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Potent and Specific Atg8-Targeting Autophagy Inhibitory Peptides from Giant Ankyrins

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Potent and Specific Atg8-Targeting Autophagy Inhibitory Peptides from Giant Ankyrins ARTICLES https://doi.org/10.1038/s41589-018-0082-8 Potent and specific Atg8-targeting autophagy inhibitory peptides from giant ankyrins Jianchao Li 1,6, Ruichi Zhu 1,6, Keyu Chen1, Hui Zheng2,3, Hongyu Zhao2,3, Chongzhen Yuan2,3, Hong Zhang2,3*, Chao Wang1,4* and Mingjie Zhang 1,5* 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 har- bored 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 inhib- ited 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. utophagy is an evolutionarily conserved degradation system process20,21,29. However, the high sequence similarities shared by in eukaryotes1,2. It is responsible for degrading intracellular mammalian Atg8 homologs and potential genetic compensation Aprotein aggregates, damaged organelles and invasive patho- complicate functional studies of each individual member. gens, and thus is essential in maintaining cellular homeostasis as Atg8s contain a short N-terminal two-helix extension followed well as responding to stress conditions3–5. Dysregulation of autoph- by a C-terminal ubiquitin-like domain. Atg8s can recognize pro- agy is associated with a variety of human diseases, including but teins containing a Φ XXΨ motif (where Φ represents tryptophan, not limited to cancers, metabolic diseases, immune disorders and tyrosine or phenylalanine, Ψ represents leucine, isoleucine or neurodegenerative diseases6–9. valine, and X represents any amino acid), also known as the LC3 Genetic screens in yeast and C. elegans have enabled research- interacting region (LIR) or Atg8 interacting motif30,31. In addition ers to identify and characterize a series of key components of the to the aromatic and the hydrophobic residues, a typical LIR usually autophagy machinery, including autophagy-related (Atg) genes contains a few N-terminal acidic residues and binds to Atg8s with 4,10–14 and ectopic P granules (Epg) genes . Among these autophagy modest Kd values ranging from micromolar to submicromolar. Two proteins, Atg8 participates in multiple steps of the autophagic pro- recent studies have reported the development of LIR-based sensors cess15–17. Atg8 attaches to the phagophore membrane via conjugation to monitor autophagy using different strategies32,33. However, these with phosphatidylethanolamine18,19. Atg8–phosphatidylethanol- LIR-based peptides also bind to Atg8s with modest affinities. Due amine can promote phagophore elongation and closure17,20,21. Atg8 to the central roles of Atg8s in autophagy, it is highly desirable to also recruits the Atg1–ULK1 complex to the phagophore to promote develop potent and selective Atg8-binding peptides for applications autophagosome formation22. In the closed autophagosome, Atg8 on such as inhibiting Atg8-mediated autophagy spatiotemporally in the outer membrane interacts with Rab effectors PLEKHM1 and living animals, delineating functions of different Atg8 members in EPG5 for fusion with late the endosome or lysosome23,24. In selec- autophagy, and monitoring autophagy by specifically recognizing tive autophagy, Atg8 on the inner membrane of the phagophore each member of the Atg8 family. We have noted that the 480 kDa interacts with autophagy receptors (for example, p62 and NBR1) to ankyrin-G (AnkG), a neuron-specific isoform of AnkG34,35, was 25,26 36 recruit targets for degradation . As a result, Atg8 and its ortho- recently reported to bind to GABARAP with Kd ≈ 10–20 nM . logs (Atg8s) are commonly used as autophagy indicators27,28, and The superior affinity of the binding between AnkG and GABARAP elimination of Atg8 function impairs autophagy. Yeast contains prompted us to ask whether it might be possible to develop super- only one Atg8, but in higher eukaryotes such as C. elegans, there are strong Atg8-binding peptides as autophagy inhibitors. two homologs, LGG-1 and LGG-2. In vertebrates, these two mem- We found that an extended AnkG-LIR peptide could bind to bers further expand into two subfamilies, namely the GABARAP GABARAP with a super-strong affinity (Kd ≈ 2.6 nM). Notably, this subfamily (GABARAPs, including GABARAP, GABARAPL1 and AnkG-LIR peptide binds to the LC3 subfamily with substantially GABARAPL2) and the LC3 subfamily (LC3s, including LC3A, LC3B weaker affinity. High-resolution crystal structures of AnkG-LIR in and LC3C)15. The two families have some non-redundant func- complex with GABARAPL1 or LC3B reveal the molecular basis for tions, and neither of them is dispensable for the overall autophagic the super-strong and selective binding of AnkG-LIR to GABARAP. 1Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. 2National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. 3College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China. 4Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China. 5Center of Systems Biology and Human Health, Institute for Advanced Study, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. 6These authors contributed equally: Jianchao Li, Ruichi Zhu. *e-mail: [email protected]; [email protected]; [email protected] 778 NATURE CHEMIcal BIOLOGY | VOL 14 | AUGUST 2018 | 778–787 | www.nature.com/naturechemicalbiology NATURE CHEMICAL BIOLOGY ARTICLES a MBD SBD Giant insertion DD ST-rich 480 kDa specific 480 kDa Giant AnkG 270 kDa 1985 2010 b AnkG human 1979 AnkG rat 1979 AnkG chicken 1978 AnkG X. trop. 1966 AnkG D. rerio 1986 ceTime (min) d Time (min) Time (min) 0 10 20 30 40 50 60 0102030405060 0102030405060 0.00 0.00 0.00 –0.20 –0.10 –0.20 –0.40 –0.20 cal/s cal/s –0.60 –0.40 cal/s –0.30 μ μ –0.80 μ –0.40 –0.60 –1.00 –0.50 K = 2.6 ± 0.7 nM K = 546 ± 38 nM K = 2,930 ± 280 nM –1.20 d –0.80 d d 0.00 0.00 0.00 –2.00 –4.00 –2.00 –4.00 –4.00 –8.00 –6.00 –12.00 –8.00 –6.00 –16.00 –10.00 –8.00 kcal/mol of injectant kcal/mol of injectant –12.00 kcal/mol of injectant 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.01.5 2.0 0.00.5 1.01.5 2.0 Molar ratio Molar ratio Molar ratio GABARAP LC3A LC3C fgN i N C N N α1 C Y5 F7 1 D1987 4 1 α 4 1 β β β β K48 R70 β3 2 β W1989 3 β3 α 2 α3 β LIR F104 E17 W1989 W1989 α2 core P30 C F1992 E1996 F1992 E1996 E1991 α2 C-helix E1991 α4 α4 C R28 K30 H27 Negative Hydrophobic Polar Positive AnkG-LIR–GABARAPL1 AnkG-LIR–LC3B h j GABARAP LC3A 10,000 R67 R67 1,000 K66 1 K66 1 β β d β4 β4 K 100 3 3 β L63 β L63 F62 α3 Y49 F62 α3 Y49 10 E1999 E1996 F60 F1992 E1999 E1996 F60 F1992 1 compared to WT A2003 A20200000000000 AA2003 A2220002000 Fold changes of 0.1 R2001 ∣19971919999977 R2001 ∣19979999797 Q59 α4 Q59 α4 0.01 D54 D54 WT C-helix I1997Q W1989RE1991RE1996R A2000QF1992V Y5F/F7Y 1985–2000 R67E/R70EF62K/K65F Fig. 1 | The super-strong affinity of selective AnkG-LIR binding to GABARAPs and its structural basis. a, Domain organizations of 270/480 kDa AnkG and the location of the LIR sequence in AnkG. MBD, Membrane Binding Domain, SBD, Spectrin Binding Domain, DD, Death Domain. b, Sequence alignment of AnkG-LIR from vertebrates. X. trop., Xenopus tropicalis; D. rerio, Danio rerio. The symbols above the sequences are defined as follows: an asterisk (*) indicates positions that have a single, fully conserved residue; a colon (:) indicates conservation between groups of strongly similar properties; a period (.) indicates conservation between groups of weakly similar properties. c–e, ITC results for GABARAP (c), LC3A (d) and LC3C (e), showing that AnkG-LIR can bind to GABARAP with nanomolar affinities and to LC3s with affinities ranging from hundreds to thousands of nM; see Supplementary Fig. 2. ITC profiles are representative of three independent experiments (see Supplementary Fig. 1). The ITC-derived dissociation constants here and throughout the manuscript are reported as value ± fitting errors. f, Combined surface (GABARAPL1) and ribbon–stick (AnkG-LIR) model showing the two hydrophobic pockets of GABARAPL1 accommodating the LIR core and the C-helix of AnkG-LIR. g, Ribbon representation of the AnkG-LIR–GABARAPL1 complex structure. Key residues critical for the binding are shown in stick model format. Salt bridges and hydrogen bonds are indicated with dashed lines. h, Stereo view showing the detailed interactions of the AnkG-LIR C-helix and GABARAPL1. i, Ribbon representation of the AnkG-LIR–LC3B complex structure. Key residues critical for binding are shown in stick model format. j, Bar graph showing the impacts on binding resulted from truncations or mutations of critical residues in the interface.
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