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Potent and specific Atg8-targeting autophagy inhibitory peptides from giant ankyrins

Nature Chemical Biology volume 14, pages 778–787 (2018)Cite this article

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Abstract

The mammalian Atg8 family proteins are central drivers of autophagy and contain six members, classified into the LC3 and GABARAP subfamilies. Due to their high sequence similarity and consequent functional overlaps, it is difficult to delineate specific functions of Atg8 proteins in autophagy. Here we discover a super-strong GABARAP-selective inhibitory peptide harbored in 270/480 kDa ankyrin-G and a super-potent pan-Atg8 inhibitory peptide from 440 kDa ankyrin-B. Structural studies elucidate the mechanism governing the Atg8 binding potency and selectivity of the peptides, reveal a general Atg8-binding sequence motif, and allow development of a more GABARAP-selective inhibitory peptide. These peptides effectively blocked autophagy when expressed in cultured cells. Expression of these ankyrin-derived peptides in Caenorhabditis elegans also inhibited autophagy, causing accumulation of the p62 homolog SQST-1, delayed development and shortened life span. Thus, these genetically encodable autophagy inhibitory peptides can be used to occlude autophagy spatiotemporally in living animals.

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Fig. 1: The super-strong affinity of selective AnkG-LIR binding to GABARAPs and its structural basis.
Fig. 2: AnkB-LIR binds to all Atg8 family members with super-strong affinities.
Fig. 3: Structures of AnkB-LIR in complex with GABARAP and LC3B reveal a consensus sequence motif for the extended LIRs with strong binding to Atg8s.
Fig. 4: Autophagy inhibition by AnkB/G-LIR peptides in COS7 cells.
Fig. 5: Expression of the AnkB peptide impairs autophagy in C. elegans.
Fig. 6: The AnkB peptide inhibits autophagy in a tissue- and time-specific manner.

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Acknowledgements

We thank the BL19U1 beamline at National Facility for Protein Science Shanghai (NFPS) and BL17U1 beamline at Shanghai Synchrotron Radiation Facility (SSRF) for X-ray beam time, and staff at the BioCRF of HKUST for assistance in MALDI-TOF analysis. The GFP-LC3-expressing HeLa stable cell line was a kind gift from Yingyu Chen, Peking University School of Basic Medical Sciences. This work was supported by National Key R&D Program of China (2016YFA0501903) and a 973 program grant (2014CB910204) from the Minister of Science and Technology of China to M.Z., grants from RGC of Hong Kong (664113, 16103614, 16100517 and AoE-M09-12) to M.Z., and grants from the National Natural Science Foundation of China (NSFC) (31421002, 31630048, 31561143001 to H.Z. and 31670734 to C.W.). C.W. is supported by CAS Pioneer Hundred Talents Program. M.Z. is a Kerry Holdings Professor in Science and a Senior Fellow of IAS at HKUST.

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  1. These authors contributed equally: Jianchao Li, Ruichi Zhu.

Authors and Affiliations

  1. Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

    Jianchao Li, Ruichi Zhu, Keyu Chen, Chao Wang & Mingjie Zhang

  2. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Hui Zheng, Hongyu Zhao, Chongzhen Yuan & Hong Zhang

  3. College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China

    Hui Zheng, Hongyu Zhao, Chongzhen Yuan & Hong Zhang

  4. Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China

    Chao Wang

  5. Center of Systems Biology and Human Health, Institute for Advanced Study, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

    Mingjie Zhang

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Contributions

J.L., R.Z., C.W. and K.C. performed structural and biochemical experiments; H. Zheng, H. Zhao and C.Y. performed worm experiments; R.Z. performed COS7 cell experiments; J.L., C.W., H. Zhang and M.Z. wrote the paper, and all authors approved the manuscript; H. Zhang and M.Z. supervised the research; M.Z. coordinated the project.

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Correspondence to Hong Zhang, Chao Wang or Mingjie Zhang.

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Li, J., Zhu, R., Chen, K. et al. Potent and specific Atg8-targeting autophagy inhibitory peptides from giant ankyrins. Nat Chem Biol 14, 778–787 (2018). https://doi.org/10.1038/s41589-018-0082-8

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