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ATM functions at the peroxisome to induce pexophagy in response to ROS

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Abstract

Peroxisomes are highly metabolic, autonomously replicating organelles that generate reactive oxygen species (ROS) as a by-product of fatty acid β-oxidation. Consequently, cells must maintain peroxisome homeostasis, or risk pathologies associated with too few peroxisomes, such as peroxisome biogenesis disorders, or too many peroxisomes, inducing oxidative damage and promoting diseases such as cancer. We report that the PEX5 peroxisome import receptor binds ataxia-telangiectasia mutated (ATM) and localizes this kinase to the peroxisome. In response to ROS, ATM signalling activates ULK1 and inhibits mTORC1 to induce autophagy. Specificity for autophagy of peroxisomes (pexophagy) is provided by ATM phosphorylation of PEX5 at Ser 141, which promotes PEX5 monoubiquitylation at Lys 209, and recognition of ubiquitylated PEX5 by the autophagy adaptor protein p62, directing the autophagosome to peroxisomes to induce pexophagy. These data reveal an important new role for ATM in metabolism as a sensor of ROS that regulates pexophagy.

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Figure 1: ATM kinase is localized at the peroxisome and activated in response to ROS.
Figure 2: PEX5 localizes ATM to the peroxisome.
Figure 3: Peroxisomal ROS activates ATM to repress mTORC1 and induce autophagy.
Figure 4: ATM phosphorylates PEX5 at Ser 141 in response to ROS.
Figure 5: Ser 141 regulates PEX5 ubiquitylation at Lys 209 in response to ROS.
Figure 6: Ubiquitylated PEX5 binds with the autophagy adaptor protein p62 in response to ROS.
Figure 7: Induction of pexophagy by ROS.
Figure 8: Working model for peroxisomal ATM signalling to the TSC signalling node to repress mTORC1, phosphorylation of PEX5 to induce ubiquitylation by PEX2/10/12 E3 ligase, and recognition of Ub–PEX5 by p62 to induce pexophagy in response to ROS.

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Acknowledgements

We thank S. Subramani (University of California, San Diego, California) for critical advice and providing the mRFP–EGFP–SKL plasmids, and M. Mancini (Baylor College of Medicine, Houston, Texas for providing the DsRed–SKL plasmid. We are also grateful for the assistance of K. Dunner (U.T.M.D. Cancer Center, Houston, Texas) and D. Townley (Baylor College of Medicine, Houston, Texas) in electron microscopy image acquisition. This work was supported by National Institutes of Health (NIH) Grant R01 CA143811 to C.L.W., a Robert A. Welch Endowed Chair in Chemistry (BE-0023) to C.L.W., and NIH grants CA129537, CA154320 and GM109768 to T.K.P. J.J. was a recipient of the China Scholarship Council (CSC).

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Authors

Contributions

J.Z., D.N.T., J.J. and C.L.W. designed research; J.Z., D.N.T., J.J., J.K., A.A., R.T.P., R.D., J.T.-M., J.-H.L., R.K.P. and V.K.C. performed research; J.Z., D.N.T., J.J., R.D., T.T.P., T.K.P., M.B.K. and C.L.W. analysed data; J.Z., D.N.T. and C.L.W. wrote the manuscript.

Corresponding author

Correspondence to Cheryl Lyn Walker.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 2 Response of ATM RQ mutant to oxidative and DNA damage.

