磷酸蛋白體學研究:(1)光合菌Rhodopseudomonas palustris的不同代謝調控(2)細胞表面ATP合成酶的反應路徑

Abstract

蛋白職磷酸化存在於原核及真核生物中，是一種重要的轉譯後修飾。為了解細胞生理的調控，系統性研究細胞內的磷酸化蛋白已成為必要的項目。在本研究中，磷酸蛋白體分析被分別應用於真核和原核系統，藉此去探討細胞內分子的調控，並利用「羥基酸修正金屬氧化物層析」(hydroxy acid-modified metal oxide chromatography, HAMMOC) 去有效的純化磷酸化胜肽。在原核生物磷酸化蛋白體學研究裡，我們探討Rhodopseudomonas palustris (R. palustris)不同的代謝調控。R. palustris 是一種紫色非硫化厭氧光合菌，具有代謝多樣性並可生長於光合與化學異營條件。此細菌也具有製造生質能源與生物分解的潛力。本研究為第一個揭開R. palustris在磷酸化蛋白體資訊，包含化學異營的100條磷酸化胜肽(54個磷酸化蛋白)及光合異營的74條磷酸化胜肽(42個磷酸化蛋白)。其中, 丙酮酸磷酸雙激酶(pyruvate phosphate dikinase, PPDK)在第487個蘇氨酸的磷酸化被發現可能參與在不同生長條件的碳代謝調控。我們在此研究中發現PPDK在PH生長中帶有較多第487個蘇氨酸磷酸化與較高的活性。在PH的條件下，我們進一步發現，過度表現的正常PPDK能比突變無法磷酸化的PPDK(T487V)更能促進脂質的製造。本研究展現了PPDK在生質柴油─脂質製造的重要性，並揭露第487個蘇氨酸磷酸化在脂質生成及PH生長的調控角色。在真核生物的磷酸化蛋白體研究中，ATP合成酶抑制劑在肺癌細胞中的反應途徑是探討的主題。ATP合成酶是一個由多種蛋白質組成的化合物並促進ATP的生成。長期以來ATP合成酶被認定只存於粒線體，但近年研究發現，ATP合成酶也會出現在特定細胞的細胞表面上(包含癌細胞)，此種合成酶被稱作異位型ATP合成酶(ecto-ATP synthase)。ATP合成酶抑制劑被認為具有成為癌症藥物的潛力，其打擊癌症的作用主要是透過有效的抑制異位型ATP合成酶，但並不會對粒線體的功能造成明顯影響。在本研究中我們利用定量磷化蛋白體學方式，分析在癌細胞及異種移植組織中ATP合成酶抑制劑所引起的分子反應。利用此方法，共2834條磷酸化胜肽(836的磷酸化蛋白)與862條磷酸化胜肽(423個磷酸化蛋白)分別被發現於癌細胞與異種移植組織。基因本體分析顯示ATP合成酶抑制劑會影響蛋白質折疊、細胞週期及細胞骨架的磷酸化調控。透過集群與網路分析時間依賴的磷酸化蛋白體資訊，我們進一步發現ATP合成酶抑制劑能立即的抑制熱休克蛋白(heat shock protein 90, HSP90AB1)在第255個絲胺酸的磷酸化，並於抑制劑處理的晚期造成有絲分裂活化蛋白質激酶(mitogen-activated protein kinase 1, MAPK1)在活化功能區(TEY domain)的去磷酸化。MAPK1參與在MAPKs/ERKs的訊息傳遞中，而此訊息傳遞已知與癌症的發展有關。我們利用HSP90在第255氨基酸的突變(HSP90-S255A)去闡明HSP90的磷酸化對MAPKs/ERKs訊息傳遞的影響，並發現HSP90-S255A的突變無法調控MAPK1的磷酸化及MAPKs/ERKs的訊息傳遞。這些研究結果顯示，利用ATP合成酶抑制劑阻斷癌細胞的異位型ATP合成酶，能透過調控HSP90磷酸化所控制的MAPKs/ERKs訊息傳遞，來達到抑制癌細胞生長的目的。本研究不僅提供了發展標靶治療的新方向，也拓展了對抑制肺腫瘤發長之反應途徑的了解。

