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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 negatively regulates autophagy.(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. Results show the quantification of endogenous LC3 puncta counted in >20 cells per experimental condition, in duplicate.
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pgen-1003007-g002: RNF5 negatively regulates autophagy.(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. Results show the quantification of endogenous LC3 puncta counted in >20 cells per experimental condition, in duplicate.

Mentions: To pursue this finding further, we monitored the effect of RNF5 on the appearance of different intracellular LC3 forms by western blotting. We monitored changes in the processing of LC3 in WT and RNF5 KO MEFs prior to, or 1, 2, and 4 h after treatment with HBSS (Figure 2A). Loss of RNF5 expression enriched in expression of the matured LC3 form, LC3-II, with the greatest difference being observed at the earlier time points (Figure 2A; control and 1 h, compared with 2 or 4 h). The latter is consistent with the finding that RNF5–ATG4B association is seen prior and at early time points following autophagy stimuli (Figure 1B). Neither GABARAP nor NBR1 expression were altered under the same conditions (Figure S6A). Notably, following LC3-II or LC3 decoration at later time points (6 and 8 h) did not reveal changes (not shown), probably since RNF5 affects to lesser degree the course of autophagy, once triggered. Inhibition of RNF5 led to the appearance of the processed LC3-II form in cells growing under normal conditions, and has increased the relative amount of LC3-II forms in cells that were subjected to starvation or after induction of ER stress with tunicamycin (Figure 2B). These data demonstrate that disruption of RNF5 expression affects LC3 processing under normal growth conditions, while also contributing to Atg4B availability following induction of autophagy. The level of LC3-II in both WT and RNF5 KO cells was elevated following treatment with the lysosomal inhibitors E64A and pepstatin A (Figure S6B). Similarly, degradation of the autophagy marker p62 was elevated upon inhibition or depletion of RNF5 expression (Figure 2A, 2B). Collectively, these data point to a role for RNF5 as a negative regulator of autophagy.


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 negatively regulates autophagy.(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. Results show the quantification of endogenous LC3 puncta counted in >20 cells per experimental condition, in duplicate.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475677&req=5

pgen-1003007-g002: RNF5 negatively regulates autophagy.(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. Results show the quantification of endogenous LC3 puncta counted in >20 cells per experimental condition, in duplicate.
Mentions: To pursue this finding further, we monitored the effect of RNF5 on the appearance of different intracellular LC3 forms by western blotting. We monitored changes in the processing of LC3 in WT and RNF5 KO MEFs prior to, or 1, 2, and 4 h after treatment with HBSS (Figure 2A). Loss of RNF5 expression enriched in expression of the matured LC3 form, LC3-II, with the greatest difference being observed at the earlier time points (Figure 2A; control and 1 h, compared with 2 or 4 h). The latter is consistent with the finding that RNF5–ATG4B association is seen prior and at early time points following autophagy stimuli (Figure 1B). Neither GABARAP nor NBR1 expression were altered under the same conditions (Figure S6A). Notably, following LC3-II or LC3 decoration at later time points (6 and 8 h) did not reveal changes (not shown), probably since RNF5 affects to lesser degree the course of autophagy, once triggered. Inhibition of RNF5 led to the appearance of the processed LC3-II form in cells growing under normal conditions, and has increased the relative amount of LC3-II forms in cells that were subjected to starvation or after induction of ER stress with tunicamycin (Figure 2B). These data demonstrate that disruption of RNF5 expression affects LC3 processing under normal growth conditions, while also contributing to Atg4B availability following induction of autophagy. The level of LC3-II in both WT and RNF5 KO cells was elevated following treatment with the lysosomal inhibitors E64A and pepstatin A (Figure S6B). Similarly, degradation of the autophagy marker p62 was elevated upon inhibition or depletion of RNF5 expression (Figure 2A, 2B). Collectively, these data point to a role for RNF5 as a negative regulator of autophagy.

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