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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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RNF5 interacts with ATG4B and mediates its ubiquitination and degradation.(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.
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pgen-1003007-g001: RNF5 interacts with ATG4B and mediates its ubiquitination and degradation.(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.

Mentions: Given the various effects of RNF5 on ER stress and innate immune pathways, processes that are influenced by autophagy, we examined the possibility that RNF5 may play a direct role in the control of autophagy. A cDNA library screen using a yeast-based functional assay for ATG4B inhibitors identified RNF5 as a candidate regulator (1 in 12 hits among 2×105 colonies; Figure S1). To confirm a possible interaction of RNF5 with ATG4B, we examined the association between exogenous and endogenously expressed proteins. Immunoprecipitation of exogenously expressed WT or activity-dead mutant (C74A) forms of ATG4B confirmed their association with exogenously expressed RNF5 (Figure S2a). ATG4B also associated with the RING mutant (RM) form of RNF5, indicating that the ubiquitin ligase activity of RNF5 was not required for this interaction. However, RNF5 lacking its membrane-spanning C-terminal domain (ΔCT) was no longer able to interact with ATG4B (Figure 1A), suggesting that the interaction takes place within the membranal domain. The latter is consistent with earlier studies showing that RNF5 interactions with, and effects upon, its substrates require membrane anchoring [36].


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)

RNF5 interacts with ATG4B and mediates its ubiquitination and degradation.(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.
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pgen-1003007-g001: RNF5 interacts with ATG4B and mediates its ubiquitination and degradation.(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.
Mentions: Given the various effects of RNF5 on ER stress and innate immune pathways, processes that are influenced by autophagy, we examined the possibility that RNF5 may play a direct role in the control of autophagy. A cDNA library screen using a yeast-based functional assay for ATG4B inhibitors identified RNF5 as a candidate regulator (1 in 12 hits among 2×105 colonies; Figure S1). To confirm a possible interaction of RNF5 with ATG4B, we examined the association between exogenous and endogenously expressed proteins. Immunoprecipitation of exogenously expressed WT or activity-dead mutant (C74A) forms of ATG4B confirmed their association with exogenously expressed RNF5 (Figure S2a). ATG4B also associated with the RING mutant (RM) form of RNF5, indicating that the ubiquitin ligase activity of RNF5 was not required for this interaction. However, RNF5 lacking its membrane-spanning C-terminal domain (ΔCT) was no longer able to interact with ATG4B (Figure 1A), suggesting that the interaction takes place within the membranal domain. The latter is consistent with earlier studies showing that RNF5 interactions with, and effects upon, its substrates require membrane anchoring [36].

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