, a balance between SIRT1 and PARP1 activity has been checked in MNNG-treated MEFs, both of which compete for NAD+ as the substrate. Since AMPK could reportedly signal through SIRT1 to favor mitochondrial biogenesis and respiration, herein we showed that MNNG challenge elevated mtDNA content in MEFs. Indeed, we proved that mitochondrial activity is required to surmount parthanatos. Importantly, we demonstrated that Atg5-/- MEFs showed rather compromised mitochondrial respiration compared to the WT counterpart, which may explain the fragility of Atg5-/- MEFs to MNNG insult. Finally, since the metabolic module of transformed cells (cancer cells) is quite different from that of normal cells, we also underlined the different response of HeLa cells to MNNG. Collectively, this study provides an insight into the bioenergetic alteration during parthanatos and suggests a function of Atg5 in mitochondrial respiration against MNNG cytotoxicity.