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PARL mediates Smac proteolytic maturation in mitochondria to promote apoptosis

Abstract

Mitochondria drive apoptosis by releasing pro-apoptotic proteins that promote caspase activation in the cytosol. The rhomboid protease PARL, an intramembrane cleaving peptidase in the inner membrane, regulates mitophagy and plays an ill-defined role in apoptosis. Here, we employed PARL-based proteomics to define its substrate spectrum. Our data identified the mitochondrial pro-apoptotic protein Smac (also known as DIABLO) as a PARL substrate. In apoptotic cells, Smac is released into the cytosol and promotes caspase activity by inhibiting inhibitors of apoptosis (IAPs). Intramembrane cleavage of Smac by PARL generates an amino-terminal IAP-binding motif, which is required for its apoptotic activity. Loss of PARL impairs proteolytic maturation of Smac, which fails to bind XIAP. Smac peptidomimetics, downregulation of XIAP or cytosolic expression of cleaved Smac restores apoptosis in PARL-deficient cells. Our results reveal a pro-apoptotic function of PARL and identify PARL-mediated Smac processing and cytochrome c release facilitated by OPA1-dependent cristae remodelling as two independent pro-apoptotic pathways in mitochondria.

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Figure 1: Proteomic identification of PARL substrates.
Figure 2: Two-step processing of Smac in mitochondria.
Figure 3: Loss of PARL confers apoptotic resistance.
Figure 4: Cytochrome c but not Smac is released from PARL-deficient mitochondria to the cytosol during apoptosis.
Figure 5: PARL cleavage allows the release of Smac and PGAM5 from mitochondria following apoptotic induction.
Figure 6: Loss of PARL prevents interaction between Smac and XIAP in the cytosol.
Figure 7: PARL-driven apoptosis depends on XIAP inhibition.
Figure 8: Cytosolic Smac promotes apoptosis in PARL−/− cells.

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Acknowledgements

We thank E. Barth for technical support in electron microscopy and other laboratory members for their support. This work was supported a Japan Society for the Promotion of Science (JSPS) Fellowship for Research Abroad to S.S. and a Reinhart Koselleck grant of the Deutsche Forschungsgemeinschaft to T.L. R.P.Z. and A.S.V. thank the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Senatsverwaltung für Wirtschaft, Technologie und Forschung des Landes Berlin and the Bundesministerium für Bildung und Forschung for financial support. We thank B. de Strooper (VIB, Leuven) for the Parl−/− MEFs and E. Shoubridge (McGill University, Montreal) for the pBABE-puro vector.

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Contributions

S.S. and T.L. designed the research. S.S. and K.U.F. performed the experiments. H.N. and M.K. performed the mass spectrometric analysis for differential proteomics. A.S.V. and R.P.Z. performed the ChaFRADIC analysis. H.K. provided essential tools and reagents. S.S. and T.L. wrote the manuscript with the comments of the other authors.

Corresponding author

Correspondence to Thomas Langer.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 PARL-based proteomics.

(a) Proteomic strategies for the identification of PARL substrate proteins in mitochondria. Left panel; determination of N-terminal peptides of mitochondrial proteins quantitative affinity purification proteomics. (b) iTRAQ based ratios were log2 transformed and biological replicates were plotted against each other. Reproducibility was determined by Pearson correlation coefficient: r > 0.4 (after filtering proteins with higher standard deviations than 99% of the complete population). Proteins that were found with extreme ratios (>0.99 quantile) are highlighted by their gene name. (c) Isolation of mitochondria from wild type (WT) and PARL−/− HEK293 cells by Percoll gradient centrifugation. Fraction 3 was collected and analysed by ChaFRADIC (Supplementary Table 1). (d) SDS-PAGE and immunoblot analysis of cellular fractions of wild type (WT) and PARL−/− HEK293 cells. p, precursor; i, intermediate; m, mature form. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 2 Characterization of Smac/DIABLO in PARL deficient cells.

