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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 regulates autophagosome formation.(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.
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pgen-1003007-g003: RNF5 regulates autophagosome formation.(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.

Mentions: To determine whether this change in LC3 processing can be associated with the effect of RNF5 on ATG4B, we monitored changes in LC3 puncta in WT or ATG4B−/− MEFs in the presence of shRNF5. As shown in Figure 3A, inhibition of RNF5 expression increased the number of LC3 puncta (three times) in WT, but not in ATG4B−/− cells. Support for ATG4B-dependent effect of RNF5 on LC3 processing comes from the use of RNF5 that is truncated in its C-terminal transmembrane domain (ΔCT), a mutant that retains its E3 ligase activity, but no longer interacts with ATG4B (Figure 1A, Figure S3C). Ectopic expression of RNF5 ΔCT (Figure S6C, S6D) in RNF5−/− MEF did not reduce the number of LC3 puncta, as seen with the WT RNF5 (Figure S6C). These findings substantiate that the negative regulation of LC3 puncta by RNF5 occurs via its regulation of ATG4B.


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 regulates autophagosome formation.(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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3475677&req=5

pgen-1003007-g003: RNF5 regulates autophagosome formation.(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.
Mentions: To determine whether this change in LC3 processing can be associated with the effect of RNF5 on ATG4B, we monitored changes in LC3 puncta in WT or ATG4B−/− MEFs in the presence of shRNF5. As shown in Figure 3A, inhibition of RNF5 expression increased the number of LC3 puncta (three times) in WT, but not in ATG4B−/− cells. Support for ATG4B-dependent effect of RNF5 on LC3 processing comes from the use of RNF5 that is truncated in its C-terminal transmembrane domain (ΔCT), a mutant that retains its E3 ligase activity, but no longer interacts with ATG4B (Figure 1A, Figure S3C). Ectopic expression of RNF5 ΔCT (Figure S6C, S6D) in RNF5−/− MEF did not reduce the number of LC3 puncta, as seen with the WT RNF5 (Figure S6C). These findings substantiate that the negative regulation of LC3 puncta by RNF5 occurs via its regulation of ATG4B.

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