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Macroautophagy/autophagy is a highly conserved metabolic process that degrades intracellular components and recycles bioenergetic substrates. SQSTM1/p62 (sequestosome 1) is a classical autophagy receptor that participates in selective autophagy to eliminate abnormal intracellular components and recycle bioenergetic substrates. In autophagy, SQSTM1 recruits ubiquitinated substrates to form SQSTM1 droplets and delivers these cargoes to phagophores, the precursors to autophagosomes. Recently, we reported a previously unidentified SQSTM1 S-acylation, which is catalyzed by S-acyltransferase ZDHHC19 and reversed by LYPLA1/APT1. S-acylation of SQSTM1 enhances the affinity of SQSTM1 droplets with the phagophore membrane, thereby promoting efficient autophagic degradation of ubiquitinated substrates. Our study uncovers the role of the S-acylation-deacylation cycle in regulating SQSTM1-mediated selective autophagy. [ABSTRACT FROM AUTHOR]

Proteolysis-targeting chimeras (PROTACs) based on the ubiquitin-proteasome system have made great progress in the field of drug discovery. There is mounting evidence that the accumulation of aggregation-prone proteins or malfunctioning organelles is associated with the occurrence of various age-related neurodegenerative disorders and cancers. However, PROTACs are inefficient for the degradation of such large targets due to the narrow entrance channel of the proteasome. Macroautophagy (hereafter referred to as autophagy) is known as a self-degradative process involved in the degradation of bulk cytoplasmic components or specific cargoes that are sequestered into autophagosomes. In the present study, we report the development of a generalizable strategy for the targeted degradation of large targets. Our results suggested that tethering large target models to phagophore-associated ATG16L1 or LC3 induced targeted autophagic degradation of the large target models. Furthermore, we successfully applied this autophagy-targeting degradation strategy to the targeted degradation of HTT65Q aggregates and mitochondria. Specifically, chimeras consisting of polyQ-binding peptide 1 (QBP) and ATG16L1-binding peptide (ABP) or LC3-interacting region (LIR) induced targeted autophagic degradation of pathogenic HTT65Q aggregates; and the chimeras consisting of mitochondria-targeting sequence (MTS) and ABP or LIR promoted targeted autophagic degradation of dysfunctional mitochondria, hence ameliorating mitochondrial dysfunction in a Parkinson disease cell model and protecting cells from apoptosis induced by the mitochondrial stress agent FCCP. Therefore, this study provides a new strategy for the selective proteolysis of large targets and enrich the toolkit for autophagy-targeting degradation. Abbreviations: ABP: ATG16L1-binding peptide; ATG16L1: autophagy related 16 like 1; ATTEC: autophagy-tethering compound; AUTAC: autophagy-targeting chimera; AUTOTAC: autophagy-targeting chimera; Baf A1: bafilomycin A1; BCL2: BCL2 apoptosis regulator; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CPP: cell-penetrating peptide; CQ: chloroquine phosphate; DAPI: 4',6-diamidino-2-phenylindole; DCM: dichloromethane; DMF: N,N-dimethylformamide; DMSO: dimethyl sulfoxide; EBSS: Earle′s balanced salt solution; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; FITC: fluorescein-5-isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HEK293: human embryonic kidney 293; HEK293T: human embryonic kidney 293T; HPLC: high-performance liquid chromatography; HRP: horseradish peroxidase; HTT: huntingtin; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFF: mitochondrial fission factor; MTS: mitochondria-targeting sequence; NBR1: NBR1 autophagy cargo receptor; NLRX1: NLR family member X1; OPTN: optineurin; P2A: self-cleaving 2A peptide; PB1: Phox and Bem1p; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; PROTACs: proteolysis-targeting chimeras; QBP: polyQ-binding peptide 1; SBP: streptavidin-binding peptide; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SPATA33: spermatogenesis associated 33; TIMM23: translocase of inner mitochondrial membrane 23; TMEM59: transmembrane protein 59; TOMM20: translocase of outer mitochondrial membrane 20; UBA: ubiquitin-associated; WT: wild type. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy is a fundamental cellular catabolic process that delivers cytoplasmic components into double-membrane vesicles called autophagosomes, which then fuse with lysosomes and their contents are degraded. Autophagy recycles cytoplasmic components, including misfolded proteins, dysfunctional organelles and even microbial invaders, thereby playing an essential role in development, immunity and cell death. Autophagosome formation is the main step in autophagy, which is governed by a set of ATG (autophagy related) proteins. ATG16L1 interacts with ATG12–ATG5 conjugate to form an ATG12–ATG5-ATG16L1 complex. The complex acts as a ubiquitin-like E3 ligase that catalyzes the lipidation of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), which is crucial for autophagosome formation. In the present study, we found that ATG16L1 was subject to S-palmitoylation on cysteine 153, which was catalyzed by ZDHHC7 (zinc finger DHHC-type palmitoyltransferase 7). We observed that re-expressing ATG16L1 but not the S-palmitoylation-deficient mutant ATG16L1C153S rescued a defect in the lipidation of LC3 and the formation of autophagosomes in ATG16L1-KO (knockout) HeLa cells. Furthermore, increasing ATG16L1 S-palmitoylation by ZDHHC7 expression promoted the production of LC3-II, whereas reducing ATG16L1 S-palmitoylation by ZDHHC7 deletion inhibited the LC3 lipidation process and autophagosome formation. Mechanistically, the addition of a hydrophobic 16-carbon palmitoyl group on Cys153 residue of ATG16L1 enhances the formation of ATG16L1-WIPI2B complex and ATG16L1-RAB33B complex on phagophore, thereby facilitating the LC3 lipidation process and autophagosome formation. In conclusion, S-palmitoylation of ATG16L1 is essential for the lipidation process of LC3 and the formation of autophagosomes. Our research uncovers a new regulatory mechanism of ATG16L1 function in autophagy.Abbreviation: ABE: acyl-biotin exchange; ATG: autophagy related; Baf-A1: bafilomycin A1; 2-BP: 2-bromopalmitate; CCD: coiled‐coil domain; co-IP: co-immunoprecipitation; CQ: chloroquine; EBSS: Earle’s balanced salt solution; HAM: hydroxylamine; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NP-40: Nonidet P-40; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PtdIns3K-C1: class III phosphatidylinositol 3-kinase complex I; PTM: post-translational modification; RAB33B: RAB33B, member RAS oncogene family; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscope; WD: tryptophan and aspartic acid; WIPI2B: WD repeat domain, phosphoinositide interacting 2B; WT: wild-type; ZDHHC: zinc finger DHHC-type. [ABSTRACT FROM AUTHOR]