探討SNAP29蛋白在細胞自噬作用之分子機制

Molecular regulation of SNAP29 during autophagy

劉俐君(Li-Jun Liu)

指導教授 ： 陳光超

國立臺灣大學/生命科學院/生化科學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201802694

摘要

當細胞面對營養壓力或處在重要的發育階段時，會進行一個自我消化的過程稱作細胞自噬(Autophagy)，此過程對於能量的平衡是重要的。在細胞自噬進行的過程中，會形成雙層膜的結構並包覆要降解的胞內物質，此完整的雙層膜結構稱作細胞自噬體，最終和溶酶體融合並藉由水解酶降解胞內物質並回收。SNAP29是一個可溶性NSF附著蛋白受體(SNARE)，參與並調控細胞自噬體和溶酶體融合的過程。此外，在營養匱乏時，SNAP29上的O端醣修飾會降低，促進SNAP29和其他可溶性NSF附著蛋白受體的結合，進而加速細胞自噬體的清除。然而，是否有其他的後轉譯修飾(PTM)調控SNAP29的功能尚未釐清。
近期，我們實驗室發現在飢餓狀態時，SNAP29會受到酪胺酸磷酸化。質譜資料顯示SNAP29有四個可能被進行酪胺酸磷酸化的位子，根據點突變分析顯示，SNAP29主要由其中一個酪胺酸影響到其酪胺酸磷酸化的狀況。深入研究後發現，當此位置的酪胺酸突變為苯丙胺酸時會影響細胞自噬的進行並增加SNAP29和其他可溶性NSF附著蛋白受體的作用。此外，我們發現調控SNAP29酪胺酸磷酸化的去磷酸酶，而激酶則存在於Src家族中。除了細胞層面，我們也進一步利用果蠅當作模型進行研究，結果顯示當酪胺酸突變為苯丙胺酸時也會影響果蠅幼蟲脂肪組織中的細胞自噬進行。綜合以上，SNAP29的酪胺酸磷酸化對於細胞和果蠅幼蟲組織中的細胞自噬都扮演重要的角色。

關鍵字

細胞自噬 ； SNAP29 ； 磷酸化 ； 可溶性NSF附著蛋白受體(SNAREs)

並列摘要

Autophagy is a self-degradative cellular process crucial for energy balance under nutrient stress and during critical developmental stages. During autophagy, cytoplasmic materials are sequestered by autophagosome, a double-membraned structure, which eventually transfers the materials to lysosomes through membrane fusion for subsequent hydrolase digestion and recycled. SNAP29 is one of the autophagic SNAREs which was shown to be involved in the control of autophagosome-lysosome fusion. Moreover, during starvation, the O-GlcNAc modification of SNAP29 is reduced to promote SNAREs interaction and therefore accelerates autophagosome clearance. However, little is known about whether other post-translational modifications (PTMs) regulate the function of SNAP29.
Recently, our lab found that SNAP29 is tyrosine phosphorylated under starvation conditions. The mass spectrometric data of purified SNAP29 showed four predicted tyrosine phosphorylation sites in SNAP29. Mutational analysis showed that one of tyrosine residues in SNAP29 is responsible for starvation-induced phosphorylation. We further found that tyrosine mutant of SNAP29 affects autophagic flux and increases the interaction with other SNAREs. Additionally, we identified tyrosine phosphatase and Src-family kinase. In Drosophila, we found that the tyrosine phosphorylation mutant of SNAP29 can also influence autophagy flux in the larval fat body. Taken together, our study indicates that the tyrosine phosphorylation of SNAP29 plays a significant role in autophagy.

並列關鍵字

Autophagy ； SNAP29 ； Phosphorylation ； SNARE proteins

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