人類粒線體酵素複合體I內NDUFV1次單元蛋白質 功能研究及其在呼吸鏈酵素複合體 組合機制中所扮演的角色

NADH dehydrogenase (ubiquinone) flavoprotein 1，簡稱NDUFV1，為粒線體酵素複合體I中由細胞核基因體組表現的核心蛋白。此蛋白具有一個高度保留的黃素單核苷酸(FMN)的結合位置，可提供菸鹼醯胺腺嘌呤二核苷酸(NADH)氧化產生的電子進入粒線體的電子傳遞鏈；另外還含有一個高度保留的半胱胺酸的模組，可幫助形成一組[4Fe-4S]的N3鐵硫中心，而這個鐵硫中心在粒線體酵素複合體I的電子傳遞過程中扮演重要的角色。NDUFV1的缺失已經被發現和一些神經相關疾病例如心理性肌退化有關，而位於FMN結合位置的點突變也被發現存在於患有Leigh症候群的病人中。另一方面，粒線體酵素複合體I~V的組合對於粒線體能量產生的功能也是相當重要。此外，粒線體酵素複合體之間會形成更大型的複合體，用以增進電子傳遞的效率。目前由酵素複合體I、III和IV所形成的呼吸體酵素複合體(respirasome)已經被發現，且其缺失會造成腦肌病變及神經退化性疾病。在本研究中，我們使用核酸干擾(RNAi)技術抑制人類胚胎腎細胞(HEK293)中NDUFV1的表現，藉此探討NDUFV1缺失後所帶來的影響。實驗結果顯示，NDUFV1受到抑制的細胞生長速度以及耗氧速率會下降，其粒線體酵素複合體I的功能亦有所降低。此外，NDUFV1的抑制也會使細胞內粒線體的膜電位降低，進而導致三磷酸腺苷(ATP)生成量的減少。同時在酵素複合體I的蛋白質分析中也發現NDUFV1的抑制會導致酵素複合體I的減少，同時也可能導致酵素複合體III和IV的減少。進一步的呼吸體酵素複合體分析顯示抑制NDUFV1也會使粒線體呼吸體酵素複合體減少，顯示著NDUFV1在調控粒線體酵素複合體的形成及穩定性上扮演著非常重要的角色。

關鍵字

粒線體

並列摘要

NADH dehydrogenase (ubiquinone) flavoprotein 1 (NDUFV1) is a nuclear-encoded subunit of mitochondrial Complex I. It provides a conserved FMN binding site and is responsible for transferring electrons from NADH to FMN to facilitate the entrance of electrons into the electron transport chain (ETC). NDUFV1 also has an iron sulfur cluster [4Fe-4S] (N3) that is the first of that kind in the Complex I. The defect in NDUFV1 could cause neural disease like psychomotor retardation, and some point mutations on FMN binding site have been found in patients with Leigh syndrome. The assembly of intact OXPHOS complexes (complex I~V) is very important in mitochondrial energy production. In addition, the assembly of several respiratory complexes to form a larger supercomplex may improve the efficiency of electron transport. The supercomplex composed by complex I, complex III and complex IV has been confirmed and the defect in supercomplex assembly is associated with encephalomyopathies and neurodegenerative disorders. In present study, we applied a RNA interference system to suppress the NDUFV1 expression in human embryonic kidney cell line (HEK293), and generated three knockdown cell lines A9, B5 and E12 with 55~70% reduction in NDUFV1 protein level. We also performed oxygen consumption assay, dynamic complex I activity assay, ATP determination assay and cell growth rate measurement to evaluate the effects of NDUFV1 suppression. The results showed that the metabolic activity and growth rate were significantly decreased in NDUFV1 knockdown cell lines. In the respiratory complex assembly study, we solubilized mitochondria and applied a high resolution clear native electrophoresis (HrCNE) approach to investigate whether NDUFV1 knockdown affects individual respiratory complex assembly. The results III indicated that NDUFV1 knockdown would significantly decrease the level of complex I, III and IV. Further investigation of OXPHOS supercomplex formation also showed that while NDUFV1 was suppressed, CI/CIII2/CIVn supercomplexes were significantly reduced in cells. These finding suggests that NDUFV1 might play an important role in the assembly/stability of mitochondrial OXPHOS complexes and supercomplexes.

並列關鍵字

mitochondrial ； NDUFV1 ； Complex I

參考文獻

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人類粒線體酵素複合體I內NDUFV1次單元蛋白質功能研究及其在呼吸鏈酵素複合體組合機制中所扮演的角色

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