Decoding three distinct states of the Syntaxin17 SNARE motif in mediating autophagosome-lysosome fusion
Decoding three distinct states of the Syntaxin17 SNARE motif in mediating autophagosome-lysosome fusion
复制标题
解码 Syntaxin17 SNARE 基序在介导自噬体与溶酶体融合中的三种不同状态
DOI：
10.1073/pnas.2006997117
复制
发表时间：
2020-09-01
影响因子：
11.1
通讯作者：
Pan, Lifeng
中科院分区：
文献类型：
--
作者：
Li, Ying;Cheng, Xiaofang;Pan, Lifeng
大自噬对于维持哺乳动物的细胞稳态和生理至关重要，它依赖于自噬体和溶酶体之间的囊泡融合，形成自噬体来降解不需要的细胞质内容物并进行循环利用。自噬体和溶酶体之间的膜融合需要ATG8家族蛋白和自噬相关的SNARE蛋白，包括Syntaxin17、VAMP8和SNAP29，但机制尚不清楚。在这项研究中，我们通过系统的生化和结构表征，揭示了关键的自噬体SNARE蛋白Syntaxin17的三种不同状态，并提供了Syntaxin17的自抑制状态及其与ATG8家族蛋白、SNAP29和VAMP8相互作用的机制见解。我们的发现对于进一步了解Syntaxin17在自噬体-溶酶体融合过程中的功能具有重要价值。Syntaxin17是自噬体n -乙烯基酰亚胺敏感因子附着蛋白受体（SNARE）的关键蛋白，可与ATG8家族蛋白SNAP29和VAMP8结合，促进哺乳动物巨噬过程中双膜自噬体和单膜溶酶体之间的膜融合过程。然而，Syntaxin17的固有特性以及Syntaxin17与其结合蛋白相互作用的机制基础在很大程度上仍然未知。本文采用生化、核磁共振和结构方法，系统表征了Syntaxin17及其与ATG8家族蛋白、SNAP29和VAMP8的相互作用。我们发现Syntaxin17单独采用由Habc结构域和Qa-SNARE基序之间的直接相互作用介导的自抑制构象。此外，我们发现Syntaxin17的Qa-SNARE区域包含一个lc3相互作用区域（LIR）基序，该基序优先结合GABARAP亚家族成员。重要的是，GABARAP结合Syntaxin17可以释放其自抑制状态。确定的Syntaxin17 LIR - GABARAP复合物的晶体结构不仅提供了Syntaxin17与GABARAP相互作用的机制见解，而且揭示了一个具有c端延伸310螺旋的非常规LIR基序，用于选择性结合ATG8家族蛋白。最后，我们还阐明了自噬Syntaxin17-SNAP29-VAMP8 SNARE核心复合物的结构排列，并揭示了其与所有其他SNAREs共同的保守生化和结构特征。总之，我们的研究结果揭示了Syntaxin17的三种不同状态，并为Syntaxin17介导的自噬体-溶酶体融合过程提供了机制见解。
Significance Macroautophagy is essential for the maintenance of cellular homeostasis and physiology in mammals, and relies on vesicle fusion between the autophagosome and the lysosome, forming the autolysosome to degrade unwanted cytosolic contents for recycling. The membrane fusion between the autophagosome and lysosome requires ATG8 family proteins and autophagy-related SNARE proteins including Syntaxin17, VAMP8, and SNAP29, but with poorly understood mechanisms. In this study, through systemic biochemical and structural characterizations, we reveal three different states of the key autophagosomal SNARE protein Syntaxin17 and provide mechanistic insights into the autoinhibited state of Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. Our findings are valuable for further understanding the functions of Syntaxin17 in the autophagosome–lysosome fusion process. Syntaxin17, a key autophagosomal N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa-SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR–GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 310 helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17–SNAP29–VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome–lysosome fusion process.