- 學位論文
探討哺乳動物肌苷酸脫氫酶與三磷酸胞苷合成酶細胞蛇於細胞代謝之⾓⾊
Investigating Roles of IMPDH and CTPS Cytoophidia in Mammalian Cell Metabolism
摘要
核苷酸不僅為RNA及DNA的原料，亦參與細胞內多條生物合成及訊息傳導路徑。由於細胞內核苷酸衡定失調可能造成細胞生理壓力，甚至可能引發細胞凋亡，核苷酸濃度的維持必需受到多層面的精準調控。三磷酸胞苷合成酶（CTPS）和肌苷酸脫氫酶（IMPDH）分別負責催化細胞內CTP及GTP生合成的速率限制步驟，亦即調控了核苷酸濃度的平衡。細胞蛇 (cytoophidium) 是一種由代謝酵素聚合形成的纖維狀次細胞結構，目前已知CTPS和IMPDH為兩種哺乳動物細胞蛇的組成份。細胞蛇結構具有極高的物種保留度，自細菌到哺乳動物，經過數十億年的演化後依然存在著。然而，我們對其生理意義卻仍鮮少了解。在本研究結果中顯示，CTPS和IMPDH可在不同刺激下分別形成獨立的細胞蛇結構。細胞內精氨酸的缺乏或是加強CTPS及IMPDH和其反應物的結合，皆可促進細胞蛇之形成，由此推測細胞蛇的功能是為反應特定細胞代謝需求。此外，本研究記錄描述了細胞蛇形成、成熟的動態過程，並以點突變調節酵素生成細胞蛇的特性後，測量表現突變酵素的細胞內核苷酸濃度變化，顯示CTPS及IMPDH之酵素活性均可能受到細胞蛇結構提升。最後，透過組織切片免疫染色，證實細胞蛇確實存在活體動物組織中，IMPDH細胞蛇表現在小鼠胰島細胞中，而CTPS細胞蛇則在肝癌等腫瘤組織中被發現，推測其可能受到組織生理狀況之調控，並參與體內細胞代謝的調節。綜合上述，本研究對CTPS及IMPDH細胞蛇於哺乳動物細胞中之特性、調控及功能進行初步分析，然其生理重要性仍待後續研究發掘。未來，吾人對CTPS及IMPDH細胞蛇的了解，將有潛力應用於特定疾病之臨床治療或診斷，更可作為其他酵素活性調控之研究模型。
並列摘要
Nucleotide is not only the building blocks of RNA and DNA, but also participate in many biosynthetic and signaling pathways. Imbalance of nucleotides may result cell stress or even induce cell death. In this respect, nucleotide homeostasis is precisely controlled by various mechanisms. The cytoophidium is a fiber-like subcellular structure composed of polymerized metabolic enzymes. Previously, cytidine triphosphate synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH) have been identified as components of mammalian cytoophidium. CTPS and IMPDH catalyze rate-limiting steps of de novo CTP and GTP synthesis, and thereby mediate the balance of nucleotides. Although the cytoophidium is conserved in bacteria to mammals, across billions of year evolution, its biological purposes are still largely in mystery. Herein, it was identified that CTPS and IMPDH actually form independent cytoophidia in response to different stimuli. Assembly of cytoophidia of both enzymes could be promoted by either intracellular glutamine deficiency or enlarged substrate pool size, suggesting the presence of cytoophidium reflects particular metabolic status. In addition, by live-cell imaging, it was revealed that cytoophidium formation initiates from assembly of many self-elongated small cytoophidia, followed by serial fusions of them to generate one or few large ones. CTPS and IMPDH mutants, which would spontaneously form the cytoophidium, were established for elucidating the putative functions of cytoophidium. As the result, assembly of cytoophidium was proposed to enhance activities of IMPDH and CTPS. Finally, The presence of cytoophidia in mouse pancreas and human cancer tissues was observed, implying cytoophidium play a role in our bodies. Future studies in this developing field are required to clarify the physiological importance of this special compartmentation. The knowledge of CTPS and IMPDH cytoophidium would not only shed the light on new strategies in clinical applications for such as cancers, infectious diseases and autoimmune diseases, but also be a model for following studies on other cytoophidium-forming metabolic enzymes.
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