Protein phosphorylation is one of the most important post-translational modifications in cells, including prokaryotes and eukaryotes. Systematic study of phosphoproteome in cells is becoming a crucial determinant for understanding the regulation of cell physiology. In the study, we applied the phosphoproteomic analyses to investigate the molecular regulation in both prokaryotic and eukaryotic systems. An efficient approach, hydroxy acid-modified metal oxide chromatography (HAMMOC), was used for phosphopeptide enrichment. In the prokaryotic phosphoproteome study, the regulation of different metabolic states in Rhodopseudomonas palustris (R. palustris) was studied. R. palustris is a purple nonsulfur anoxygenic phototrophic bacterium with metabolic versatility and is able to grow under photoheterotrophic and chemoheterotrophic states. It also has the potential for bioenergy production and biodegradation. This study is the first to identify the phosphoproteome of R. palustris including 100 phosphopeptides from 54 phosphoproteins and 74 phosphopeptides from 42 phosphoproteins in chemoheterotrophic and photoheterotrophic growth conditions, respectively. In the identified phosphoproteome, phosphorylation at the threonine residue, Thr487, of pyruvate phosphate dikinase (PPDK, RPA1051) was found to participate in the regulation of carbon metabolism. Here, we show that PPDK enzyme activity is higher in photoheterotrophic growth, with Thr487 phosphorylation as a possible mediator. Under the same photoheterotrophic conditions, R. palustris with overexpressed wild-type PPDK showed an enhanced accumulation of total lipids than those with mutant PPDK (T487V) form. This study reveals the role of the PPDK in the production of biodiesel material, lipid content, with threonyl-phosphorylation as one of the possible regulatory events during photoheterotrophic growth in R. palustris. In the eukaryotic phosphoproteomic study, the response pathways of ATP synthase inhibitor in lung cancer cells were investigated. ATP synthase is a multimeric protein complex that catalyzes the synthesis of ATP. For a long time, animal ATP synthase was believed to be found only in mitochondria; however, in recent studies, ATP synthase was also found on the extracellular surface of some cell types including cancer cells, named as ectopic ATP synthase. ATP synthase inhibitor is a potential drug candidate for fighting cancer by blocking the ectopic ATP synthase without obvious damages to mitochondrial function. In this study, we performed quantitative phosphoproteomics to elucidate the molecular response to ectopic ATP synthase inhibition in both cell and xenograft systems. A total of 2834 phosphopeptides covering 836 phosphoproteins and 862 phosphopeptides containing 423 phosphoproteins were identified in cells and xenograft phosphoproteome, respectively. The gene ontology analysis showed that ATP synthase inhibitor treatment had the impacts on phosphorylated proteins involved in protein folding, cell cycle, and cytoskeleton. Clustering and network analysis of time-dependent phosphorylation profiles further revealed that inhibiting ectopic ATP synthase could immediately down-regulate the phosphorylation of heat shock protein 90 (HSP90AB1), and reduce the function of mitogen-activated protein kinase 1 (MAPK1) in the late-response by dual de-phosphorylation of a TEY activation motif, which is involved in the MAPKs/ERKs cascade associated with cancer progression. We next found that the dephosphorylated HSP90-S255A with alanine substitution of the identified phosphorylated residue Ser255 was unable to mediate the phosphorylation of MAPK1 and the activity of MAPKs/ERKs signaling. These results imply that inhibiting ecto-ATP synthase might suppress the cancer growth through the de-phosphorylation of HSP90-regulated MAPKs/ERKs signaling. This study provides the new insight into the development of novel therapeutic strategies that exploit response pathways on lung tumor suppression.