利用蛋白質體探討粒線體動態於ATP合成酶移動至細胞膜的變化

Proteomics Investigation of Mitochondrial Dynamics Leading ATP Synthase toward Cell Surface

蔡漢萱(Han-Hsuan Tsai)

指導教授 ： 阮雪芬

國立臺灣大學/生命科學院/生命科學系/碩士(2019年)

https://doi.org/10.6342/NTU201903522

摘要

已知三磷酸腺苷合成酶 (ATP synthase）通常位於粒線體內膜上，主要功能為產ATP。許多研究顯示ATP合成酶也在各種癌細胞的膜上表達，即所謂「異位表達ATP合成酶」。異位表達ATP合成酶與癌細胞增殖和遷移有關，是癌細胞的生物標記，因此有可能成為癌症治療的靶點。然而，細胞如何運送ATP合成酶至細胞膜上仍未解，於是我們想釐清ATP合成酶的運送機制。實驗結果顯示，利用粒線體分裂抑制劑 (Mdivi-1) 破壞粒線體動態平衡，細胞膜上的ATP合成酶會減少，因此我們推測粒線體動態平衡跟ATP合成酶的運送是有關的。此外，相較於正常細胞，我們發現抑制粒線體分裂會造成癌細胞的存活率下降。因此我們推測粒線體過度分裂會使細胞傾向運送更多ATP合成酶至細胞膜上，進而增加癌細胞存活率。為了進一步探討異位ATP合成酶傳運的機制，我們利用蛋白質體學和磷酸化蛋白質體學分析神經母細胞瘤細胞加藥前後變化。我們總共鑑定了3070種蛋白質，其中124個蛋白表現上升，142個表現下降，還有1945磷酸化位點相當於 880種磷酸化蛋白質，其中34個蛋白表現上升，39個表現下降。在蛋白質體學分析中，共有266種差異顯著的蛋白質主要參與“細胞分裂和增殖”，此外，在差異性磷酸化位點參與的功能中亦發現NF-κB信號傳導概括其中，目前研究已知NF-κB在細胞分化和存活中具有調節作用，這些種種證據顯示破壞線粒體動態平衡會導致細胞週期的變化。接著，我們整合蛋白質體學與磷酸化蛋白質體學結果，發現5個重要的磷酸化位點，如VAPAS214，DYNC1L1S207，AKPA13S1929可能參與NF-κB信號傳導。最後，我們預測Mdivi-1處理後的蛋白調控網絡，發現泛素–蛋白酶體系統（Ubiquitin-proteasome system）會參與粒線體動態變化，值得注意的是，PSMA3S250和RANBP2S1456可能是通過降解無用蛋白質進行異位ATP合酶轉運的關鍵磷酸位點。這項研究揭示了參與線粒體裂變的分子可能與ATP合酶移向細胞表面有關。

關鍵字

異位表達ATP合成酶 ； 粒線體動態變化 ； Mdivi-1抑制劑 ； 蛋白質體學 ； 蛋白質運輸 ； 磷酸化蛋白質體學

並列摘要

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.

並列關鍵字

Ectopic ATP synthases ； Mitochondrial dynamics ； Protein transport ； Mdivi-1 ； Proteomics ； Phosphoproteomics

參考文獻

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