FBXL14和FBXL8在調控粒線體動態中所扮演的角色

Abstract

為了迎合細胞的需求，細胞裡的粒線體一直在融合與分裂。這些需求包括粒線體內容物的混合，提供ATP，準備進入凋亡階段和粒線體自噬等等。我們之前的實驗結果顯示當一個F-box 家族的蛋白質FBXL14過度表現在Cos-7細胞裡時，它會跟粒線體重合，並且使粒線體碎裂和聚集在細胞核附近。同樣地，當FBXL14過度表現在皮質神經細胞裡時，也會使粒線體比控制組的粒線體的短。當FBXL14的F-box domain被剔除掉之後，會無法形成SCF 複合物，來對目標蛋白質泛素化。然而，它依然會跟粒線體重合，並且粒線體還是呈碎裂的形態，說明FBXL14對粒線體的作用並非需要SCF 複合物的形成。影響粒線體形狀的因素很多，其中包括調控MFN1、MFN2或DRP1的因子、粒線體基因的完整性、鈣離子的調控、粒線體自噬以及粒線體膜電位的變化。當在Cos-7細胞裡過度表現粒線體融合蛋白MFN1或MFN2，或者降低粒線體分裂蛋白DRP1與過度表現FBXL14時，因為粒線體的融合增加，我們觀察到粒線體的形狀有部分恢復。儘管我們觀察不到粒線體基因表現量的降低，但我們能觀察到粒線體膜電位的變化、粒線體的呼吸作用的變化、粒線體移動的速率減弱、粒線體內活性氧的提升以及細胞凋亡的現象（尤其在低氧的條件下）。以上種種結果說明了FBXL14對調控粒線體具有多向性。 如今有多項實驗發現了多組相互拮抗作用的F-box蛋白共同來調控多種細胞內的作用。在F-box蛋白家族裡，FBXL14和FBXL8的F-box domain最為相似，因此我們想要探討FBXL8是否會與FBXL14一起調控粒線體。然而，與Fbxl14不同的是，在Cos-7細胞裡面FBXL8並不會與粒線體重合，它反而會部分與溶酶體、內質網與高爾基體重合。不過當我們同時過度表現FBXL14和FBXL8的時候，FBXL8和FBXL14會高度重合，粒線體的形態也會沒那麼破裂，細胞凋亡率也跟著降低（同樣也在低氧的條件下）。但有趣的是，儘管Co-IP的實驗顯示FBXL14和FBXL8並不會相互作用。以上的觀察告訴我們FBXL8會與FBXL14相互調控粒線體，但是通過什麼樣的機制，我們還無從得知。

Mitochondria undergo constant fusion and fission processes to meet the requirements of multiple cellular functions, such as mitochondria content mixing, meeting cellular ATP demand, preparation for apoptosis or mitophagy etc. Our previous data show that FBXL14, a member of the F-box protein family, co-localised with mitochondria while transiently expressed in Cos-7 cells. This co-localisation can cause mitochondria to undergo fragmentation and perinuclear aggregation. Likewise, dissociated cortical neurons transiently expressing FBXL14-GFP showed significant reduction in mitochondria length compared with those of neurons transfected with Mock-GFP. Deletion of the F-box domain of FBXL14, which disrupts SCF complex formation and thus disables ubiquitination of FBXL14’s target proteins, could not prevent FBXL14 from localization in the mitochondria and mitochondrial fragmentation, suggesting that FBXL14-mediated mitochondrial fragmentation is not through functional FBXL14SCF. Mitochondria morphology can be affected by multiple factors, which include molecules that regulate mitochondria dynamics such as MFN1/2 and DRP1, mitochondria DNA integrity, calcium regulation, mitophagy and altered mitochondria membrane potential. Our study reveals that overexpressing MFN1, MFN2 or downregulating DRP1 in co-transfected Cos-7 cells could not prevent FBXL14 from causing mitochondria fragmentation. Despite the fact that there is no decrease in mtDNA level, FBXL14 overexpression results in decreased mitochondria connectivity, depolarized mitochondria membrane potential, elevated ROS levels, and promoted apoptosis in response to cell stress especially under hypoxia conditions, all of which suggest pleiotropic effect of FBXL14 on/or through mitochondria. Current studies imply reciprocal or collaborative antagonistic regulation of biological activity by paired F-box family members in multiple cellular processes. Given that FBXL8 is the closest relative of FBXL14 in phylogenetic tree of the F-box protein family, we investigate whether FBXL8 is functionally related to FBXL14 in modulating mitochondria dynamics. Different from FBXL14, FBXL8 did not share the same localisation with mitochondria when transiently expressed in Cos-7 cells, but is found partially locating on lysosome, Golgi, and ER. However, FBXL8 and FBXL14 are highly co-localised when co-expressed in Cos-7 cells and partially located on peroxisome, ER, lysosome and Golgi. Co-expression of FBXL8 and FBXL14 resulted in less mitochondria fragmentation and lower apoptotic percentage, also under hypoxia condition. Intriguingly, FBXL8 and FBXL14 did not form protein complex as shown by co-immunoprecipitation assay. All the data suggest that FBXL8 cross-talks with FBXL14 via an indirect, yet-to-be-defined molecular mechanism in the regulation of mitochondria morphology and function.