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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 affects engulfment of GAS in autophagosomes.(A) Immunoblotting analysis of LC3 and p62 in RNF5 WT and KO macrophages maintained in normal or HBSS-containing media. (B) Subcellular localization of LC3 and ATG4B in macrophages of RNF5 WT and KO mice. Cell extracts were prepared from bone marrow-derived macrophages and fractionated using sucrose gradient ultracentrifugation. Fractions (40 µg protein/lane) enriched for endoplasmic reticulum (ER), mitochondria (Mito), or cytosolic (Cyt) were resolved and analyzed by western blotting with the indicated antibodies. Markers representing each of the organelles are shown. (C) Confocal microscopic images of GFP-labeled GAS (green) and immunostained LC3 (red) depicts representative images of GAS contained in LC3-positive compartments (arrowheads) in bone marrow-derived macrophages from WT and RNF5 KO mice (DNA labeling by DAPI in blue). Bone marrow-derived macrophages were seeded on glass coverslips (5×104/well in 24-well plates) and the following day infected with GAS (MOI = 10) for 2.5 h before being washed and fixed in 3% PFA. Cells were processed for immunofluorescence microscopy using affinity-purified rabbit anti-LC3 IgG, followed by goat anti-rabbit Alexa-488 F(ab′)2, and DAPI to stain the bacterial and cellular DNA. (D) Quantification of GAS associated with LC3-positive autophagosomes in macrophages of WT and RNF5 KO mice. RNF5 KO macrophages contain more GAS engulfed by LC3-decorated structures per cell (I, n = 20, P = 0.077) and more LC3 puncta per cell (II, n = 20, P<0.05). Graphs show the mean ± SE, P value was calculated by Student t-test. Images were acquired on an Olympus FluoView 1000 confocal microscope using a 100× oil immersion lens. (E) EM-based ultrastructural analysis of GAS-infected macrophages from WT (I) and RNF5 KO (II) mice. Within 2.5 h after macrophage infection, most GAS are engulfed within autophagosome-like vacuoles (arrowheads). Scale bar = 2 µm (I).
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pgen-1003007-g006: RNF5 affects engulfment of GAS in autophagosomes.(A) Immunoblotting analysis of LC3 and p62 in RNF5 WT and KO macrophages maintained in normal or HBSS-containing media. (B) Subcellular localization of LC3 and ATG4B in macrophages of RNF5 WT and KO mice. Cell extracts were prepared from bone marrow-derived macrophages and fractionated using sucrose gradient ultracentrifugation. Fractions (40 µg protein/lane) enriched for endoplasmic reticulum (ER), mitochondria (Mito), or cytosolic (Cyt) were resolved and analyzed by western blotting with the indicated antibodies. Markers representing each of the organelles are shown. (C) Confocal microscopic images of GFP-labeled GAS (green) and immunostained LC3 (red) depicts representative images of GAS contained in LC3-positive compartments (arrowheads) in bone marrow-derived macrophages from WT and RNF5 KO mice (DNA labeling by DAPI in blue). Bone marrow-derived macrophages were seeded on glass coverslips (5×104/well in 24-well plates) and the following day infected with GAS (MOI = 10) for 2.5 h before being washed and fixed in 3% PFA. Cells were processed for immunofluorescence microscopy using affinity-purified rabbit anti-LC3 IgG, followed by goat anti-rabbit Alexa-488 F(ab′)2, and DAPI to stain the bacterial and cellular DNA. (D) Quantification of GAS associated with LC3-positive autophagosomes in macrophages of WT and RNF5 KO mice. RNF5 KO macrophages contain more GAS engulfed by LC3-decorated structures per cell (I, n = 20, P = 0.077) and more LC3 puncta per cell (II, n = 20, P<0.05). Graphs show the mean ± SE, P value was calculated by Student t-test. Images were acquired on an Olympus FluoView 1000 confocal microscope using a 100× oil immersion lens. (E) EM-based ultrastructural analysis of GAS-infected macrophages from WT (I) and RNF5 KO (II) mice. Within 2.5 h after macrophage infection, most GAS are engulfed within autophagosome-like vacuoles (arrowheads). Scale bar = 2 µm (I).

Mentions: Given the demonstration that RNF5 controls basal autophagy activity, and the fact that autophagy levels are associated with altered susceptibility to intracellular bacterial replication [8], we next asked if RNF5 WT and KO mice exhibit differences in their response to bacterial challenge. In these studies, we employed the leading human pathogen GAS, previously identified to be susceptible to autophagy-mediated intracellular clearance [9]. Bone marrow-derived macrophages isolated from RNF5−/− mice demonstrated increased levels of LC3, ATG4B, and decreased level of p62 compared to RNF5+/+ macrophages (Figure 6A), suggesting they had a higher basal level of autophagy. Furthermore, levels of ATG4B and LC3 were elevated in mitochondrial and ER-enriched fractions of RNF−/− macrophages compared to RNF5+/+ macrophages (Figure 6B). These observations are consistent with membrane anchoring of RNF5 and suggest that RNF5 limits ATG4B-dependent LC3 processing in both mitochondrial and ER membranes. Autophagy-deficient (ATG5−/−) cells have been shown to be more susceptible to GAS intracellular proliferation [9]. In agreement with those findings, we found higher numbers of LC3 puncta in in RNF5−/− macrophages infected with GAS than RNF5+/+ macrophages (Figure 6C, 6D), pointing to a more efficient bacterial processing by autophagy in the absence of RNF5. Ultrastructural analysis of GAS-infected macrophages using EM confirmed that higher numbers of bacteria were engulfed in RNF5−/− macrophages than in WT cells (Figure 6E).


