- 學位論文
辨析細胞自噬作用時LC3B蛋白鍵結的PE種類
Identification of PE species conjugated by LC3B protein during autophagy
摘要
細胞自噬作用是真核生物內一種高度保留的自我吞噬機制,可以將細胞內 的大分子(例如:蛋白質堆積物)或胞器分解,用於循環再利用。在細胞自噬 作用的過程中,細胞質內會形成一種雙層膜的 autophagosome (自噬小體)並 將不需要的物質包覆在其內,當 autophagosome 與 lysosome (溶酶體)融合 後,autophagosome 中所包覆的物質就會被 lysosome 內的酵素給分解掉。在 autophagosome 形成的過程中,LC3/GABARAP(Atg8 蛋白在哺乳動物內的同 源蛋白)與 autophagosome 膜上的 PE (磷脂醯乙醇胺)產生鍵結的反應扮演 了重要角色,因為可以促進 phagophore(吞噬泡,為自噬小體的前趨物)膜的 延長。PE 的結構可分為三個部分,包含:甘油骨架.一或兩個脂醯鏈和一個磷 酸乙醇胺的頭部,不同的 PE 差別在於所接的脂醯鏈有所不同,且 PE 也會因為 所接的脂醯鏈不同而形成不同的形狀(柱狀或錐狀)。近來有研究指出, LC3/GABARAP 傾向於和高曲度膜上的 PE 形成鍵結,由於錐狀形 PE (PE 若 接有不飽和的脂醯鏈,形狀上像錐形)會使膜的排列不整齊而增加膜的曲度, 因此我們認為當細胞自噬作用發生時,LC3/GABARAP 會優先和 autophagosome 膜上的錐形 PE 形成鍵結。在本篇研究中,我們想找出當 HeLa cell(一種子宮頸癌細胞株)因飢餓而誘發細胞自噬作用時,其胞內的 LC3B 蛋 白會和哪些種類的 PE 形成鍵結。實驗過程中,首先我們將 HeLa cell 養在缺乏 養分的 EBSS 培養液中,且為了避免 LC3B-II 蛋白被分解掉,我們也在培養液 中加入了 lysosome 抑制物(E64d 和 pepstatin A)。接著我們透過免疫沈澱的方 式將細胞內的 LC3B-II 蛋白分離出來,並且利用重組的 Atg4B(一種已知的半 胱氨酸蛋白酶,可將 LC3B 和 PE 間的鍵結給切斷)進行切割反應。最後我們將溶液中的脂質萃取出來並加入 LiOH 去改變脂質的電性,再透過負離子模式 的電噴撒質譜進行偵測。從我們的實驗結果得知 PE 的脂肪酸鏈並不會影響到 其在電噴撒時的游離效率。此外,相較於營養充足的細胞,飢餓細胞內總體 PE 的種類及含量並不會有明顯的改變。如果未來我們能夠成功辨析出 LC3B 蛋白 鍵結的 PE 種類,我們就能夠更加了解脂質在 autophagosome 形成過程中所扮演 的角色為何。
並列摘要
Macroautophagy, hereafter called autophagy, is a highly conserved self-digestion process for macromolecules (ex: protein aggregates) and organelles in eukaryotic cells. During autophagy, double-membrane autophagosome forms and sequesters selected cargoes, subsequently fusing with lysosome. Afterwards, the selected cargoes are degraded by hydrolases inside lysosome. Conjugation of mammalian Atg8 homologues, LC3/GABARAP, to phosphatidylethanolamine (called lipidated II form) on autophagosome membrane is critical for autophagosome formation, facilitating phagophore (autophagosome precursor) elongation. Phosphatidylethanolamine (PE) contains three building blocks including a glycerol backbone, one or two fatty acyl chains and a phosphoethanolamine headgroup. Due to the distinct fatty acyl composition, PEs can be classified into different molecular species and form specific shape (cylinder- or cone-shaped). Recent studies using in vitro assays have shown that LC3/GABARAP lipidation prefers to occur on highly curved membrane. Because PEs with unsaturated fatty acyl chains can form cone-shaped lipids and contribute to strident membrane curvature, we suggest that LC3/GABARAP preferentially conjugate to cone-shaped PEs. In our study, we attempted to find out PE species conjugated by LC3B protein in HeLa cells during starvation-induced autophagy. We cultured HeLa cells in EBSS medium with lysosome inhibitors for two hours to induce autophagy and accumulate LC3B-II proteins. Then, we pulled down LC3B-II proteins by immunoprecipitation and cut the bonding between LC3B and PE using recombinant Atg4B protein, an already known Cysteine protease for LC3B-II cleavage. Eventually, we extracted lipids from the above solution and analyzed by negative-mode ESI-MS after the addition of a small amount of LiOH. Our results showed that the fatty acyl chains of PEs do not affect their ionization efficiency. Also, the overall PE species pattern does not change in starved HeLa cells compared to nutrient-rich ones. If PE species conjugated by LC3B protein can be identified in the future, we can know more about the roles of membrane lipids in the regulation of autophagosome biogenesis.