Image-based genome-wide siRNA screen identifies selective autophagy factors.
Image-based genome-wide siRNA screen identifies selective autophagy factors.
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DOI：
10.1038/nature10546
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发表时间：
2011-12-01
期刊：
影响因子：
64.8
通讯作者：
Levine, Beth
中科院分区：
文献类型：
--
作者：
Orvedahl, Anthony;Sumpter, Rhea, Jr.;Xiao, Guanghua;Ng, Aylwin;Zou, Zhongju;Tang, Yi;Narimatsu, Masahiro;Gilpin, Christopher;Sun, Qihua;Roth, Michael;Forst, Christian V.;Wrana, Jeffrey L.;Zhang, Ying E.;Luby-Phelps, Katherine;Xavier, Ramnik J.;Xie, Yang;Levine, Beth
选择性自噬涉及识别和定位特定的货物，例如受损的细胞器、错误折叠的蛋白质或入侵的病原体以破坏溶酶体。酵母基因筛查已经确定了不同形式的选择性自噬所需的蛋白质，包括细胞质到空泡靶向、错位吞噬和有丝分裂吞噬，哺乳动物的基因筛查已经确定了自噬调节所需的蛋白质。然而，目前还没有系统的方法来确定哺乳动物细胞中选择性自噬的分子决定因素。为了确定选择性自噬所需的哺乳动物基因，我们进行了高含量、基于图像的全基因组siRNA筛选，以检测Sindbis病毒衣壳蛋白与自噬溶酶体共存所需的基因。我们确定了141个病毒自噬所需的候选基因，这些基因丰富了与mRNA处理、干扰素信号转导、囊泡运输、细胞骨架运动功能和新陈代谢相关的细胞通路。其中96个基因也是Parkin介导的有丝分裂吞噬所必需的，这表明共同的分子决定因素可能参与了病毒核衣壳的自噬靶向和受损线粒体的自噬靶标。缺乏这些基因产物之一的小鼠胚胎成纤维细胞，含有C2结构域的蛋白SMurf1，在针对Sindbis和单纯疱疹病毒的自噬靶向和清除受损线粒体方面存在缺陷。此外，S-1基因缺陷的小鼠在心脏、大脑和肝脏中显示出受损线粒体的积累。因此，我们的研究确定了选择性自噬的候选决定因素，并将SMurf1定义为新发现的病毒自噬和有丝分裂吞噬的中介。
Selective autophagy involves the recognition and targeting of specific cargo, such as damaged organelles, misfolded proteins, or invading pathogens for lysosomal destruction. Yeast genetic screens have identified proteins required for different forms of selective autophagy, including cytoplasm-to-vacuole targeting, pexophagy, and mitophagy, and mammalian genetic screens have identified proteins required for autophagy regulation. However, there have been no systematic approaches to identify molecular determinants of selective autophagy in mammalian cells. To identify mammalian genes required for selective autophagy, we performed a high-content, image-based, genome-wide siRNA screen to detect genes required for the colocalization of Sindbis virus capsid protein with autophagolysosomes. We identified 141 candidate genes required for viral autophagy, which were enriched for cellular pathways related to mRNA processing, interferon signaling, vesicle trafficking, cytoskeletal motor function, and metabolism. Ninety-six of these genes were also required for Parkin-mediated mitophagy, indicating that common molecular determinants may be involved in autophagic targeting of viral nucleocapsids and autophagic targeting of damaged mitochondria. Murine embryonic fibroblasts lacking one of these gene products, the C2-domain containing protein, Smurf1, are deficient in the autophagosomal targeting of Sindbis and herpes simplex viruses and in the clearance of damaged mitochondria. Moreover, Smurf1-deficient mice display an accumulation of damaged mitochondria in heart, brain, and liver. Thus, our study identifies candidate determinants of selective autophagy, and defines Smurf1 as a newly recognized mediator of both viral autophagy and mitophagy.
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通讯作者：
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