核苷二磷酸激酶3與粒線體泛素連接酶1相互拮抗調控粒線體之型態

Abstract

核苷二磷酸激酶3 (NME3) 屬於核苷二磷酸激酶 (NDPK) 家族，其家族能夠透過組氨酸激酶 (histidine kinase) 活性催化乒乓反應 (Ping-pong reaction)，使磷酸根 (phosphate) 從核苷三磷酸轉移至核苷二磷酸。我們先前的研究發現，NME3參與在粒線體融合蛋白 (Mitofusin) 所調控粒線體融合的過程中。NME3表現量的抑低會導致粒線體呈現片段狀的型態。NME3的過量表現則會造成粒線體簇集於細胞核周圍，顯示NME3的蛋白質表現量對於粒線體形態的重要性。本研究中，我證明NME3是一個容易經由泛素化所降解的高度不穩定蛋白質。我也發現，引導NME3座落於粒線體外膜的N端氨基酸對於此降解過程是必要的。更進一步的研究發現，位於粒線體外膜上的粒線體泛素連接酶1 (MUL1) 負責NME3的泛素化及蛋白質降解。MUL1的E3連接酶活性在低氧時受到氧化壓力的影響而下降，使NME3的表現量上升最終導致粒線體簇集於細胞核周圍。有趣的是，NME3也會透過組胺酸激酶活性磷酸化MUL1的H319位點來抑制其活性，顯示兩個蛋白質之間有互相調控的作用。因此，喪失酵素活性的NME3 H135Q突變型比野生型NME3具有更短的蛋白質半衰期。根據以上結果，NME3和MUL1之間具有相互拮抗的關係，在粒線體型態的調控中扮演相反的角色。

NME3, a member of nucleoside diphosphate kinase (NDPK) family, is capable of transferring a phosphate from a nucleoside triphosphate to a nucleoside diphosphate by a ping-pong reaction via its histidine kinase activity. Our laboratory has previously demonstrated that NME3 has a function in stimulation of MFN-mediated mitochondrial fusion. Knockdown of NME3 increases fragmented mitochondria while overexpression of NME3 leads to mitochondrial clustering, suggesting the importance of expression level of NME3 in maintaining mitochondrial dynamics. Using an inducible system, I presented evidence that NME3 is a highly unstable protein degraded by ubiquitin-proteasome system dependent on its N-terminus-mediated mitochondrial anchoring. MUL1, an E3 ligase localized on outer membrane of mitochondria, was identified to be responsible for ubiquitination and proteolysis of NME3. In response to hypoxia, NME3 is upregulated to mediate mitochondrial clustering. The E3 ligase activity of MUL1 is reduced by hypoxia-induced oxidative stress. Most interestingly, NME3 also suppresses MUL1 activity through phosphorylation of H319 via histidine kinase activity. As such, catalytic-dead mutant has a shorter half-life than wild-type of NME3. Therefore, the reciprocal regulation between NME3 and MUL1 exerts an opposite control in mitochondrial morphology.