The Cys-N-degron pathway modulates pexophagy through the N-terminal oxidation and arginylation of ACAD10

Abstract

In the N-degron pathway, N-recognins recognize cognate substrates for degradation via the ubiquitin (Ub)-proteasome system (UPS) or the autophagy-lysosome system (hereafter autophagy). We have recently shown that the autophagy receptor SQSTM1/p62 (sequestosome 1) is an N-recognin that binds the N-terminal arginine (Nt-Arg) as an N-degron to modulate autophagic proteolysis. Here, we show that the N-degron pathway mediates pexophagy, in which damaged peroxisomal fragments are degraded by autophagy under normal and oxidative stress conditions. This degradative process initiates when the Nt-Cys of ACAD10 (acyl-CoA dehydrogenase family, member 10), a receptor in pexophagy, is oxidized into Cys sulfinic (Cys^O2) or sulfonic acid (Cys^O3) by ADO (2-aminoethanethiol (cysteamine) dioxygenase). Under oxidative stress, the Nt-Cys of ACAD10 is chemically oxidized by reactive oxygen species (ROS). The oxidized Nt-Cys2 is arginylated by ATE1-encoded R-transferases, generating the RC^OX N-degron. RC^OX-ACAD10 marks the site of pexophagy via the interaction with PEX5 and binds the ZZ domain of SQSTM1/p62, recruiting LC3⁺-autophagic membranes. In mice, knockout of either Ate1 responsible for Nt-arginylation or Sqstm1/p62 leads to increased levels of peroxisomes. In the cells from patients with peroxisome biogenesis disorders (PBDs), characterized by peroxisomal loss due to uncontrolled pexophagy, inhibition of either ATE1 or SQSTM1/p62 was sufficient to recover the level of peroxisomes. Our results demonstrate that the Cys-N-degron pathway generates an N-degron that regulates the removal of damaged peroxisomal membranes along with their contents. We suggest that tannic acid, a commercially available drug on the market, has a potential to treat PBDs through its activity to inhibit ATE1 R-transferases.Abbreviations: ACAA1, acetyl-Coenzyme A acyltransferase 1; ACAD, acyl-Coenzyme A dehydrogenase; ADO, 2-aminoethanethiol (cysteamine) dioxygenase; ATE1, arginyltransferase 1; CDO1, cysteine dioxygenase type 1; ER, endoplasmic reticulum; LIR, LC3-interacting region; MOXD1, monooxygenase, DBH-like 1; NAC, N-acetyl-cysteine; Nt-Arg, N-terminal arginine; Nt-Cys, N-terminal cysteine; PB1, Phox and Bem1p; PBD, peroxisome biogenesis disorder; PCO, plant cysteine oxidase; PDI, protein disulfide isomerase; PTS, peroxisomal targeting signal; R-COX, Nt-Arg-CysOX; RNS, reactive nitrogen species; ROS, reactive oxygen species; SNP, sodium nitroprusside; UBA, ubiquitin-associated; UPS, ubiquitinproteasome system.

Keywords: Acyl-CoA dehydrogenase family, member 10; N-degron pathway; oxidative stress; peroxisomal biogenesis disorders; peroxisome; pexophagy.

Figure 1.

The Nt-Cys generates an autophagic degron via oxidation and arginylation for pexophagy. (A) Generation of antibodies that detect the RC^OX motif. (B) Dot blot analysis of RC^OX antibodies using 100 ng antigen peptides (red: strong signals, blue: weak signals). (C) HeLa cells treated with 200 µm sodium nitroprusside (SNP) were subjected to immunostaining with anti-RC^OX antibody. Scale bar: 10 μm. (D and E) HeLa cells were treated with 400 nM bafilomycin A₁ for 16 h and/or 1 mM N-acetyl-cysteine (NAC) (D) or 40 µm tannic acid (TA) (E) for 24 h, followed by immunostaining with anti-RC^OX antibody. Scale bar: 10 μm. (F) HeLa cells treated with bafilomycin A₁ were analyzed using immunostaining with anti-RC^OX antibody. Scale bar: 10 μm. (G) HeLa cells treated with bafilomycin A₁ were subjected to co-immunostaining using antibodies to RC^OX proteins in comparison with ABCD3 (left panel). Scale bar: 10 μm. The colocalization was quantified using the Pearson’s correlation coefficient (middle panel) or the Manders’ colocalization coefficient (right panel) (n = 4, ***P < 0.001).

