Characterization of the molecular mechanism of the autophagy-related Atg8-Atg3 protein interaction in Toxoplasma gondii
Characterization of the molecular mechanism of the autophagy-related Atg8-Atg3 protein interaction in Toxoplasma gondii
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弓形虫自噬相关 Atg8-Atg3 蛋白相互作用的分子机制表征
DOI：
10.1074/jbc.ra118.002614
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发表时间：
2018-09-14
影响因子：
4.8
通讯作者：
Tan, Feng
中科院分区：
文献类型：
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作者：
Liu, Shuxian;Zhang, Fangfei;Tan, Feng
弓形虫病是由一种专性细胞内寄生虫引起的，即原生动物弓形虫。抗T.弓形虫感染可规避现有药物的毒性，弓形虫感染可使T.弓形虫对当前治疗的抵抗力。自噬相关蛋白8（Atg 8）-Atg 3相互作用在T.由于其对调节Atg 8脂化的重要性，弓形虫是一种有前途的药物靶点。我们以前报道过TgAtg 8和TgAtg 3直接相互作用。在这里，我们验证了与Atg 3中的Atg 8家族相互作用基序（AIM）相互作用的TgAtg 8保守残基的取代破坏了TgAtg 8-TgAtg 3相互作用，并减少了TgAtg 8脂化和自噬体形成。这些发现与先前报道的疟原虫Atg 8的结果一致，表明弓形虫和疟原虫中Atg 8的功能保守。此外，使用肽和AlphaScreen测定，我们鉴定了结合TgAtg 8的TgAtg 3中的AIM序列。我们确定核心TgAtg 3 AIM含有Phe 239-Ala 240-Asp 241-Ile 242（239 FADI 242）特征，其不同于疟原虫Atg 3的AIM中的105 WLLP 108特征。此外，丙氨酸扫描分析表明，T.弓形虫还强烈依赖于核心TgAtg 3 AIM周围的几个残基，如Asn 238、Asp 243和Cys 244。这些结果表明，不同的AIM在Atg 3弓形虫和疟原虫Atg 8-Atg 3之间的相互作用的差异。通过阐明TgAtg 8-TgAtg 3相互作用中涉及的关键残基，我们的工作为发现潜在的抗弓形虫病药物铺平了道路。本文开发的定量和直接的AlphaScreen测定法可以高通量筛选破坏TgAtg 8-TgAtg 3相互作用的小分子。
Toxoplasmosis is caused by an obligate intracellular parasite, the protozoan Toxoplasma gondii. Discovery of novel drugs against T. gondii infection could circumvent the toxicity of existing drugs and T. gondii resistance to current treatments. The autophagy-related protein 8 (Atg8)–Atg3 interaction in T. gondii is a promising drug target because of its importance for regulating Atg8 lipidation. We reported previously that TgAtg8 and TgAtg3 interact directly. Here we validated that substitutions of conserved residues of TgAtg8 interacting with the Atg8 family–interacting motif (AIM) in Atg3 disrupt the TgAtg8–TgAtg3 interaction and reduce TgAtg8 lipidation and autophagosome formation. These findings were consistent with results reported previously for Plasmodium Atg8, suggesting functional conservation of Atg8 in Toxoplasma and Plasmodium. Moreover, using peptide and AlphaScreen assays, we identified the AIM sequence in TgAtg3 that binds TgAtg8. We determined that the core TgAtg3 AIM contains a Phe239-Ala240-Asp241-Ile242 (239FADI242) signature distinct from the 105WLLP108 signature in the AIM of Plasmodium Atg3. Furthermore, an alanine-scanning assay revealed that the TgAtg8–TgAtg3 interaction in T. gondii also depends strongly on several residues surrounding the core TgAtg3 AIM, such as Asn238, Asp243, and Cys244. These results indicate that distinct AIMs in Atg3 contribute to differences between Toxoplasma and Plasmodium Atg8–Atg3 interactions. By elucidating critical residues involved in the TgAtg8–TgAtg3 interaction, our work paves the way for the discovery of potential anti-toxoplasmosis drugs. The quantitative and straightforward AlphaScreen assay developed here may enable high-throughput screening for small molecules disrupting the TgAtg8–TgAtg3 interaction.