| Summary: | 碩士 === 國立臺灣大學 === 生命科學系 === 107 === Adenosine triphosphate synthase (ATP synthase) is known for generating ATP and is typically located on mitochondria inner membrane. Recently, many reports showed that ATP synthase also expresses on the plasma membrane of various cancer cell lines, so-called "ectopic ATP synthase". Ectopic ATP synthase is associated with cancer cell proliferation and migration, so it is potential to be a target for cancer therapy. However, the transportation mechanism of ectopic ATP synthase remains unclear. In our study, we found mitochondrial dynamics is essential for ectopic ATP synthase expression through mitochondrial division inhibitor 1 (Mdivi-1) treatment. The lower frequency mitochondria fission happened, the less ectopic ATP synthase was expressed on the cell surface. Additionally, we found cancer cell proliferation decreased after inhibited ectopic ATP synthases transportation. Hence, mitochondrial hyper-fission enabled cells to synthesize more ATP synthase toward the plasma membrane and resulted in a higher survival rate of cancer cells. To advance our understanding of their biology of ectopic ATP synthase transportation, we applied mass spectrometry-based quantitative proteomics and phosphoproteomics in Mdivi-1-treated SK-N-BE(2)C. We identified 3070 proteins with 124 up-regulated and 142 down-regulated as well as 1945 phosphosites corresponding to 880 phosphoproteins with 34 up-regulated and 39 down-regulated. In proteomics, a total of 266 differentially expressed proteins mainly involved in “cell division and proliferation”. These differentially expressed proteins indicated that the perturbation of mitochondrial dynamics might lead to the change of cell cycle. Moreover, in our phosphoproteomic data revealed that “NF-κB signaling” were enriched in the annotation of GO terms in our biological process analysis. NF-κB is known for its regulatory role in cell differentiation and survival. This conforms to the high correlation that we observed the relative level between mitochondrial dynamics and cell division. Moreover, we integrated proteomic and phosphoproteomic data, and found 45 proteins with 58 phosphosites which had no change in protein abundance but significantly changed in phosphorylation level. We discovered 5 important phosphorylation sites such as VAPAS214, DYNC1L1S207, and AKPA13S1929 might participate in NF-κB signaling. The results indicated that not only cell division-associated proteins but also several phosphosites would be involved in mitochondrial dynamics regulating cell division. Ultimately, we assessed the functional network, and discovered the ubiquitin-proteasome system (UPS system) was regulated after Mdivi-1 treatment. Remarkably, PSMA3S250 and RANBP2S1456 may be the critical phosphosites for ectopic ATP synthase transportation via degrading useless proteins. This study revealed the molecules participated in mitochondria fission leading ATP synthase toward cell surface.
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