Synthesis of Autophagosomal Marker Protein LC3-II under Detergent-Free Conditions
Synthesis of Autophagosomal Marker Protein LC3-II under Detergent-Free Conditions
复制标题
无去污剂条件下合成自噬体标记蛋白 LC3-II。
DOI：
10.1002/anie.201209523
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发表时间：
2013-01-01
影响因子：
16.6
通讯作者：
Liu, Lei
中科院分区：
文献类型：
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作者：
Huang, Yi-Chao;Li, Yi-Ming;Liu, Lei
自噬体然后与溶酶体融合以形成自溶酶体，其中螯合的物质被溶酶体水解酶降解。自噬作为一种重要的细胞管家系统，在细菌和病毒感染、细胞死亡、抗原提呈等多种生理过程中发挥着重要作用。自噬功能障碍涉及许多病理学，如肝脏炎症、神经退行性疾病和癌症。[2]由于这些原因，在分子水平上了解自噬的机制越来越受到关注。自噬体膜的形成是自噬过程中的关键事件。在此过程中，胞质微管相关蛋白1A/1B-轻链3（LC 3-I）通过C-末端残基Gly 120的酰胺键与1，2-二硬脂酰-sn-甘油基-3-磷酸乙醇胺（DSPE）偶联。[3]由此产生的脂质锚定蛋白被称为LC 3-II。它促进吞噬细胞（自噬体前体）的伸长和细胞质物质的吞噬，靶向降解。随后，LC 3-II通过半胱氨酸蛋白酶Atg-4酶促转化回LC 3-I，或通过溶酶体蛋白酶降解。[4]先前的研究表明，细胞LC 3-II水平可以连接到自噬活性。[5]因此，LC 3-II是研究自噬机制的良好标志物。虽然传统的方法如蛋白质印迹法可以用来监测LC 3-II，[6]更先进的自噬研究需要制备量的LC 3-II，理想的情况下，报告基团和标签纳入。先前生产LC 3-II的尝试依赖于使用E1-和E2-样酶Atg 7和Atg 3体外缀合LC 3-I与PE。[7]不幸的是，这种酶促方法只能提供蛋白质-脂质混合物和低微克规模的LC 3-II。在这里，我们报告的第一个合成同质LC 3-II的实际数量，使用的策略表达蛋白连接。[8]这项研究是我们正在进行的努力，以阐明自噬调控机制的一部分。[9]以前化学合成已用于制备具有连接的类异戊二烯脂质[10]和糖基磷脂酰肌醇的脂化蛋白质。[11]然而，我们认识到LC 3-II呈现出甚至更具挑战性的合成靶标，因为PE尾对于先前的去污剂辅助连接方法来说过于疏水。为了解决这个问题，我们开发了一种新的方法，用于合成脂化蛋白质使用可去除的增溶侧链。通过这种方法，脂化蛋白质可以在无洗涤剂的条件下制备，而无需费力地筛选洗涤剂和溶剂。该策略不仅使得LC 3-II的制备具有良好的生物相容性，
Autophagosomes then fuse with lysosomes to form autolysosomes where the sequestered materials are degraded by the lysosomal hydrolases. As a pivotal cellular housekeeping system, autophagy plays important roles in many physiological processes including bacterial and viral infection, cell death, and antigen presentation. Dysfunction of autophagy is involved in a number of pathologies such as hepatic inflammation, neurodegenerative diseases, and cancer.[2] Owing to these reasons, there has been growing interest to understand the mechanisms of autophagy at the molecular level. The formation of autophagosomal membranes is a key event in autophagy. During this process, a cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3-I) is conjugated to 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) through an amide bond with the C-terminal residue, Gly120.[3] The resulting lipid-anchored protein is called LC3-II. It promotes the elongation of the phagophore (the autophagosome precursor) and engulfment of cytoplasmic materials targeted for degradation. Later on LC3-II is either enzymatically converted back to LC3-I by a cysteine protease, Atg-4, or degraded by the lysosomal proteases.[4] Previous studies have shown that the cellular LC3-II level can be connected to the autophagic activity.[5] Accordingly, LC3-II is a good marker for the study of the machinery of autophagy. Although traditional methods such as Western blotting can be used to monitor LC3-II,[6] more advanced studies on autophagy require preparative amounts of LC3-II, ideally with reporter groups and tags incorporated. Previous attempts to produce LC3-II have relied on in vitro conjugation of LC3-I with PE using E1-and E2-like enzymes, Atg7 and Atg3.[7] Unfortunately, this enzymatic approach can only afford LC3-II in a protein–lipid mixture and in the low microgram scale. Herein, we report the first synthesis of homogeneous LC3-II in practical quantities, using the strategy of expressed protein ligation.[8] This study is part of our on-going efforts to elucidate the mechanism of autophagy regulation.[9] Previously chemical synthesis has been used for the preparation of lipidated proteins with attached isoprenoid lipids [10] and glycosylphosphatidylinositols.[11] However, we recognized that LC3-II presents an even more challenging synthetic target, because the PE tail is too hydrophobic for the previous detergent-assisted ligation methods. To solve this problem, we developed a new method for the synthesis of lipidated proteins using a removable solublizing side chain. With this approach, lipidated proteins can be prepared under detergent-free conditions, without the laborious screening of detergents and solvents. This strategy not only enables the preparation of LC3-II with good