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

<div><p>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 <em>RNF5^−/−</em> 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 <em>RNF5^−/−</em> 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 <em>Caenorhabditis elegans</em> increases the level of LGG-1/LC3::GFP puncta. <em>RNF5^−/−</em> mice are more resistant to group A <em>Streptococcus</em> infection, associated with increased autophagosomes and more efficient bacterial clearance by <em>RNF5^−/−</em> macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.</p> </div

RNF5 affects engulfment of GAS in autophagosomes.

<p>(A) Immunoblotting analysis of LC3 and p62 in RNF5 WT and KO macrophages maintained in normal or HBSS-containing media. (B) Subcellular localization of LC3 and ATG4B in macrophages of RNF5 WT and KO mice. Cell extracts were prepared from bone marrow-derived macrophages and fractionated using sucrose gradient ultracentrifugation. Fractions (40 µg protein/lane) enriched for endoplasmic reticulum (ER), mitochondria (Mito), or cytosolic (Cyt) were resolved and analyzed by western blotting with the indicated antibodies. Markers representing each of the organelles are shown. (C) Confocal microscopic images of GFP-labeled GAS (green) and immunostained LC3 (red) depicts representative images of GAS contained in LC3-positive compartments (arrowheads) in bone marrow-derived macrophages from WT and RNF5 KO mice (DNA labeling by DAPI in blue). Bone marrow-derived macrophages were seeded on glass coverslips (5×10⁴/well in 24-well plates) and the following day infected with GAS (MOI = 10) for 2.5 h before being washed and fixed in 3% PFA. Cells were processed for immunofluorescence microscopy using affinity-purified rabbit anti-LC3 IgG, followed by goat anti-rabbit Alexa-488 F(ab′)2, and DAPI to stain the bacterial and cellular DNA. (D) Quantification of GAS associated with LC3-positive autophagosomes in macrophages of WT and RNF5 KO mice. RNF5 KO macrophages contain more GAS engulfed by LC3-decorated structures per cell (I, n = 20, P = 0.077) and more LC3 puncta per cell (II, n = 20, P<0.05). Graphs show the mean ± SE, P value was calculated by Student t-test. Images were acquired on an Olympus FluoView 1000 confocal microscope using a 100× oil immersion lens. (E) EM-based ultrastructural analysis of GAS-infected macrophages from WT (I) and RNF5 KO (II) mice. Within 2.5 h after macrophage infection, most GAS are engulfed within autophagosome-like vacuoles (arrowheads). Scale bar = 2 µm (I).</p

RNF5 co-localizes with—and limits—ATG4B at the membrane compartment.

<p>(A) RNF5/LC3 immunofluorescence images in HeLa cells expressing GFP-sec61b. Magnified view of the white square region is shown on the bottom right panels; white arrows denote LC3 foci that colocalized with ER-localized RNF5. Scale bar, 10 microns. (B) ATG4B and RNF5 co-fractionate in ER and autophagosomes. 293T cells were maintained under normal medium or HBSS starvation (3 h) were collected and passed through a 25-gauge needle 10 times and the membrane fractions were enriched and loaded to 10–50% linear sucrose gradient and subjected to ultracentrifugation (160,000 g for 2.5 h). Twelve fractions (1 ml each) were collected from the top to the bottom, and analyzed by western blots as indicated. Calnexin, ER membrane marker. (C) 293T cells were starved using media supplemented with HBSS, and the cells were collected at the indicated time points. Membrane fractions were separated by sucrose gradient ultracentrifugation. Proteins (40 µg/lane) were resolved and immunoblotted with the indicated antibodies. Top, ER-associated GFP-LC3 puncta; Middle, cytosolic GFP-LC3 puncta. (D) PC3 cells stably expressing shRNF5 were maintained in HBSS (3 h) or regular medium. Membrane fractions were separated by sucrose gradient ultracentrifugation. Proteins (40 µg/lane) were resolved and immunoblotted with the indicated antibodies.</p

RNF-5 regulates autophagy in <i>C. elegans</i>.

<p>(A–C) Representative images of seam cells from L3 larvae expressing the GFP::LGG-1 transgene, and the average number of GFP::LGG-1 puncta in seam cells. Three to four independent experiments were performed for each condition. Error bars are ± SEM. P value was calculated using an unpaired two-tailed t-test. (A) <i>rnf-5(tm794)</i> larvae had an average of 5.53±0.49 puncta/cell compared to 2.11±0.30 puncta/cell in WT (36 cells from 9 <i>rnf-5(tm794)</i> larvae, and 46 cells from 10 WT larvae, p<0.0001). (B) <i>rnf-5(RNAi)</i>-treated worms had an average of 4.42±0.47 puncta/cell compared to 1.83±0.31 puncta/cell in control animals (77 cells from 15 <i>rnf-5(RNAi)</i> larvae and 64 cells from 17 control larvae, p<0.0001). (C) Animals grown constantly at 25°C: <i>hsp-16p</i>::<i>rnf-5</i> larvae had an average of 1.71±0.24 puncta/cell compared to 3.75±0.34 puncta/cell in the non-transgenic population (69 cells from 18 <i>hsp-16p</i>::<i>rnf-5</i> larvae, and 68 cells from 15 non-transgenic larvae, p<0.0001).</p

RNF5 interacts with ATG4B and mediates its ubiquitination and degradation.

