Regulation of Autophagy-related proteins: Roles of post-translational modifications

Abstract

Autophagy is a cell renovation system that directs almost any type of cell contents for lysosomal degradation and recycling of building blocks. It is fundamental for cellular homeostasis and for determining cell fate in response to stress. Hence, understanding the regulation of autophagy-related proteins is of great importance. This study focuses on the effect of posttranslational modifications on the autophagy proteins LC3B and TP53INP2, and importance of molecular interactions for ATG4B mediated cleavage and delipidation of the ATG8 family proteins. In the first paper, we shed light on the effect of phosphorylation of LC3B on selective autophagy. LC3B is the most recognized member of the ATG8 family proteins and is used as a readout for autophagy activity. LC3B is enriched in the growing autophagosomal membrane, where it recruits cargo and cargo receptors to the autophagosomes via direct LC3 interacting region (LIR)-LC3 docking site (LDS) interactions. We identified four serine-threonine kinases that phosphorylate LC3B at the threonine 50 (T50) residue. In vivo and in vitro data show that they interact with LC3B via LIR-LDS mediated interactions. The LC3B T50 residue is adjacent to the LDS motif in LC3B. Importantly, we found that the phospho-mimicking LC3B T50E mutant inhibited the interaction of LC3B with core autophagy proteins and negatively regulated selective autophagy. We showed that NEK9 phosphorylate LC3B T50 and that depletion of NEK9 facilitated autophagic flux. This study thus points to NEK9 as a regulator of selective autophagic flux via phosphorylation of LC3B T50. In the second paper, we show that subcellular localization of the autophagy protein TP53INP2 is regulated by acetylation. TP53INP2 is previously recognized as a regulator of autophagy, providing nuclear export of LC3B and autophagosome formation by interactions with the ATG8s and VMP1. Here we show that upon mTOR inactivation, nuclear import of TP53INP2 is impaired via acetylation of K187, while its degradation is facilitated by acetylation of K165 and K204. Hence, mTOR dependent re-localization of TP53INP2 is due to enhanced nuclear degradation and cytoplasmic retention. The functional role of the cysteine protease ATG4B for cleavage and delipidation of ATG8 homologues are well known. In the third paper, we unravel essential residues involved in the ATG4B-ATG8s functional interactions. We identified a canonical LIR motif at C- terminal part of ATG4B and solved the crystal structure of GABARAPL1 in complex with a peptide containing this LIR motif. Our in vivo and in vitro data showed that the ATG4B C- terminal LIR is important for efficient cleavage of LC3B. Furthermore, it was found to mediate stabilization of unlipidated GABARAP and GABARAPL1, protecting them from proteasomal degradation.