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  • 學位論文

細胞自噬蛋白Atg8表面的功能區域之定性分析

Characterization of different functional sites of Atg8 in Saccharomyces cerevisiae

指導教授 : 黃偉邦

摘要


細胞自噬作用是一種在演化上高度守衡的細胞內大分子物質分解機制,所有真核生物,從單細胞的酵母菌到哺乳動物細胞都會進行細胞自噬。當外在環境改變,例如養份供應匱乏時,細胞會藉由在細胞質內產生稱為自噬小泡的雙層膜狀構造來包裹一部分細胞質和大分子物質,甚至是整個胞器,並且運送至溶酶體或是酵母菌的液泡中進行分解。在這過程中所釋放出來的小分子,例如胺基酸等,會被用來合成新的蛋白質以幫助細胞適應改變後的環境。同時,細胞自噬作用也是維持真核細胞內部蛋白質正常代謝的重要機制。目前已經知道細胞自噬與許多退化性神經疾病亦有關聯,例如亨丁頓氏舞蹈症中的聚麩醯胺酸沉澱會誘發細胞產生自噬小泡來包裹並分解之,而失衡的細胞自噬活性與不正常累積的蛋白質聚合物造成的毒性可能是這類退化性神經疾病中神經元大量死亡的主因之一。此外細胞自噬作用也被發現和多細胞生物的胚胎發育、程式性細胞死亡、或是癌症的病理機制等皆有密切的關連。 在目前已知的31種細胞自噬相關蛋白中,Atg8是細胞自噬小泡生成過程中所必需的一種調控蛋白。它在細胞質中被合成之後會藉由一系列的後轉錄修飾作用最後在末端接上一個磷脂質。此外研究發現,細胞自噬的專一性運送物質Ape1的受體Atg19也和Atg8有直接的交互作用,顯示Atg8很可能也參與了細胞自噬作用中的被分解物篩選作用。但不論是Atg8所參與的自噬小泡生合成途徑,或被分解物篩選的確切機制皆尚不清楚。在本篇研究中,我利用定點突變的方法分離出六個Atg8表面的重要胺基酸殘基,並且將它們對應到所負責的生理功能上。其中,殘基Arg28、Tyr49和Leu50組成一個和Atg19結合的區位,同時殘基Tyr49和Leu50也是Atg8在合成後進行後轉錄修飾作用中所必須的。此外殘基Phe79會被蛋白酶Atg4所辨識來調控該修飾作用中的第一步驟,殘基Leu55則是參與在Atg4所調控的另一步驟:去脂質修飾反應。另外一個殘基,Lys48則很可能參與在Atg8所調控的自噬小泡生成作用中。

並列摘要


Autophagy is a highly conserved membrane trafficking pathway, which is evoked during stress condition, such as nutrient starvation. Excess or abnormal intracellular macromolecules are sequestered by the double-membrane vesicle, autophagosome, and transported to the vacuole/lysosome for degradation and recycling of nutrient. The released amino acids are used in synthesis of proteins required for cells to adapt to the changed environment. Atg8 is an essential regulator for autophagosome biogenesis. It is post-translationally conjugated to lipid at its C-terminus and is proposed to be the membrane modifier that may be functionally similar to coat proteins. Besides, Atg8 also interacts directly with the autophagic cargo receptor, Atg19. We are interested in whether the Atg8-mediated vesicle expansion process and cargo sorting are coupled. Here we identified 6 residues on Atg8 surface that are required for the modification and/or physiological function of it. Residue Arg28 is specific for the cargo receptor binding; Tyr49, Leu50, Leu55, and Phe79 are involved in different steps of its post-translational modification; and Lys48 is important for perhaps the biogenesis of autophagosome.

並列關鍵字

autophagy Atg8 cargosorting lipidation

參考文獻


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