(a) Fractionation of wild type (WT) and PARL−/− HEK293 cells into post nuclear (PNS), mitochondrial (mito), microsomal and cytosolic fractions. Samples were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature. (b) siRNA-mediated downregulation of Smac/DIABLO in wild type (WT) and PARL−/− HEK293 cells for 72 h (or with control siRNA). Cell lysates were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature. (c) Transient expression of Smac/DIABLOHA in wild type (WT) and PARL−/− (KO) HEK293 cells for 24 h (reagent 1, GeneJuice; reagent 2, Lipofectamine 2000). Cell lysates were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature. (d) Quantification of different Smac/DIABLO forms in wild type (WT) and PARL−/− HEK293 cells expressing PARLFLAG or PARLS277A−FLAG when indicated. Left panel; cell lysates were analysed by SDS-PAGE and immunoblotting. Right panel; quantification of fluorescence intensities using the infrared Odyssey System (n = 3 independent experiments; mean values ± SD). p, precursor; i, intermediate; m, mature. (eg) SDS-PAGE and immunoblot analysis of cell extracts of wild type (WT) and PARL-deficient (KO) MEFs (e), HeLa cells (f), HCT116 (g) cells. Different clones of PARL−/− HeLa and HCT116 cells were analysed. ae, OPA1 forms, p, precursor; i, intermediate; m, mature form. unspecific cross-reaction. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 3 Processing of Smac/DIABLO.

(a) Hydropathy plot of the N-terminal 90 amino acids of Smac/DIABLO. The red bar indicates a predicted transmembrane domain (W42–A60). (b) Accumulation of the intermediate form of Smac/DIABLO upon downregulation of MPP in wild type (WT) and PARL−/− HEK293 cells. siRNAs directed against α- and β-Mpp (or control siRNA) were transfected for 72 h. Cell lysates were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature form. unspecific cross-reaction. (c) siRNA-mediated downregulation of MPP in wild type (WT) and PARL−/− HEK293 cells expressing Smac/DIABLOHA (for 24 h) when indicated. Cell lysates were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature form. unspecific cross-reaction. (d) Recombinant Smac/DIABLOHA (synthesized in rabbit reticulocyte lysate) was incubated for 1 h at 30 °C with mitochondrial extracts of wild type (WT) and PARL−/− HEK293 cells in the absence or presence of CCCP, o-phenanthroline, DCI, PMSF or cOmplete inhibitor mix. Samples were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature form. (e,f) Downregulation of (e) IMMPL1/2L or (f) HTRA2/Omi in PARL wild type (WT) and PARL−/− HEK293 cells for 72 h (or with control siRNA). Cell lysates were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature form. unspecific cross-reaction. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 4 Increased apoptotic resistance of PARL-deficient MEFs and HeLa cells.

(a) Total RNA was extracted from wild type (WT) and PARL−/− HeLa or HCT116 cells complemented with PARLFLAG or PARLS277A−FLAG and subjected to RT and real-time PCR analysis of human PARL mRNA. (n = 3 independent experiments; mean values ± SD). (b) Wild type (WT) and Parl−/− MEFs were treated for 8 h with staurosporine (STS, 1 μM), actinomycin D (Act D, 1 μM), or a combination of TNFα (10 ng ml−1) and CHX (10 μg ml−1). Cell lysates were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature form. unspecific cross-reaction. (c) Quantification of PARP cleavage of experiments in b (n = 3 independent experiments; mean values ± s.d.). P values were calculated by one-way ANOVA and indicated in figure. A P value of <0.05 was considered statistically significant. (d) Viability of wild type and Parl−/− MEFs (complemented with PARLFLAG when indicated) after incubation for 8 h with staurosporine (STS, 0.1 μM) (n = 3 independent experiments; mean values ± s.d.). Cell viability was assessed monitoring cellular ATP levels. P values were calculated by one-way ANOVA and indicated in figure. A P value of <0.05 was considered statistically significant. (e) Relative confluency of wild type (WT) and PARL−/− HeLa cells in the presence of actinomycin D (Act D, 1 μM). Images of the cells after 16 h Act D treatment are shown in the lower panel. Scale bars, 100 μm. Unprocessed original scans of blots are shown in Supplementary Fig. 9. Statistical source data can be found in Supplementary Table 3.

Supplementary Figure 5 The loss of PARL does not affect mitochondrial morphology.