Figure 2.

ACAD10 modulates pexophagy through oxidation and arginylation of its Nt-Cys2. (A and B) Immunoblotting of HeLa cells treated with tannic acid (A) or transfected with ATE1 siRNA for 48 h (B). (C and D) Immunostaining of HeLa cells treated with tannic acid (C) or transfected with ATE1 siRNA for 48 h (D). Scale bar: 10 μm. Quantification of ABCD3 intensity (n = 30, **P < 0.01). (E) HeLa cells overexpressing either of mouse ATE1 isoforms, ATE1-1A7A, ATE1-1A7B ATE1-1B7A, or ATE1-1B7B for 48 h were analyzed using immunoblotting. (F and G) Immunoblotting analyses of HeLa cells transiently expressing ATE1^K417A mutant (F) or ATE1-1A7A for 48 h in the presence of bafilomycin A₁ (G). (H) Schematic diagram of screening 350 Met-Cys proteins. Cells were transfected with 93 different siRNAs targeting Nt-Cys2 proteins, followed by immunostaining with RC^OX antibody and confocal microscopy. (I) Sequences of JUNB, GPR22, MOXD1, and ACAD10. (J-L) Immunoblotting analyses of cells treated with SNP for indicated time (J), 20 μM MG132 and/or bafilomycin A₁ for 6 h (K), or tannic acid for 24 h (L). Quantification of ACAD10 intensity (n = 4, *P < 0.05; **P < 0.01; ***P < 0.001).

Figure 3.

The Nt-Cys2 of ACAD10 is a sensor of O₂ and ROS in pexophagy. (A) Immunoblotting analyses of HeLa cells transfected with ADO and CDO1 siRNA for 48 h. Quantification of band intensities (n = 4, *P < 0.05). (B) HeLa cells transfected with ADO siRNA in the presence of cycloheximide (CHX) were analyzed using immunoblotting. Shown below is the relative quantification of band intensities (n = 3, *P < 0.05). (C) HeLa cells transfected with ADO or CDO1 siRNA in the presence of SNP were subject to immunoblotting. Shown below is the relative quantification of band intensities (n = 3, **P < 0.01). (D) Immunoblotting analyses of HeLa cells treated with SNP for 1 h in the presence of NAC. (E and F) Immunoblotting analyses of cells treated with SNP for 1 h in the presence of tannic acid (E) or ATE1 siRNA (F). (G) Immunoblotting analyses of HeLa cells overexpressing either of mouse ATE1 isoforms, ATE1-1A7A, ATE1-1A7B, ATE1-1B7A, or ATE1-1B7B for 48 h in the presence of bafilomycin A₁. (H and I) Immunoblotting analyses of HeLa cells treated with SNP for 1 h in the presence of bafilomycin A₁ (H) or MG132 (I). (J-L) HeLa cells were transfected with wild type (WT) or ACAD10^C2V in the presence of cycloheximide (CHX) (J), tannic acid (K), or bafilomycin A₁ (L), followed by immunoblotting analyses. (M-O) Immunoblotting analyses of peroxisomal proteins in cells overexpressing ACAD10 for 48 h in the absence (M and N) or presence of 40 μM tannic acid (O) or 400 nM bafilomycin A₁ (P). (Q) HeLa cells were transfected with two different siRNAs targeting ACAD10, followed by immunoblotting. (R and S) HeLa cells were transfected with pCMV14 empty vector, wild type ACAD10, or ACAD10^C2V for 48 h in the presence of 40 μM tannic acid (R) or 400 nM bafilomycin A₁ (S), followed by immunoblotting.

Figure 4.

