ATG4B和LC3-I交互作用對細胞自噬的影響

ATG4B/LC3-I interactions in autophagy regulation

林佩璇(Pei-Shuan Lin)

指導教授 ： 楊維元

國立臺灣大學/生命科學院/生化科學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.01377

摘要

細胞自噬作用是一種大分子物質的分解機制，細胞藉由自噬體其雙層膜狀構造包圍細胞質的物質，並和溶小體融合以分解物質，其整體過程受一組稱為ATGs的基因控制著。同時，細胞自噬作用在細胞存活、生長調控和蛋白質代謝中，扮演十分重要的角色，更維持細胞物質合成、分解和循環的平衡。酵母菌ATG8在哺乳類細胞內的同源蛋白LC3，是一種類泛素蛋白，其間介自噬體膜的連結與半融和，並在自噬體的形成具有無可取代的重要性。游離於細胞質的LC3-I與自噬體膜上的磷脂醯乙醇胺(Periyasamy-Thandavan, Jiang et al.)共價結合形成LC3-II，並調節自噬體膜的形成與延伸。另一方面，半胱胺酸蛋白酶ATG4B在自噬作用中是一個關鍵的酵素，其可和兩種LC3作用，首先，ATG4B 可切割LC3前驅蛋白的C端片段產生LC3-I，另外，A TG4B也會和自噬體膜外的LC3-II作用，LC3-II被移除並轉換回LC3-I，使細胞自噬作用得以進行。因此，ATG4B調控著整個自噬作用的過程，細胞到底是如何嚴格的控管ATG4B活性呢?
觀察ATG4B和LC3-I複合體的結構，我們推測LC3-I可能會和ATG4B結合。為證實ATG4B和LC3-I的交互作用，我們利用等溫滴定微量熱法證明ATG4B和LC3-I的結合，以西方墨點法觀察與定量內生性ATG4B、LC3-I濃度的變化驗證細胞內的反應，再以酵素動力學探討LC3-I和ATG4B的關係，另外在螢光顯微鏡下，觀測細胞內ATG4B對GFP-LC3分佈的影響。
實驗發現， 除LC3前驅物，ATG4B也可與LC3-I結合，且親和力更好，ATG4B作用的速率隨著LC3-I濃度的升高，逐漸下降，透過實驗結果得到LC3- I對ATG4B有競爭型抑制作作用，證明LC3-I和ATG4B在細胞自噬的相互作用。此外，在細胞過量表現ATG4B也使GFP-LC3產生在細胞質聚集的現象，因此，我們推論在正常環境下，游離在細胞質的LC3-I會和ATG4B交互作用，抑制ATG4B的活性；當細胞遇到生存壓力誘發細胞自噬作用時， LC3-I轉換成LC3-II的比例增高，使細胞質的LC3-I減少，激活ATG4B移除自噬體膜上的LC3-II，促使自噬體與溶小體融合。

關鍵字

細胞自噬 ； 半胱胺酸蛋白酶ATG4B ； 細胞微管相關蛋白1輕鏈3

並列摘要

Autophagy is a degradation system involving the formation of double-membrane autophagosome, which encloses the cellular components and fuses with lysosome for content degradation. This pathway is governed by a group of genes called ATGs and plays a critical role in cell survival, growth control and homeostasis, which maintains a balance between the synthesis, degradation and recycling of cellular components. LC3, a homologue of yeast ATG8, is a ubiquitin-like protein required for autophagosome formation and mediates membrane tethering and hemi-fusion. Cytosolic LC3-I (termed LC3-I) is conjugated to phosphatidylethanolamine to produce LC3-II, located on the autophagosomal membrane, to regulate autophagosome formation. ATG4B, on the other hand, is a key cysteine protease required for two forms of LC3 processing. First, ATG4B is required for cleaving the C-terminal fragment from LC3 precursor protein to produce LC3-I. Secondly, ATG4B deconjugates LC3-II from autophagosomal membranes into LC3-I to allow autophagy to proceed. Therefore, ATG4B participates in the control of the rate of autophagy degradation. How does the cell regulate the ATG4B activity tightly?
The complex structure of ATG4B and LC3-I, has suggested that LC3-I and ATG4B could bind to each other. To explore the relationship between LC3-I and ATG4B, we used isothermal titration calorimetry to prove ATG4B bind to LC3-I .Than quantify the changes in protein concentration of the endogenous ATG4B and LC3 I with Western blotting. In addition, enzyme kinetics helped us to study the interaction between LC3-I and ATG4B. Furthermore, we examined whether ATG4B could regulate LC3-I in living cell by over expressing ATG4B and fluorescent LC3.
We found that ATG4B bind to LC3-I. In addition, the binding affinity of LC3-I is better than LC3 precursor. Furthermore, the activity of ATG4B reduced when the concentration of LC3-I increased. Besides, GFP-LC3 was distributed in the cytoplasm when ATG4B was overexpressed. Therefore, we propose that cytosolic LC3-I can suppress the activity of ATG4B in normal condition. The conversion of LC3-I to LC3-II is promoted while autophagy is induced. The reduction of LC3-I is than able to activate ATG4B to deconjugate LC3-II from autophagosomal membranes, thereby promoting the fusion of autophagosome and lysosome.

並列關鍵字

Autophagy ； ATG4B ； LC3

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