Abstract
Selective autophagy sequesters cytoplasmic cargo for lysosomal degradation via the binding of autophagy receptors to Atg8 (autophagy-related 8) family proteins on the autophagic membrane. The sole yeast Atg8 gene has six mAtg8 (mammalian Atg8) homologs, including the MAP1LC3 (microtubule-associated protein-1 light chain 3) family and the GABA receptor-associated proteins. Selective autophagy receptors interact with two conserved hydrophobic pockets (termed the W-site and L-site) of mATG8 proteins through a linear motif called the LC3-interacting region (LIR) with the general composition (W/F/Y)XX(I/L/V). To address a lack in our knowledge regarding LIR peptide specificity toward each mATG8 homolog, here we used competitive time-resolved FRET to sensitively and quantitatively characterize the interactions between LIRs and mAtg8. We report that 14 representative LIR-containing peptides display differential binding affinities toward the mAtg8 proteins and identified the LIR domain peptide of TP53INP1 as exhibiting high affinity for all six mATG8 proteins. Using peptide truncation studies, we found that both N- and C-terminal acidic residues, as well as the C-terminal Cys residue of the TP53INP1 LIR peptide, are required for its high-affinity binding to LC3A and LC3B, whereas binding to the GABARAP subfamily proteins was facilitated by residues either N-terminal or C-terminal to the core motif. Finally, we used NMR chemical shift perturbation analysis to gain molecular insights into these findings. Collectively, our results may aid in the development of molecules that selectively disrupt specific mATG8-LIR interactions to dissect the biological roles of the six mATG8 homologs for potential therapeutic applications.
Highlights
Selective autophagy sequesters cytoplasmic cargo for lysosomal degradation via the binding of autophagy receptors to Atg8 family proteins on the autophagic membrane
We found that both N- and C-terminal acidic residues, as well as the C-terminal Cys residue of the TP53INP1 LC3-interacting region (LIR) peptide, are required for its high-affinity binding to LC3A and LC3B, whereas binding to the GABARAP subfamily proteins was facilitated by residues either N-terminal or C-terminal to the core motif
Consistent with the literature, all six mATG8 proteins interact with the p62 peptide with Kd in the range of 61–271 nM (Fig. 1D), establishing the TR-FRET assay as a sensitive and quantitative method to investigate the relative selectivity of LIR sequences toward Atg8 family proteins
Summary
Proteins have demonstrated that selective autophagy receptors interact with two conserved hydrophobic pockets (termed the W-site and L-site) of mATG8 proteins through a linear motif called the LC3-interacting region (LIR).. Studies have indicated that the aromatic residue appears to be the most crucial binding determinant, both N-terminal and C-terminal residues of the core LIR motif may play a role in regulating binding affinity and selectivity between the mAtg proteins, a possibility that has not been fully explored in LIR-containing proteins [18, 19]. We found that the LIR domain peptide of TP53INP1 displays a high affinity for all six mATG8 proteins Utilizing this peptide as a model, we sought to identify key residues that control selectivity toward Atg family members. Our results will facilitate the development of molecules that selectively disrupt specific mATG8 –LIR interactions for dissecting the biological roles of the six mATG8 homologs and for therapeutic applications
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