The RC^OX of ACAD10 is required for peroxisomal targeting during oxidative stress. (A) HeLa cells treated with SNP and bafilomycin A₁ for 6 h were fractionated. Whole cell lysates (WCL) in comparison with membrane (including mitochondria), cytosolic, and peroxisomal fractions were analyzed by immunoblotting (upper panel). Quantification was shown (lower panel) (n = 3, **P < 0.01). (B) Immunostaining analyses of HeLa cells treated with SNP-treated HeLa cells (upper) or ACAA1 plasmid transfected cells (lower), followed by immunostaining. Scale bar: 10 μm. (C) Quantification of colocalization data shown in (B) using the Pearson’s correlation coefficient (left panel) or the Manders’ colocalization coefficient (right panel) (n = 40, **P < 0.01). (D) HeLa cells transfected with wild type ACAD10, ACAD10^K1052 G or ACAD10^C2V were fractionated. Whole cell lysates in comparison with membrane (including mitochondria), cytosolic, and peroxisomal fractions were analyzed by immunoblotting (upper panel). Quantification was shown (lower panel) (n = 3, **P < 0.01; ***P < 0.001). (E) Immunostaining analyses of HeLa cells were transfected with wild type or C2V mutant of ACAD10 in the presence of SNP. Scale bar: 10 μm. (F) Quantification of colocalization data shown in (E) using the Pearson’s correlation coefficient (left panel) or the Manders’ colocalization coefficient (right panel) (n = 40, **P < 0.01, ns, not significant).

Figure 5.

ACAD10 binds PEX5 via RC^OX and PTS1 domain during oxidative stress. (A) HEK293 cell lysates were subjected to affinity-isolation assay with X-ACAD10 peptides (X = RC^O3, C^O3, C, or V), followed by immunoblotting (upper panel). Quantification of interacting PEX5 that binds to peptides for pull down (lower panel) (n = 3, **P < 0.01). (B) HeLa cells treated with 200 μM SNP or 10 μM hydroxychloroquine for 48 h, followed by immunoprecipitation with IgG or PEX5 antibody. Quantification was shown (lower panel) (n = 3, *P < 0.05; ***P < 0.001). (C) HeLa cells were transfected with wild type or K1052G mutant of ACAD10 for 48 h in the presence of SNP and hydroxychloroquine, followed by immunoprecipitation with Flag antibody. Quantification was shown (lower panel) (n = 3, **P < 0.01) (D) Schematic diagram of wild type (WT) PEX5 in comparison with TPR domain deleted mutant (∆TPR) PEX5. (E) HeLa cells were transfected with wild type or ∆TPR mutant of PEX5 for 48 h followed by immunoprecipitation with Flag or MYC antibody (left panel). Quantification of ACAD10-Flag bound to PEX5 (n = 3, *P < 0.05). (F) Peroxisomal fractions were subjected to protease protection assay, followed by immunoblotting with ACAD10 antibody detecting amino acid 1–85 at the N-terminal region. (G and H) Peroxisomal fractions of HeLa cells transfected with ACAD10-Flag for 48 h were subjected to protease protection assay (G) or alkaline sodium carbonate extraction assay (H), followed by immunoblotting. T, total; S, supernatant; P, pellet.

Figure 6.

