Abstract
Autophagy is critical for maintaining cellular homeostasis during times of stress, and is thought to play important roles in both tumorigenesis and tumor cell survival. Formation of autophagosomes, which mediate delivery of cytoplasmic cargo to lysosomes, requires multiple autophagy-related (ATG) protein complexes, including the ATG12–ATG5-ATG16L1 complex. Herein, we report that a molecular ATG5 “conjugation switch”, comprised of competing ATG12 and ubiquitin conjugation reactions, integrates ATG12–ATG5-ATG16L1 complex assembly with protein quality control of its otherwise highly unstable subunits. This conjugation switch is tightly regulated by ATG16L1, which binds to free ATG5 and mutually protects both proteins from ubiquitin conjugation and proteasomal degradation, thereby instead promoting the irreversible conjugation of ATG12 to ATG5. The resulting ATG12–ATG5 conjugate, in turn, displays enhanced affinity for ATG16L1 and thus fully stabilizes the ATG12–ATG5-ATG16L1 complex. Most importantly, we find in multiple tumor types that ATG5 somatic mutations and alternative mRNA splicing specifically disrupt the ATG16L1-binding pocket in ATG5 and impair the essential ATG5-ATG16L1 interactions that are initially required for ATG12–ATG5 conjugation. Finally, we provide evidence that ATG16L2, which is overexpressed in several cancers relative to ATG16L1, hijacks the conjugation switch by competing with ATG16L1 for binding to ATG5. While ATG16L2 stabilizes ATG5 and enables ATG12–ATG5 conjugation, this endogenous dominant-negative inhibitor simultaneously displaces ATG16L1, resulting in its proteasomal degradation and a block in autophagy. Thus, collectively, our findings provide novel insights into ATG12–ATG5-ATG16L1 complex assembly and reveal multiple mechanisms wherein dysregulation of the ATG5 conjugation switch inhibits autophagy.
Highlights
Macroautophagy is a highly conserved catabolic process that maintains cellular homeostasis by targeting excess or damaged organelles, large protein aggregates, invading pathogens, and nonselective portions of the cytoplasm for lysosomal degradation via double-membrane vesicles, termed autophagosomes
While assessing basal autophagic flux in classical prostate cancer (PCa) cell lines, we found that DU145 cells had strikingly higher basal levels of p62 compared to LNCaP and PC-3 cells (Fig. 1a)
Inhibition of autophagic flux with Bafilomycin A1 (Baf A1) treatment triggered a significant build-up of lipid-conjugated LC3B (LC3B-II) in both LNCaP and PC-3 cells, whereas no LC3B-II was detected in DU145 cells (Fig. 1a)
Summary
Macroautophagy (hereafter referred to as autophagy) is a highly conserved catabolic process that maintains cellular homeostasis by targeting excess or damaged organelles, large protein aggregates, invading pathogens, and nonselective portions of the cytoplasm for lysosomal degradation via double-membrane vesicles, termed autophagosomes. Reverting the mutant splice site allele to wild type in DU145 cells using CRISPR/Cas[9] rescued full-length ATG5 mRNA expression (Fig. 1e, lane 4), as well as ATG12–ATG5 conjugation, functional ATG12–ATG5-ATG16L1 complex formation and autophagic flux, as determined by LC3 lipidation and p62 degradation (Fig. 1h, lanes 3 and 4).
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have