NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) is one of the core nucleus-encoded subunits existing in human mitochondrial complexⅠ. It contains one iron sulfur cluster ([2Fe-2S] binuclear cluster N1a), which may serve as an electron reservoir and play an important role in the prevention of oxidative damage. Mutations of NDUFV2 have been implicated in Parkinson's disease, bipolar disorder and schizophrenia. To verify the mitochondrial metabolic function of NDUFV2, we applied the RNA interference technology in the T-REx293 cells. We found that the suppression of NDUFV2 decreased the complex I activity and reduced oxygen consumption rate of complex I, but had no significantly effect on ATP generation, reactive oxygen species (ROS) formation in normal cell culture conditions. While exposure to oxidative stress, the deficiency of NDUFV2 in the knockdown cells resulted in decreasing the oxygen consumption rate, reducing the ATP generation and increasing the ROS formation. These data suggested that NUDFV2 are essential for electron-transport and normal energy production under oxidative stress. In addition, in an attempt to identify the iron-sulfur cluster of NDUFV2 for complex I function, the NDUFV2 cysteine mutation cell lines were established. We found that the cysteine mutation of NDUFV2 led to a decline of oxygen consumption rate but caused no significant change in targeting NDUFV2 to mitochondria. While exposure to oxidative stress, the cysteine mutation cell lines also diminished oxygen consumption rate. Taken these data together, we suggested that intact iron-sulfur cluster of NDUFV2 is essential for mitochondrial respiratory chain function. In this report, we proved that the NDUFV2 plays a critical role in energy generation process of mammalian cells and the iron-sulfur cluster of NDUFV2 is involved in conducting the electron transfer in the mitochondrial complex I.

Table of contents
中文摘要. i
Abstracts. ii
Abbreviations v
Introduction 1
Materials and methods 11
Results……... 20
A. The functional studies of NDUFV2………………………………………... 20
1. Reduced expression of NDUFV2 in T-REx293 cells………………… 20
2. The complex I activity was reduced in NDUFV2 knock-down cells.. 20
3. Oxygen consumption rate of respiratory chain was reduced in NDUFV2 suppression cells…………………………………………... 21
4. NDUFV2 suppression could affect ROS generation in cells under oxidative stress……………………………………………………….. 22
5. NDUFV2 suppression cells had no effect on ATP generation……… 23
B. The functional studies of NDUFV2 cysteine mutations…………………... 24
1. Establishment of cysteine mutations in NDUFV2…………………... 24
2. NDUFV2 cysteine mutation(s) cell lines did not affect the NDUFV2 targeting to mitochondria……………………………………………. 25
3. Each NDUFV2 cysteine mutation cell line varied in NDUFV2 gene expression level……………………………………………………….. 26
4. Cysteine mutation cell lines reduced the oxygen consumption rate of the respiratory chain………………………………………………… 27
Discussion 28
A. The functional studies of NDUFV2……………………………………….. 28
B. Characterization of iron-sulfur cluster of NDFUV2……………………… 30
Table…………… 32
Figures…… 34
Reference 45
Appendixes 49

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