(a) Subcellular fractionation of Zellweger (GM13267) fibroblasts demonstrating the localization of phospho-ATM (S1981) and ATM in response to H2O2 (0.4 mM, 1 h) in the indicated subcellular compartments. LDH, Lamin A/C and β-integrin were used as markers for cytosol (C), nuclear (N) and membrane (M) fractions, respectively. WCE, whole cell extract. (b) AT (GM05849) fibroblasts were transfected with either wild type (WT) or peroxisome localization-deficient R3047Q (RQ) mutant Flag-tagged ATM and treated with H2O2 (0.4 mM) for 1 h. ATM was immunoprecipitated using anti-Flag antibody and activation determined using phosphoserine S1981 specific antibody. (c and d) ATM kinase assay with MR complex, Nbs1, and DNA were performed with 0.2 nM dimeric ATM, 50 nM GST-p53 substrate, 96 nM MR, 195 nM Nbs1, and 140 nM linear double stranded DNA. Phosphorylation of ATM was detected by SDS-PAGE and western blotting with an antibody to p53 phospho-Ser15. (e) Frequency of γ-H2AX foci in AT cells (AT5, GM05849) and HepG2 cells + siATM at different time points post irradiation of 2 Gy. 100 cells were analyzed for each time point in each experiment (Mean ± s.d., n = 3 independent experiments, student’s t test). The mean number of foci is plotted against time. P < 0.05, P < 0.01. (f) Representative figures of γ-H2AX foci. A: Untreated cells; B: AT5 (GM05849) + ATM cells 30 min post irradiation; C: AT5 cells 30 min post irradiation; D: AT5 cells 180 min post irradiation; E: AT5 + ATM cells 180 min post irradiation; F: AT5 + ATM-R3047Q cells 180 min post irradiation. Scale bar, 10 μm. (g) Representative images of chromosome aberrations at metaphase. A: AT5 + ATM control metaphase; B: AT5 + ATM showing dicentric (big arrow) and breaks (small arrow); C: AT5 showing radials (big arrow) and breaks (small arrow); D: AT5 + ATM-R3047Q showing radials (big arrow) and breaks (small arrow). (h) Chromosome aberrations at metaphase post irradiation of 3 Gy in AT cells (AT5, GM05849) and HepG2 cells + siATM. For each group 50 metaphases were scored in each experiment (mean ± s.d., n = 3 independent experiments, student’s t test). P < 0.01. Uncropped images of western blots are shown in Supplementary Fig. 9. Statistical source data for Supplementary Fig. 1e, h can be found in Supplementary Table 1.

Supplementary Figure 3 Peroxisomal activation of ATM signaling to AMPK-TSC2 to repress mTORC1 and induce autophagy.

(a) FAO cells treated with clofibrate (0.25 mM) for 48 h activated PPARα to increase the mRNA level of EHHADH by RT–PCR analysis. The data shown is representative of two independent experiments. (b) FAO cells treated with clofibrate (0.25 mM) for 48 h activated PPARα to increase the ACAA1 mRNA levels as demonstrated by RT–PCR analysis. (mean ± s.d., n = 3 independent experiments, Student’s t test). P < 0.05, P < 0.01. (c) Western analysis of FAO cells treated with clofibrate at 0.25 mM for indicated times with ACAA1 and EHHADH antibodies. (d) MCF7 cells stably transfected with GFP–LC3 were treated with clofibrate (0.25 mM) for indicated times, and activation of ATM-AMPK-TSC2 signaling and induction of autophagy monitored by western analysis using pATM (S1981), ATM, pAMPK (T172), AMPK, pACC (S79), ACC, pS6K (T389), S6K, pS6 (S235/236), S6, p62 and LC3. (e) Representative images of GFP–LC3 puncta from MCF7 cells stably expressing GFP–LC3 and treated with clofibrate (0.25 mM) for 6 h. Scale bar, 10 μm. (f) HepG2 cells treated with 1 mM clofibrate for indicated times, were monitored for ATM-AMPK-TSC2 signaling, suppression of mTORC1, and induction of autophagy by western analysis using pATM (S1981), ATM, pAMPK (T172), AMPK, pACC (S79), ACC, pS6K (T389), S6K, pS6 (S235/236), S6, p4E-BP1 (T37/46), 4E-BP1, pULK1 (S757), pULK1 (S317), ULK1, p62 and LC3. (g) TSC2+/+ and TSC2−/− MEF cells were treated with clofibrate for 6 h at the indicated doses, and suppression of mTORC1 monitored by western analysis using pS6 (235/236), and S6. (h) Western analysis of FAO cells pre-incubated in the presence or absence of Bafilomycin A1 (Baf A1, 200 nM) for 1 h before treatment with 1 mM clofibrate for 3 h using anti-p62 and LC3-II antibodies. Uncropped images of western blots are shown in Supplementary Fig. 9, statistic source data for Supplementary Fig. 2a, b can be found in Supplementary Table 1.

