- 學位論文
核苷二磷酸激酶3對粒線體泛素連接酶1在微氧環境下的調控
The NME3-dependent regulation of MUL1 in hypoxia
摘要
核苷二磷酸激酶3 (NME3)是核苷二磷酸激酶 (NDPK)家族的一員。本實驗室先前研究發現NME3基因缺陷的新生兒於出生時就表現出肺部呼吸通氣量不足的症狀並於三個月內死亡。病人組織解剖分析中出現小腦及大腦皮質空泡化。為了瞭解NME3基因缺乏和微氧環境 (hypoxic stress)的關係及其致病機制，本研究探討NME3在微氧環境誘發的粒線體自噬 (mitophagy)中的角色。本研究中發現NME3表現量的抑低會減少微氧環境誘發的粒線體自噬，同時也會增加泛素化蛋白累積在粒線體上。重要的是，移除粒線體泛素連接酶1 (MUL1)降低細胞內的泛素化累積以及回復粒線體自噬作用。此外，我也發現NME3透過組氨酸135位點的磷酸化作用來抑制MUL1所調控的粒線體蛋白質泛素化。進一步研究，證實NME3與MUL1的交互作用受到氧化壓力的影響而增加。有趣的是，MUL1半胱氨酸位點突變也會增加其與NME3的交互作用。這些結果顯示，NME3會抑制MUL1造成的粒線體泛素化作用，進而有利於微氧環境下誘發的粒線體自噬作用。
並列摘要
A previous study from our laboratory demonstrated histopathological alterations in the cerebellum and cerebrum in a neonatal death patient deficient of NME3, a member of the nucleoside diphosphate kinase (NDPK) family. The patient was born with hypotonia symptoms. To understand the pathogenesis resulted from NME3 deficiency in relation to hypoxic stress, this study investigated the role of NME3 in hypoxia-induced mitophagy. Here, I found that NME3 knockdown impaired hypoxia-induced mitophagy with concurrent increases in ubiquitinated protein aggregation. Importantly, knockdown of MUL1, a mitochondrial E3 ligase, rescued mitophagy and diminished ubiquitinated aggresomes in cells. Furthermore, I found that NME3 suppressed MUL1-mediated mitochondrial ubiquitinated aggregates dependent on histidine 135 phosphorylation. Using NME3-GFP / -HA knock-in cells, I presented evidence that hypoxia increases the interaction between NME3 with MUL1 dependent on hypoxia-induced oxidative stress. Intriguingly, mutation of the cysteine residues of MUL1 increased the interaction. In summary, these data imply the critical role of NME3 in hypoxia-induced mitophagy through regulating MUL1-mediated ubiquitination.