探討三磷酸胞苷合成酶細胞蛇於哺乳動物細胞內之角色

Abstract

核苷酸不僅作為DNA、RNA與細胞膜磷脂質的原料，更參與於細胞內多種代謝反應及訊息傳遞路徑之中。因此為了維持細胞正常的生理功能，核苷酸的生合成必須受到嚴密精確的調控。三磷酸胞苷合成酶（CTPS）於細胞內的CTP新合成（de novo synthesis）反應中負責催化速率限制步驟，所以對於細胞內核苷酸濃度的平衡相當重要。自2010年以來，多個研究團隊證實CTPS在特定情況下能夠於果蠅、細菌、酵母細胞、哺乳動物細胞內聚集形成一種稱為細胞蛇（cytoophidium）的獨特絲狀結構，在其他研究中亦經證實，超過20種以上的蛋白質可形成細胞蛇結構，然其功能與相關機制則尚未釐清。由於細胞蛇普遍存在於原核細胞與真核細胞中，因而被認定為是在演化上被高度保留的次細胞構造。此外，於多種癌症組織當中除了發現CTPS的酵素活性受到高度提升，也被驗證有CTPS細胞蛇的存在，顯示細胞蛇可能參與了癌症細胞代謝的調控。再者，純化之人類CTPS1已被發表能透過形成細胞蛇的結構來提高酵素活性，因此細胞蛇可能是細胞作為調控代謝與維持細胞內物質動態平衡的手法之一。然而，目前對於細胞蛇於哺乳動物細胞內的生理意義仍無全面的瞭解。在本研究中，首先發現降低細胞內產物CTP的濃度能促使CTPS聚合形成細胞蛇結構，接著回升CTP濃度能使細胞蛇瓦解變回擴散狀態，表示CTP的濃度是影響細胞蛇形成的重要因素之一。除此，CTPS的兩個異構體CTPS1與CTPS2均參與於絲狀結構的聚合，然CTPS2無法獨自形成細胞蛇。接著，利用點突變CTPS1R294D產生失去形成聚合結構能力之細胞株，並觀察細胞蛇所扮演的角色，結果顯示CTPS的酵素活性能夠受到絲狀結構結構提升。此外，由另外一持續維持細胞蛇結構之點突變CTPS1E494K，發現此絲狀結構能提高蛋白質的穩定性。最後，經由組織切片免疫染色，觀察到大量細胞蛇存在於小鼠胸線內正在增殖的細胞當中，不僅證實了細胞蛇是一自然狀態下存在的結構，並與細胞的增生特性有很大的關連。綜合以上，本研究對CTPS細胞蛇於哺乳動物細胞內的調控及功能進行基礎的分析，然而其生理意義仍須進一步實驗探討。由於CTPS已是多項疾病治療的標靶之一，CTPS細胞蛇結構提供了一全新探討方向，更有潛力應用於未來臨床疾病的診斷與治療。

CTP is essential for nucleic acids and membrane phospholipids synthesis, which participates in considerable biosynthetic and signaling pathways. The CTP synthase (CTPS), an enzyme responsible for converting UTP to CTP, was identified recently with ability of organizing into a filamentous structure in several organisms, termed cytoophidium. Later, more than 20 different proteins have been shown to form filaments in yeasts, however, the function and mechanisms remain to be determined. Higher activity and filamentation of CTPS were found in various cancers. The rates of population growth and nutrients supply are abnormal in cancer cells and tumors; thereby, CTPS cytoophidium may serve as a novel indicator for certain metabolic status of cells. Previous study revealed that filamentation could enhance activity of purified human CTPS1. Therefore, forming fiber-like structures might be a potential approach to modulate metabolism and maintain robust cellular homeostasis. However, the physiological roles of CTPS cytoophidium in mammalian cells are still unclear. In this study, basic characteristics of CTPS cytoophidium were examined in cultured human HEK 293T cells. Glutamine supplementation or elevated levels of CTP would induce disassembly of CTPS cytoophidia formed in response to glutamine deprivation, suggesting CTPS filamentation may result from an incomplete CTP synthesis reaction. On the other hand, both isozymes CTPS1 and CTPS2 involved in cytoophidium structures, but CTPS2 could not form the filaments independently. Secondly, by using CTPS1R294D mutant cells, which lack of filamentation capability, the roles of CTPS cytoophidium in cell metabolism were investigated further. Compared to wild type, the mutant cells did not form the CTPS filaments and displayed lower CTPS1 protein expression level. Moreover, mutants produced CTP slower than the wild type, indicating cytoophidium might upregulate CTPS activity. Furthermore, the degradation rate of CTPS1E494K, another mutant that constitutively polymerizes, was slower than wild type CTPS1, suggesting cytoophidium may enhance CTPS stability. Lastly, CTPS filaments were found in the outer cortex of mouse thymus, which is colocalized with rapidly proliferative cells. Thus, these data reveal that the presence of CTPS cytoophidium probably reflects a more proliferative state of cells. Since CTPS has been considered as a drug target for several diseases, these findings may provide more information for the possibility of applications in disease therapy or prognosis.