Stapled finger1/6/2024 Impaired autophagy is a hallmark of many human diseases. The cysteine residues and linkers are highlighted in blue, and lowercase c denotes d-cysteine. (c) Sequences of autophagy-inducing peptides, with conserved hot spot residues highlighted in red. (44) The segment shown in black corresponds to the sequence of the Beclin 1-derived portion of Tat-11mer, and the red residues correspond to the required hot spots for Tat-11mer activity. (b) Crystal structure of the ECD of human Beclin 1. Bafilomycin A1 (Baf A1) is a known inhibitor of the lysosomal fusion step. Peptide inducers appear to act during the induction phase (red dashed arrows). Following lysosomal fusion, autophagosomes and their contents, including the autophagy adaptor protein p62, are broken down and recycled. This maturation process is controlled by a different Beclin 1 complex, involving the regulator UVRAG. The autophagosome matures, fully engulfs the cargo, and is trafficked to the lysosome where it fuses with the lysosomal membrane. The lipidated form, LC3-II, is then incorporated in autophagosome membranes. The microtubule-associated protein light chain I (LC3-I) becomes lipidated with phosphatidylethanolamine (PE). In response to upstream signaling, Beclin 1 and its PI3K complex (which includes lipid kinase subunit Vps34 and regulatory subunit Atg14) nucleate membrane formation. (a) Overall schematic of the process of autophagy. The autophagic pathway and peptide inducers of autophagy. More broadly, diversity-oriented stapling may provide a promising alternative to polycationic sequences as a means for rendering peptides more cell-penetrant.įigure 1. These new, cell-penetrant autophagy inducers and their molecular details are critical advances in the effort to understand and control autophagy. We also developed a novel assay for cell penetration that reports exclusively on cytosolic access and used it to quantitatively compare the cell penetration of DD5-o and other autophagy-inducing peptides. Unexpectedly, the solution structure of the most potent stapled peptide, DD5-o, revealed an α-helical conformation in methanol, stabilized by an unusual ( i, i+3) staple which cross-links two d-amino acids. These peptides induce autophagy at micromolar concentrations in vitro, have aggregate-clearing activity in a cellular model of Huntington’s disease, and induce autophagy in vivo. Here, we report the use of a diversity-oriented stapling approach to produce autophagy-inducing peptides that are intrinsically cell-penetrant. Induction of autophagy is a promising approach for treating diverse human diseases, including neurodegenerative disorders and infectious diseases. Autophagy is an essential pathway by which cellular and foreign material are degraded and recycled in eukaryotic cells.
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