(a) Immunofluorescence analysis of wild type (WT) and PARL−/− HEK293 cells using antibodies directed against ATP5β (green) and TOMM20 (red), and examined by confocal microscopy. Higher-magnification views of the boxed areas were shown. Scale bars, 10 μm or 2 μm (magnified image). (b,c) Immunofluorescence analysis of wild type (WT) and PARL−/− HCT116 cells (b), and HeLa cells (c) using antibodies directed against TOMM20 (green) and ATP5β (red), and examined by confocal microscopy. Higher magnifications of the boxed areas are shown. Scale bars, (b), 10 μm or 5 μm (magnified image); (c) 20 μm or 5 μm (magnified image). (d) Transmission electron microscopic analysis of wild type (WT) and PARL−/− HeLa cells (complemented with PARLFLAG when indicated). Scale bars, 1 μm. (e) BAX oligomerization in PARL-deficient HCT116 cells. Wild type (WT) and PARL−/− HCT116 cells were incubated with TNFα (20 ng ml−1) and CHX (10 μg ml−1) for 6 h. Mitochondria were isolated and analysed by BN-PAGE or SDS-PAGE, followed by immunoblotting with the indicated antibodies. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 6 Quantification of XIAP mRNA and protein in human and mouse cell lines.

(a) Immunoblot analysis of cell lysates of wild type (WT) or PARL−/− cells using the indicated antibodies. unspecific cross-reaction. (b) Quantification of experiments in c (n = 3 independent experiments; mean values ± s.d.). P values were calculated by one-way ANOVA and indicated in figure. A P value of <0.05 was considered statistically significant. Statistical source data can be found in Supplementary Table 3. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 7 Depletion of XIAP restores apoptosis in PARL-deficient cells.

(a) After transfection with Xiap (or with control) siRNA for 48 h, wild type (WT) and PARL−/− (KO) HCT116 cells were incubated with TNFα (20 ng ml−1) and CHX (10 μg ml−1). Cell lysates were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature form. unspecific cross-reaction. (b) Quantification of PARP cleavage in experiments described in a (n = 3 independent experiments); mean values ± s.d.). P values were calculated by one-way ANOVA and indicated in figure. A P value of <0.05 was considered statistically significant. Unprocessed original scans of blots are shown in Supplementary Fig. 9. Statistical source data can be found in Supplementary Table 3.

Supplementary Figure 8 Cytosolic, mature Smac/DIABLO is sufficient to drive apoptosis in PARL−/− cells.

(a, left) Wild type (WT) and PARL−/− HCT116 cells were incubated with TNFα (20 ng ml−1) and CHX (10 μg ml−1) for 6 h in the absence or presence of the Smac/DIABLO mimetic BV6 (1 μM). (a, right) Wild type (WT) and PARL−/− HeLa cells were incubated with staurosporine (STS, 0.1 μM) for 6 h in the absence or presence of the Smac/DIABLO mimetic BV6 (2.5 μM). p, precursor; i, intermediate; m, mature form. unspecific cross-reaction. Unprocessed original scans of blots are shown in Supplementary Fig. 9. (b) Smac/DIABLO-deficient HeLa cells were generated by CRISPR/Cas9-mediated genome editing. Variants of Smac/DIABLO predicted to be expressed are shown. The red asterisk indicates the CRISPR/Cas9 targeted site. (c) Domain organization of Ubiquitin-Smac/DIABLO (Δ55)-DsRed and Ubiquitin-Smac/DIABLO (Δ59)-DsRed. (d) Two pathways for the release of pro-apoptotic proteins from mitochondria. BAX/BAK-induced apoptosis requires PARL-mediated processing and cytosolic translocation of Smac/DIABLO and the release of cytochrome c, which is facilitated by OPA1-dependent cristae remodeling and cardiolipin oxidation.

Supplementary information

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Supplementary Table 1

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Supplementary Information (XLSX 31 kb)

Act D treated HeLa cells (related to Supplementary Fig. 4e).

Relative confluency of wild type (WT) HeLa cells in the presence of actinomycin D (Act D, 1 μM). Images were taken every 5 min for 16 h. (MOV 7923 kb)

Act D treated PARL−/− HeLa cells (related to Supplementary Fig. 4e).

Relative confluency of PARL−/− (KO) HeLa cells in the presence of actinomycin D (Act D, 1 μM). Images were taken every 5 min for 16 h. (MOV 10865 kb)

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Saita, S., Nolte, H., Fiedler, K. et al. PARL mediates Smac proteolytic maturation in mitochondria to promote apoptosis. Nat Cell Biol 19, 318–328 (2017). https://doi.org/10.1038/ncb3488

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