Establishing Allotopic Expression System to Study the Effect of Mitochondrial DNA Mutation in Human Cells

• Main Authors: Hsin-Hui Huang, 黃心慧
• Other Authors: Hsin-Chen Lee
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/72936656953652666434

碩士 === 國立陽明大學 === 藥理學研究所 === 101 === Mitochondria provide the primary energy in most eukaryotic cells, and mitochondrial DNA is important to maintain mitochondrial function. However, mtDNA is more susceptible to oxidative damage, and the mutation rate of mtDNA s is ten times higher than that of nuclear DNA. Approximately sixty percent of cancers can be found to harbor somatic point mutations of mtDNA, and breast cancer is the common one. In our previous data, it was found that there are many types of potentially harmful missense mtDNA mutations in breast cancer, and it may be linked to the Warburg effect. To investigate the effect of specific mtDNA mutation in breast cancer, I attempted to establishing allotopic expression system. I ligated mitochondrial targeting signals (MTS) from either the subunit VIII of human cytochrome c oxidase (COX VIII) or the P1 isoform of the subunit c of human ATP synthase (P1 isoform) with the recoded MTATP6 (rMTATP6) gene and then inserted into pDsRed2 or pcDNA3.1/ myc-His vectors. Unexpectedly, rMTATP6 was not expressed in HEK293T cells. Moreover, the result is not because of protein degradation or low expression of rMTATP6. I further checked the pellet from protein extraction, but no rMTATP6 protein in unsoluble fraction. Next, I found the mRNA of rMTATP6 was detectable, suggesting these constructs are able to be transcripted. In addition, I found that the recoded mutated mtDNA ND5 12418insA construct was not expressed in HEK293T but its mRNA was detectable. I ligated different lengthes of mutated ND5 with mitochondrial sirt3, and found these fusion proteins were detectable but the expression levels were lower than control. These results suggest that the sequence of mtDNA per se might suppress it’s translation when it locates in cytoplasma.