Supplementary Figure 4 Pexophagy in FAO cells with clofibrate.

Representative electron microscopy images of FAO cells treated with vehicle or clofibrate. (a) vehicle 6 h, (b) clofibrate 6 h (1 mM) (c) vehicle 24 h, (d) clofibrate 24 h (0.25 mM), (e) clofibrate 48 h (0.25 mM), and (f) clofibrate 72 h (0.25 mM). Scale bar, 0.5 μm. Autophagosomes containing peroxisomes are represented by yellow arrows and shown enlarged in the boxed area (Scale bar, 0.2 μm).

Supplementary Figure 5 PEX5 contains ATM S*/T*Q phosphorylation motif.

(a) Prediction of PEX5 phosphorylation site and ubiquination site by ScanSite and PhosphoSite. (b) Conserved S141 (ATM phosphorylation site) sequence in PEX5. (c) WT fibroblasts (GM08399) and AT fibroblasts (GM05849) were transfected with Myc-PEX5-WT and treated with H2O2 for 1 h. The complex was immunoprecipitated with anti-Myc and immunoblotted with phospho-(S/T) ATM substrate antibody. Inputs were immunoblotted using the indicated antibodies. Uncropped images of western blots are shown in Supplementary Fig. 9.

Supplementary Figure 6 ROS-induced PEX5 ubiquitination is ATM-dependent.

(a) HEK293 cells expressing Myc-PEX5-WT or PEX5 mutants (K28R, K52R, K170R, K204R, K209R, K292R) and HA-Ub constructs were co-immunoprecipitated using anti-HA and blotting with an anti-Myc antibody. Input lysates were immunoblotted using the indicated antibodies. (b) WT (GM08399) and AT (GM05849) fibroblasts were treated with H2O2 for 6 h and immunoprecipitated with anti-PEX5 and immunoblotted with anti-Ub antibodies. Input lysates were immunoblotted using the indicated antibodies. (c) HEK293 cells transfected with Myc-PEX5-WT and HA-Ub for 24 h following a prior siRNA knockdown of ATM for 48 h, were treated with H2O2 for 6 h and immunoprecipitated with anti-HA and immunoblotted with anti-Myc antibodies. Input lysates were immunoblotted using the indicated antibodies. (d) HEK 293 cells were transfected with SiRNA for PEX2, PEX10 and PEX12, and treated with H2O2 for 6 h. Immunoprecipitation was performed with an anti-PEX5 antibody followed by immunoblotting for endogenous ubiqintin. RT- PCR was performed to confirm siRNA knockdown of PEX5, PEX10 and PEX12. (e) HEK293 cells transfected with Myc-PEX5-WT/Myc-PEX5-K209R and HA-Ub constructs for 24 h following a prior siRNA knockdown of PEX2/10/12 for 48 h were immunoprecipitated with anti-HA and immunoblotted with anti-Myc antibodies. Input lysates were immunoblotted using the indicated antibodies. Uncropped images of western blots are shown in Supplementary Fig. 9.

Supplementary Figure 7 K209 is responsible for PEX5 interaction with p62 in response to ROS.

(a) Representative image of HepG2 cells treated with or without H2O2 for 3 h and immunostained for FIP200 or ATG16L (green) and peroxisomal marker PMP70 (red). Scale bar, 10 μm. High-magnification images of boxed areas showed co-localization between FIP200 or ATG16L and PMP70 (Scale bar, 3 μm). (b) HEK293 cells were transfected with Myc-PEX5-WT and HA-p62, and treated with 0.4 mM of H2O2 for indicated times. The complex of HA-p62 and Myc-PEX5 was immunoprecipitated with anti-HA and immunoblotted with anti-Myc. Inputs were immunoblotted using the indicated antibodies. (c) HEK293 cells were transfected with HA-p62 and Myc-PEX5-WT or Myc-PEX5-K209R. The complex of HA-p62 and Myc-PEX5 was immunoprecipitated with anti-HA and immunoblotted with anti-Myc antibodies. Input lysates were immunoblotted using the indicated antibodies. Uncropped images of western blots are shown in Supplementary Fig. 9.

