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

Nature Chemical Biologyvolume 14pages778787 (2018) | Download Citation

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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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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Author notes

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

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.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Hong Zhang or Chao Wang or Mingjie Zhang.

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https://doi.org/10.1038/s41589-018-0082-8