Regulation of ATG4B Stability by RNF5 Limits Basal Levels of Autophagy and Influences Susceptibility to Bacterial Infection

Abstract

Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with—and regulation of its ubiquitination and stability by—RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5−/− MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5−/− using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5−/− mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5−/− macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.

Role for Streptococcal Collagen-Like Protein 1 in M1T1 Group A Streptococcus Resistance to Neutrophil Extracellular Traps

“…J774 cells were seeded at 2.5 ϫ 10 5 cells/well in 24-well plates 1 day prior to infection. Bone-marrow derived macrophages (BMDMs) were isolated from 8-to 10-week-old C57BL/6 WT or cathelicidin-related antimicrobial peptide (CRAMP)-deficient mice as previously described (34) and seeded at 5 ϫ 10 5 cells/well in 24-well plates 1 day prior to infection. Prior to bacterial infection, cells were treated with 10 g/ml cytochalasin D (cyt D) (Sigma) for 10 min, 100 nM butylated hydroxyanisole (BHA) (Sigma) for 15 min, 0.2 to 0.5 kU/ml catalase (Sigma) for 20 min, or 100 M 4-aminobenzoic acid hydrazide (ABAH) (Fisher Scientific) for 20 min.…”

Regulation of autophagy by protein post-translational modification

“…Ubiquitination of DEPTOR by Skp-cullin-F-box protein (SCF) in growth factor-rich conditions releases MTORC1 from DEPTOR inhibition and attenuates autophagy (73;74). ATG4B ubiquitination by RING finger protein 5 (RNF5) led to ATG4B degradation, which in turn affects LC3 turnover and limits autophagy (75). ULK1 ubiquitination by TRAF6 stabilizes the active ULK1 complex, therefore promoting autophagy (76).…”

The functional and pathologic relevance of autophagy proteases

“…The E3 ligase RNF5 spatially controls ATG4B stability by inducing its ubiquitination and proteasome-mediated degradation at membrane domains like the phagophore. This mechanism thus represents a new regulatory layer of basal autophagy, blocking LC3 priming at the beginning of the process (70). In yeast, an Atg18-Atg21 complex is a key regulator during the phagophore formation (71), recruiting and protecting Atg8-PE.…”