Supplementary Figure 8 Regulation of pexophagy by ROS is ATM-dependent.

(a) Frame capture from time course imaging of pexophagy in live RPE cells stably expressing GFP–LC3 and DsRed-SKL following H2O2 (0.4 mM) treatment. Scale bar, 10 μm. (b) Western analysis of HepG2 cells pre-incubated in the presence or absence of Bafilomycin A1 (Baf A1, 200 nM) for 1 h before treatment with 0.4 mM H2O2 for 3 h using anti-PEX1, anti-PEX14, anti-p62 and LC3-II antibodies. (c) Quantification of PEX1 and PEX14 intensity normalized to GAPDH from Supplementary Fig. 7b (mean ± s.d., n = 3 independent experiments, Student’s t test). P < 0.05, P < 0.01, P < 0.001, NS = not significant. (d) Western analysis of whole cell extracts (WCE) and peroxisome fractions (P) of HepG2 cells treated with Baf A1 (200 nM) for 4 h immunoblotted using anti-ATM, anti-pPEX5(S141) and PEX5 antibodies. (e) Western analysis of HepG2 cells transfected with or without sip62 for 72 h and treated with 0.4 mM H2O2 for 3 h using anti-PEX1, anti-PEX14, anti-pATM, anti-ATM, anti-p62 and LC3-II antibodies. (f) Quantification of PEX1 and PEX14 intensity normalized to GAPDH from Supplementary Fig. 7e (mean ± s.d., n = 3 independent experiments, Student’s t test). P < 0.01, P < 0.001, NS = not significant. (g) Representative images of wild type (WT, GM08399) and ataxia telengiectasia (AT, GM05849) patient fibroblasts treated with H2O2 for 6 h, and immunostained with PEX14 antibody. Scale bar, 15 μm. (h) Quantification of PEX14 puncta per cell was performed from Supplementary Fig. 7g from n = 4 independent experiments, 100 cells were analyzed in each experiment. All error bars represent s.d., (Student’s t test) P < 0.001, NS = not significant. (i) Western analysis of AT (GM05849) fibroblasts transfected with Flag-ATM WT or Flag-ATM KD (kinase dead) mutant and treated with 0.4 mM H2O2 for 6 h using anti-PEX1, anti-PEX14, anti-pATM, anti-Flag and GAPDH antibodies. (j) Western analysis of HEK293 cells transfected with Pex2/10/12 siRNA for 72 h and treated with H2O2 (0.4 mM) for 6 h using anti-PEX1, anti-PEX14, anti-pATM, ATM and GAPDH antibodies. Uncropped images of western blots are shown in Supplementary Fig. 9. Statistic source data for Supplementary Fig. 7c, f, h can be found in Supplementary Table 1.

Supplementary Figure 9 Pexophagy in HepG2 cells with H2O2.

Representative electron microscopy images of HepG2 cells treated with vehicle or H2O2. (a) Vehicle 6 h, (b,c and d) H2O2 6 h (0.4 mM). Scale bar, 0.5 μm. Autophagosomes containing peroxisome are represented by yellow arrows, and shown enlarged in the boxed area (Scale bar, 0.25 μm).

Supplementary Table 1 Source data table.

Supplementary information

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Formation of autophagosomes over peroxisome.

RPE cells stably expressing GFP–LC3 and DsRed-SKL and treated with H2O2 (0.4 mM, from 30 min to 3 h) imaged by time-lapse microcopy. (AVI 188 kb)

Induction of pexophagic flux with H2O2.

MCF7 cells transfected with mRFP-EGFP-SKL and treated with H2O2 (0.4 mM, from 30 min to 3 h) imaged by time-lapse microcopy. (AVI 873 kb)

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Zhang, J., Tripathi, D., Jing, J. et al. ATM functions at the peroxisome to induce pexophagy in response to ROS. Nat Cell Biol 17, 1259–1269 (2015). https://doi.org/10.1038/ncb3230

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