<p>(A) Membrane-bound RNF5 interacts with ATG4B. RNF5 WT, C-terminal TM-deleted mutant (dCT), and RING domain mutant (RM) constructs were co-transfected with Flag-ATG4B. The cell lysates were immunoprecipitated with anti-Flag antibodies conjugated to beads, then immunoblotted with the indicated antibodies. (B) Dynamic interaction of ATG4B and RNF5 during starvation-induced autophagy. The interaction between endogenous ATG4B and RNF5 expressed in HeLa cells was monitored at the indicated time points prior to and following HBSS-induced starvation. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (C) In vivo ATG4B ubiquitination by RNF5. HeLa cells were co-transfected with Flag-ATG4B, HA-ubiquitin (Ub), and WT or RM RNF5 plasmids. After 24 h, cells were treated with MG132 (10 µM) for 4 h and lysed with buffer containing 1% SDS. The lysates were immunoprecipitated with anti-Flag antibody in the presence of 0.1% SDS, followed by immunoblotting with anti-HA antibodies. (D) ATG4B in vitro ubiquitination by RNF5. Purified His6-tagged ATG4B was bound to nickel beads and incubated for 1 h at 37°C with RNF5 in the presence of the indicated in vitro ubiquitination reagents. The bead were washed three times with PBS containing 0.1% SDS and 0.2% Triton X-100 and then immunoblotted with antibodies to ATG4B and Ub. (E) RNF5 reduces ATG4B stability. Half-life of ATG4B in RNF5 WT and KO MEFs under normal growth conditions or after HBSS treatment was determined by addition of cycloheximide (CHX) (40 µg/ml) for the indicated times. Cell extracts were subjected to immunoblot analysis using anti-ATG4B and anti-tubulin antibodies. Quantitation of ATG4B levels based on band intensity was measured using the LICOR system, and is shown as the mean of duplicate experiments.</p

Resistance to GAS infection in RNF5 KO mice.

<p>(A) Intracellular killing of GAS is enhanced in <i>RNF5^−/−</i> bone marrow-derived macrophages compared to WT macrophages. Data represent three independent experiments. Differences between groups were analyzed using an unpaired Student's t-test with two-tailed P values. (B) Enhanced killing of GAS by <i>RNF5^−/−</i> macrophages is abolished by knockdown of ATG4B. Bone marrow-derived macrophages were transduced with lentivirus-based nonsense (NS), ATG4B, or ATG5 shRNA and intracellular killing of bacteria was subsequently analyzed. One representative of three independent experiments is shown and differences between groups were analyzed using one-way ANOVA with the Tukey-Kramer post-test. **P<0.01, ***P<0.001. (C) Survival of WT and <i>RNF5^−/−</i> mice (n = 11 for each group from two individual experiments) was monitored over 10 days after intraperitoneal infection with 2×10⁷ cfu of GAS. Comparison of survival curves was performed with the log-rank (Mantel-Cox) test. (D) Bacterial load in the kidney, liver, and spleen of WT and <i>RNF5^−/−</i> mice (n = 11 for each group from two individual experiments), 3 days post-infection with GAS. Data are shown with mean and 95% confidence intervals, and differences between groups were analyzed using an unpaired Student's t-test with two-tailed P values.</p

RNF5 regulates autophagosome formation.

<p>(A) ATG4B is required for RNF5-mediated endogenous LC3 puncta. ATG4B WT and KO MEFs were transfected with mCherry harboring RNF5 or control shRNA, and 48 h later cells were fixed and immunostained with anti-LC3 antibody. Shown are the mean values for LC3-positive puncta in >20 cells per experimental condition. SC, scrambled shRNA. The right column depicts merged LC3 (green) and RNF5 (mCherry) and DAPI channels. (B) Immunofluorescence images of mRFP-GFP-LC3 in RNF5 WT and KO MEFs. The plasmid ptfLC3 expressing the LC3 WT or LC3 mutant containing a pre-cleaved LC3 TFG were transfected into RNF5 WT and KO MEFs. After 24 h, the cells were fixed and the number of LC3-positive red or yellow puncta was counted in >20 cells. Quantification was confirmed by an independent analyst who was blinded to the sample identities.</p

RNF5 negatively regulates autophagy.

<p>(A) Immunoblot analysis of LC3 and p62. RNF5 WT and KO MEFs were maintained in HBSS for 1 to 4 h before cells were lysed, and proteins were resolved and analyzed by immunoblotting with the indicated antibodies. (B) Scrambled and shRNF5-transduced PC3 cells were maintained in normal medium, serum-starved overnight, or treated with tunicamycin (TM, 5 µg/ml, 8 h). (C) Amount of endogenous LC3 puncta is affected by RNF5. RNF5 WT and KO MEFs grown in normal medium, starved with HBSS (2 h), or treated with DTT (5 mM, 8 h) were fixed and immunostained with anti-LC3 antibody. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003007#s2" target="_blank">Results</a> show the quantification of endogenous LC3 puncta counted in >20 cells per experimental condition, in duplicate.</p