2013-01-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/678763摘要：真核細胞的溶小體/液泡藉由細胞自噬（autophagy）途徑，分解細胞內物質以提供缺乏外源養分的細胞維持生命所需之能量，並確保胞內環境的恆定。異常的細胞自噬功能與許多疾病相關，例如在腫瘤與組織退化疾病的細胞中常伴隨出現過低或過高的細胞自噬活性，細胞自噬功能低下則會導致老化提早發生。因此，研究調控細胞自噬的分子機制，有助於認識細胞自噬在疾病發生中所扮演的角色，並具以發展新的療法。在出芽酵母菌中確認的細胞自噬基因已超過30個，其中許多在包括人類的多細胞生物中發現有同源基因存在，且其功能與調控細胞自噬的機轉與酵母菌細胞相似，因此利用酵母菌生物模式系統研究細胞自噬機制，具有操作便利性與多細胞生物的應用價值。本計畫預計以五年時間，以系統生物學的角度來研究細胞自噬蛋白作用的分子機制。我們將利用突變酵母菌株集合庫（nonessential gene knockout yeast library與temperature-sensitive mutant yeast library）為材料，藉由高通量自動螢光影像記錄系統來篩選新的選擇性細胞自噬調控基因；利用質譜儀分析細胞自噬缺失對酵母菌代謝途徑的影響；利用基因晶片與次世代定序技術研究細胞自噬基因表現的訊息調控；利用蛋白體學方法分析細胞自噬蛋白的作用機制。最後，酵母菌系統的實驗結果將在動物細胞中加以驗證。<br> Abstract: Autophagy, as a conserved pathway in eukaryotic cells, plays a critical role in maintaining cellular homeostasis by delivering cytoplasmic materials to lysosomes/vacuoles for degradation. It is known for a long time that autophagy pathway mediates non-selective degradation of macromolecules to sustain supplies of energy under nutrient starvation conditions. Recent studies, on the other hand, have revealed many important functions of selective autophagy pathways in multi-cellular organisms. In mammalian cells, superfluous and dysfunctional organelles are selectively degraded by autophagy. Both innate and acquired immunities are regulated by selective autophagy pathways. Abnormal autophagy activities are associated with several human diseases, such as diabetes, neurodegenerative diseases, and cancers. Aging process is found accelerated by compromised autophagy functions in many model organisms, including worms and plants. All these observation leads to the possibilities of manipulating autophagy for therapeutic applications. To achieve this purpose, detailed information on the molecular mechanisms of autophagy regulation is essential. Because many of the autophagy genes found in the budding yeast Saccharomyces cerevisiae have orthologues in higher organisms and their functions are very well conserved, I propose to study autophagy mechanisms in both yeast and mammalian cells using systematic approaches. Despite the fact that autophagy is known to be controlled by the actions of more than 30 Atg and many other proteins involved in nutrient signaling and endo-membrane trafficking, how autophagy pathways are regulated in details remains elusive. We will screen additional genes involved in selective autophagy regulation from the temperature-sensitive mutant yeast libraries and the nonessential gene knockout yeast libraries using the high-throughput automatic imaging system. Metabolic pathway changes in autophagy mutant cells will be analyzed by mass spectrometry. Signaling mechanisms regulate ATG gene expressions will be studied by microarray and second-generation DNA sequencing techniques. Molecular mechanisms of Atg protein actions will be examined by proteomic and cell biology approaches. Finally, the data acquired from the yeast model will be further tested in animal cell culture systems.細胞自噬細胞膜運輸腫瘤細胞死亡出芽酵母菌autophagymembrane traffickingcancerprogrammed cell deathSaccharomyces cerevisiae