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Regulation of ATG4B stability by RNF5 limits basal levels of autophagy and influences susceptibility to bacterial infection.

Kuang E, Okumura CY, Sheffy-Levin S, Varsano T, Shu VC, Qi J, Niesman IR, Yang HJ, López-Otín C, Yang WY, Reed JC, Broday L, Nizet V, Ronai ZA - PLoS Genet. (2012)

Bottom Line: RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 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.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction and Cell Death Programs, Sanford-Burnham Medical Research Institute, La Jolla, California, USA.

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.

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Resistance to GAS infection in RNF5 KO mice.(A) Intracellular killing of GAS is enhanced in RNF5−/− 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 RNF5−/− 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 RNF5−/− 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 RNF5−/− 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.
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pgen-1003007-g007: Resistance to GAS infection in RNF5 KO mice.(A) Intracellular killing of GAS is enhanced in RNF5−/− 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 RNF5−/− 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 RNF5−/− 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 RNF5−/− 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.

Mentions: GAS intracellular survival was significantly reduced in RNF5−/− macrophages compared to WT controls (Figure 7A). However, the enhanced intracellular bacterial clearance by RNF5−/− macrophages was abolished when cells were transfected with ATG4B shRNA (Figure 7B). These results specifically link the enhanced bacterial clearance in the RNF5−/− macrophages to increased basal autophagy in these cells. Using a murine model of systemic GAS infection, at a challenge dose that produced 75% mortality in WT mice, almost all RNF5−/− mice survived (Figure 7C). The bacterial loads in mouse organs during the early stages of infection were higher in RNF5+/+ mice compared to RNF5−/− mice (Figure 7D). Taken together, our data show that the negative regulation of autophagy by RNF5 influences the outcome of GAS infection, and demonstrate for the first time a protective role for autophagy in an in vivo model of this important human infectious disease.


Regulation of ATG4B stability by RNF5 limits basal levels of autophagy and influences susceptibility to bacterial infection.

Kuang E, Okumura CY, Sheffy-Levin S, Varsano T, Shu VC, Qi J, Niesman IR, Yang HJ, López-Otín C, Yang WY, Reed JC, Broday L, Nizet V, Ronai ZA - PLoS Genet. (2012)

Resistance to GAS infection in RNF5 KO mice.(A) Intracellular killing of GAS is enhanced in RNF5−/− 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 RNF5−/− 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 RNF5−/− 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 RNF5−/− 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.
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getmorefigures.php?uid=PMC3475677&req=5

pgen-1003007-g007: Resistance to GAS infection in RNF5 KO mice.(A) Intracellular killing of GAS is enhanced in RNF5−/− 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 RNF5−/− 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 RNF5−/− 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 RNF5−/− 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.
Mentions: GAS intracellular survival was significantly reduced in RNF5−/− macrophages compared to WT controls (Figure 7A). However, the enhanced intracellular bacterial clearance by RNF5−/− macrophages was abolished when cells were transfected with ATG4B shRNA (Figure 7B). These results specifically link the enhanced bacterial clearance in the RNF5−/− macrophages to increased basal autophagy in these cells. Using a murine model of systemic GAS infection, at a challenge dose that produced 75% mortality in WT mice, almost all RNF5−/− mice survived (Figure 7C). The bacterial loads in mouse organs during the early stages of infection were higher in RNF5+/+ mice compared to RNF5−/− mice (Figure 7D). Taken together, our data show that the negative regulation of autophagy by RNF5 influences the outcome of GAS infection, and demonstrate for the first time a protective role for autophagy in an in vivo model of this important human infectious disease.

Bottom Line: RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 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.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction and Cell Death Programs, Sanford-Burnham Medical Research Institute, La Jolla, California, USA.

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.

Show MeSH
Related in: MedlinePlus