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 affects engulfment of GAS in autophagosomes.(A) Immunoblotting analysis of LC3 and p62 in RNF5 WT and KO macrophages maintained in normal or HBSS-containing media. (B) Subcellular localization of LC3 and ATG4B in macrophages of RNF5 WT and KO mice. Cell extracts were prepared from bone marrow-derived macrophages and fractionated using sucrose gradient ultracentrifugation. Fractions (40 µg protein/lane) enriched for endoplasmic reticulum (ER), mitochondria (Mito), or cytosolic (Cyt) were resolved and analyzed by western blotting with the indicated antibodies. Markers representing each of the organelles are shown. (C) Confocal microscopic images of GFP-labeled GAS (green) and immunostained LC3 (red) depicts representative images of GAS contained in LC3-positive compartments (arrowheads) in bone marrow-derived macrophages from WT and RNF5 KO mice (DNA labeling by DAPI in blue). Bone marrow-derived macrophages were seeded on glass coverslips (5×104/well in 24-well plates) and the following day infected with GAS (MOI = 10) for 2.5 h before being washed and fixed in 3% PFA. Cells were processed for immunofluorescence microscopy using affinity-purified rabbit anti-LC3 IgG, followed by goat anti-rabbit Alexa-488 F(ab′)2, and DAPI to stain the bacterial and cellular DNA. (D) Quantification of GAS associated with LC3-positive autophagosomes in macrophages of WT and RNF5 KO mice. RNF5 KO macrophages contain more GAS engulfed by LC3-decorated structures per cell (I, n = 20, P = 0.077) and more LC3 puncta per cell (II, n = 20, P<0.05). Graphs show the mean ± SE, P value was calculated by Student t-test. Images were acquired on an Olympus FluoView 1000 confocal microscope using a 100× oil immersion lens. (E) EM-based ultrastructural analysis of GAS-infected macrophages from WT (I) and RNF5 KO (II) mice. Within 2.5 h after macrophage infection, most GAS are engulfed within autophagosome-like vacuoles (arrowheads). Scale bar = 2 µm (I).
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pgen-1003007-g006: RNF5 affects engulfment of GAS in autophagosomes.(A) Immunoblotting analysis of LC3 and p62 in RNF5 WT and KO macrophages maintained in normal or HBSS-containing media. (B) Subcellular localization of LC3 and ATG4B in macrophages of RNF5 WT and KO mice. Cell extracts were prepared from bone marrow-derived macrophages and fractionated using sucrose gradient ultracentrifugation. Fractions (40 µg protein/lane) enriched for endoplasmic reticulum (ER), mitochondria (Mito), or cytosolic (Cyt) were resolved and analyzed by western blotting with the indicated antibodies. Markers representing each of the organelles are shown. (C) Confocal microscopic images of GFP-labeled GAS (green) and immunostained LC3 (red) depicts representative images of GAS contained in LC3-positive compartments (arrowheads) in bone marrow-derived macrophages from WT and RNF5 KO mice (DNA labeling by DAPI in blue). Bone marrow-derived macrophages were seeded on glass coverslips (5×104/well in 24-well plates) and the following day infected with GAS (MOI = 10) for 2.5 h before being washed and fixed in 3% PFA. Cells were processed for immunofluorescence microscopy using affinity-purified rabbit anti-LC3 IgG, followed by goat anti-rabbit Alexa-488 F(ab′)2, and DAPI to stain the bacterial and cellular DNA. (D) Quantification of GAS associated with LC3-positive autophagosomes in macrophages of WT and RNF5 KO mice. RNF5 KO macrophages contain more GAS engulfed by LC3-decorated structures per cell (I, n = 20, P = 0.077) and more LC3 puncta per cell (II, n = 20, P<0.05). Graphs show the mean ± SE, P value was calculated by Student t-test. Images were acquired on an Olympus FluoView 1000 confocal microscope using a 100× oil immersion lens. (E) EM-based ultrastructural analysis of GAS-infected macrophages from WT (I) and RNF5 KO (II) mice. Within 2.5 h after macrophage infection, most GAS are engulfed within autophagosome-like vacuoles (arrowheads). Scale bar = 2 µm (I).
Mentions: Given the demonstration that RNF5 controls basal autophagy activity, and the fact that autophagy levels are associated with altered susceptibility to intracellular bacterial replication [8], we next asked if RNF5 WT and KO mice exhibit differences in their response to bacterial challenge. In these studies, we employed the leading human pathogen GAS, previously identified to be susceptible to autophagy-mediated intracellular clearance [9]. Bone marrow-derived macrophages isolated from RNF5−/− mice demonstrated increased levels of LC3, ATG4B, and decreased level of p62 compared to RNF5+/+ macrophages (Figure 6A), suggesting they had a higher basal level of autophagy. Furthermore, levels of ATG4B and LC3 were elevated in mitochondrial and ER-enriched fractions of RNF−/− macrophages compared to RNF5+/+ macrophages (Figure 6B). These observations are consistent with membrane anchoring of RNF5 and suggest that RNF5 limits ATG4B-dependent LC3 processing in both mitochondrial and ER membranes. Autophagy-deficient (ATG5−/−) cells have been shown to be more susceptible to GAS intracellular proliferation [9]. In agreement with those findings, we found higher numbers of LC3 puncta in in RNF5−/− macrophages infected with GAS than RNF5+/+ macrophages (Figure 6C, 6D), pointing to a more efficient bacterial processing by autophagy in the absence of RNF5. Ultrastructural analysis of GAS-infected macrophages using EM confirmed that higher numbers of bacteria were engulfed in RNF5−/− macrophages than in WT cells (Figure 6E).

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