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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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RNF-5 regulates autophagy in C. elegans.(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) rnf-5(tm794) 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 rnf-5(tm794) larvae, and 46 cells from 10 WT larvae, p<0.0001). (B) rnf-5(RNAi)-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 rnf-5(RNAi) larvae and 64 cells from 17 control larvae, p<0.0001). (C) Animals grown constantly at 25°C: hsp-16p::rnf-5 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 hsp-16p::rnf-5 larvae, and 68 cells from 15 non-transgenic larvae, p<0.0001).
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pgen-1003007-g004: RNF-5 regulates autophagy in C. elegans.(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) rnf-5(tm794) 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 rnf-5(tm794) larvae, and 46 cells from 10 WT larvae, p<0.0001). (B) rnf-5(RNAi)-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 rnf-5(RNAi) larvae and 64 cells from 17 control larvae, p<0.0001). (C) Animals grown constantly at 25°C: hsp-16p::rnf-5 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 hsp-16p::rnf-5 larvae, and 68 cells from 15 non-transgenic larvae, p<0.0001).

Mentions: We next asked if RNF5 also regulates autophagy in C. elegans. To accomplish this, we used rnf-5(tm794) deletion mutant that encodes a 36 amino-acid protein lacking the RNF5 RING domain, and thus predicted to be inactive [34]. rnf-5(tm794) L3 larvae expressing the autophagy marker GFP::LGG-1 had 2.6-fold more puncta in their seam cells compared to larvae on the WT background (Figure 4A, p<0.0001). This increase in GFP::LGG-1 puncta was also detected in worms treated with rnf-5(RNAi) (Figure 4B, 2.4-fold, p<0.0001). Conversely, animals expressing RNF-5 under the heat shock promoter [34] that were grown at 25°C (Figure 4C, conditions that increase the number of puncta in WT worms) resulted in decreased number of GFP::LGG-1 puncta in the seam cells of animals with elevated RNF-5 levels compared to the control animals at 25°C (2.2-fold; p<0.0001). These data suggest that RNF5 is a negative regulator of autophagy in C. elegans. Notably, degree of ER stress, measured via the hsp-4::gfp transcriptional reporter [47], confirmed that depletion of rnf-5does not exhibit increased hsp-4 transcription (Figure S8).


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)

RNF-5 regulates autophagy in C. elegans.(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) rnf-5(tm794) 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 rnf-5(tm794) larvae, and 46 cells from 10 WT larvae, p<0.0001). (B) rnf-5(RNAi)-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 rnf-5(RNAi) larvae and 64 cells from 17 control larvae, p<0.0001). (C) Animals grown constantly at 25°C: hsp-16p::rnf-5 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 hsp-16p::rnf-5 larvae, and 68 cells from 15 non-transgenic larvae, p<0.0001).
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pgen-1003007-g004: RNF-5 regulates autophagy in C. elegans.(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) rnf-5(tm794) 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 rnf-5(tm794) larvae, and 46 cells from 10 WT larvae, p<0.0001). (B) rnf-5(RNAi)-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 rnf-5(RNAi) larvae and 64 cells from 17 control larvae, p<0.0001). (C) Animals grown constantly at 25°C: hsp-16p::rnf-5 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 hsp-16p::rnf-5 larvae, and 68 cells from 15 non-transgenic larvae, p<0.0001).
Mentions: We next asked if RNF5 also regulates autophagy in C. elegans. To accomplish this, we used rnf-5(tm794) deletion mutant that encodes a 36 amino-acid protein lacking the RNF5 RING domain, and thus predicted to be inactive [34]. rnf-5(tm794) L3 larvae expressing the autophagy marker GFP::LGG-1 had 2.6-fold more puncta in their seam cells compared to larvae on the WT background (Figure 4A, p<0.0001). This increase in GFP::LGG-1 puncta was also detected in worms treated with rnf-5(RNAi) (Figure 4B, 2.4-fold, p<0.0001). Conversely, animals expressing RNF-5 under the heat shock promoter [34] that were grown at 25°C (Figure 4C, conditions that increase the number of puncta in WT worms) resulted in decreased number of GFP::LGG-1 puncta in the seam cells of animals with elevated RNF-5 levels compared to the control animals at 25°C (2.2-fold; p<0.0001). These data suggest that RNF5 is a negative regulator of autophagy in C. elegans. Notably, degree of ER stress, measured via the hsp-4::gfp transcriptional reporter [47], confirmed that depletion of rnf-5does not exhibit increased hsp-4 transcription (Figure S8).

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