The Nt-Cys2 of ACAD10 is an activating ligand to SQSTM1 that induces SQSTM1 oligomerization to mark the sites of pexophagy. (A and B) HeLa cells were transfected with SQSTM1/p62 siRNA (A), and wild type or C2V mutant of ACAD10 in the presence of SNP and bafilomycin A₁ (B). The cells were subjected to immunoblotting or immunostaining (left panel). Scale bar: 10 μm. Colocalization shown in immunostaining was quantified using the Pearson’s correlation coefficient (middle panel) or the Manders’ colocalization coefficient (right panel) (n = 40, **P < 0.01, ns, not significant). (C) HeLa cells were treated with H₂O₂ and bafilomycin A₁, followed by immunoprecipitation with ACAD10 or SQSTM1 antibody. ACAD indicates ACAD10. (D) HeLa cells were transfected with wild type or C2V mutant of ACAD10 in the presence of H₂O₂ and bafilomycin A₁, followed by immunoprecipitation with Flag or SQSTM1 antibody. CV indicates C2V mutant of ACAD10. (E) HEK293 cell lysates were subjected to an affinity-isolation assay with X-ACAD10 peptides (X = RC^O3, C^O3, C, or V), followed by immunoblotting (upper panel). Quantification of an affinity-isolation assay (lower panel) (n = 3, ***P < 0.001; ****P < 0.0001). (F-H) Hela cells expressing wild type, serially deleted SQSTM1 mutants (D1, D2, D3, or D4) (F), ZZ domain deletion mutant (△ZZ) (G), or ZZ domain point mutant (H) were subjected to affinity-isolation assay with RC^O3-ACAD10 peptides, followed by immunoblotting. The asterisk indicates a band with unknown nature. (I) HEK293 cell lysates transiently expressing SQSTM1 were subjected to affinity-isolation assays with X-ACAD10 peptides (X = RC^O3, C^O3, C, or V), followed by immunoblotting.

Figure 7.

Inhibition of the RC^OX-ACAD10-SQSTM1 pathway increase the levels of peroxisomes in PBDs cells and mice. (A and B) Immunohistochemical staining of E12.5 embryos from wild type or ate1^−/− mice (A), and the livers of wild type or sqstm1/p62^−/− mice (B). Scale bars: 20 µm and 40 µm (left panel) and 20 µm and 10 µm (right panel). (C) Wild type and PEX1^G843D PBD fibroblast cells expressing GFP-PTS1 were treated with tannic acid or bafilomycin A₁ for 24 h and analyzed using confocal microscopy (left) or immunoblotting (right). Scale bar: 10 μm. (D and E) Cells were transfected with siRNAs targeting ATE1 (D) or ACAD10 (E), followed by immunostaining or immunoblotting. Scale bar: 10 μm. (F) PBD cells were transfected with the pCMV14 vector in comparison with wild type or C2V mutant of ACAD10 in the presence of tannic acid, followed by immunostaining. Scale bar: 10 μm. (G) C57BL/6 mice were injected with 2 mg/kg tannic acid 3 times per week for one month. Livers were harvested and analyzed using immunohistochemical staining. Scale bars: 50 µm and 20 µm.

Figure 8.

The ACAD10-SQSTM1 circuit is a predominant pathway underlying pexophagy. (A) HeLa cells were transfected with Sqstm1/p62 siRNA in the presence of bafilomycin A₁, followed by immunostaining with anti-RC^OX and anti-SQSTM1 antibodies (left panel). Scale bar: 10 μm. Quantification of colocalization data was shown (right panel) (n = 30, ***P < 0.001). (B and C) Immunoblotting analyses of HeLa cells treated with siRNA targeting ATE1 and/or NBR1 (B) or ATE1 and/or Sqstm1/p62 (C). The asterisk indicates a band with unknown nature. (D) HeLa cells were transfected with ATE1-1A7A or its K417A mutant (KA) in combination with Sqstm1/p62 siRNA, followed by immunoblotting.

Figure 9.

Modulation of pexophagy by N-terminal oxidation and arginylation of ACAD10. The Nt-Cys2 of ACAD10 in the cytosol is oxidized by ADO and, under oxidative stress, ROS as well. The oxidized Nt-Cys2 is arginylated by ATE1 R-transferases, generating the RC^OX N-degron. The RC^OX induces the translocalization of ACAD10 to the cytosolic surface of peroxisomes, on which the RC^OX recruits SQSTM1, leading to lysosomal degradation. Unlike mitochondrial ACAD10, cytosolic ACAD10 exposes Nt-Cys and increases peroxisomal targeting by oxidative stress. Thus, the Nt-Cys2 of ACAD10 not only represents a sensor of both O₂ and oxidative stress in pexophagy but also acts as a receptor that recruits autophagy membranes to the sites of pexophagy.