The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independently of the WD domain required for LC3-associated phagocytosis

Abstract

Macroautophagy/autophagy delivers damaged proteins and organelles to lysosomes for degradation, and plays important roles in maintaining tissue homeostasis by reducing tissue damage. The translocation of LC3 to the limiting membrane of the phagophore, the precursor to the autophagosome, during autophagy provides a binding site for autophagy cargoes, and facilitates fusion with lysosomes. An autophagy-related pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosome and endosome membranes during uptake of bacterial and fungal pathogens, and targets LC3 to swollen endosomes containing particulate material or apoptotic cells. We have investigated the roles played by autophagy and LAP in vivo by exploiting the observation that the WD domain of ATG16L1 is required for LAP, but not autophagy. Mice lacking the linker and WD domains, activate autophagy, but are deficient in LAP. The LAP −/mice survive postnatal starvation, grow at the same rate as littermate controls, and are fertile. The liver, kidney, brain and muscle of these mice maintain levels of autophagy cargoes such as LC3 and SQSTM1/p62 similar to littermate controls, and prevent accumulation of SQSTM1 inclusions and tissue damage associated with loss of autophagy. The results suggest that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis. Further deletion of glutamate E230 in the coiled-coil domain required for WIPI2 binding produced mice with defective autophagy that survived neonatal starvation. Analysis of brain lysates suggested that interactions between WIPI2 and ATG16L1 were less critical for autophagy in the brain, which may allow a low level of autophagy to overcome neonatal lethality.

The WD and linker domains of ATG16L1 required for non-canonical autophagy limit lethal respiratory infection by influenza A virus at epithelial surfaces

“…This was consistent with lack of elevated cytokines in lungs prior to infection (see day 0 in Fig. 3A), and our previous work showing that serum levels of IL-1β, IL-12p70, IL-13, and TNF-α in δWD mice are the same as in littermate controls at 8-12 and 20-24 weeks 21 . The exaggerated 20 inflammatory response to IAV did not therefore result from a raised pro-inflammatory threshold or dysregulated IL-1β responses in the lung.…”

“…Importantly, and unlike other mouse models of non-canonical autophagy 4,20 , the mice grow normally, do not have pro-inflammatory phenotype and maintain tissue homeostasis 20 21 .…”

“…We have shown that the WD domain of ATG16L1 is required for LAP during uptake of yeast in vitro and for non-canonical conjugation of LC3 to endo-lysosome membranes following osmotic imbalance induced by lysosomotropic drugs 2 . This prompted us to generate a mouse lacking the WD domain of ATG16L1 to study the role 10 played by non-canonical autophagy in vivo 21 . The mouse model (WD [Atg16L1 wd/wd ]) carries a stop codon after the coiled coil domain which removes amino acid residues at 266 and 319 in the linker region and conserved phenylalanine residues at positions 467 and 490 in the WD domain required for lipid binding of ATG16L1 and subsequent recruitment of ATG5-ATG12 to endo-lysosome membranes 2,22 .…”

“…The mouse model (WD [Atg16L1 wd/wd ]) carries a stop codon after the coiled coil domain which removes amino acid residues at 266 and 319 in the linker region and conserved phenylalanine residues at positions 467 and 490 in the WD domain required for lipid binding of ATG16L1 and subsequent recruitment of ATG5-ATG12 to endo-lysosome membranes 2,22 . As a consequence the 15 mice are unable to conjugate LC3 to endo-lysosome membrane compartments 21 , but the mutation preserves the N-terminal ATG5-binding domain and glutamates at positions 226 and 230 in the CCD of ATG16L1 that are required for WIPI2 binding and autophagy 23 .…”

Autophagy-Independent Functions of the Autophagy Machinery

“…(RB1CC1, best known as FIP200), which are all involved in autophagy, but relies on RUBCN, NAPDH oxidase 2, and the WD domain of ATG16L1, which are all dispensable for autophagy (Fletcher et al, 2018;Martinez et al, 2015Martinez et al, , 2016Rai et al, 2018). Thus, the deletion of Becn1, Atg5, or Atg7 (but not Rb1cc1) from myeloid cells drives an autoimmune disorder similar to human systemic lupus erythematosus (SLE), which can be recapitulated by the whole-body knockout of Rubcn but not by that of Ulk1 (Martinez et al, 2016).…”