A20 binding and inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1): a novel modulator of mitochondrial autophagy

Abstract

A20 binding inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1), a polyubiquitin-binding protein, is a signal-induced autophagy receptor that attenuates NF-κB-mediated inflammation and cell death. The present study aimed to elucidate the potential role of ABIN-1 in mitophagy, a biological process whose outcome is decisive in diverse physiological and pathological settings. Microtubule-associated proteins 1A/1B light chain 3B-II (LC3B-II) was found to be in complex with ectopically expressed hemagglutinin (HA)-tagged-full length (FL)-ABIN-1. Bacterial expression of ABIN-1 and LC3A and LC3B showed direct binding of ABIN-1 to LC3 proteins, whereas mutations in the LC3-interacting region (LIR) 1 and 2 motifs of ABIN-1 abrogated ABIN-1/LC3B-II complex formation. Importantly, induction of autophagy in HeLa cells resulted in colocalization of ABIN-1 with LC3B-II in autophagosomes and with lysosomal-associated membrane protein 1 (LAMP-1) in autophagolysosomes, leading to degradation of ABIN-1 with p62. Interestingly, ABIN-1 was found to translocate to damaged mitochondria in HeLa-mCherry-Parkin transfected cells. In line with this observation, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated deletion of ABIN-1 significantly inhibited the degradation of the mitochondrial outer membrane proteins voltage-dependent anion-selective channel 1 (VDAC-1), mitofusin-2 (MFN2), and translocase of outer mitochondrial membrane (TOM)20. In addition, short interfering RNA (siRNA)-mediated knockdown of ABIN-1 significantly decreased lysosomal uptake of mitochondria in HeLa cells expressing mCherry-Parkin and the fluorescence reporter mt-mKEIMA. Collectively, our results identify ABIN-1 as a novel and selective mitochondrial autophagy regulator that promotes mitophagy, thereby adding a new player to the complex cellular machinery regulating mitochondrial homeostasis.

Keywords: LC3-interacting region; mitochondrial outer membrane proteins; mitophagy; selective autophagy receptor.

Graphical abstract

Figure 1.

A20 binding and inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1) binds 1A/1B light chain 3B (LC3B) via. LC3-interacting region (LIR) motifs. A: schematic representation of the domain architecture of ABIN-1. The alignment of LIR motifs with known autophagy receptors is illustrated underneath. LIR sequences are emphasized in bold. B: HeLa cells transiently transfected with hemagglutinin (HA)-tagged hABIN-1 (hABIN-1-HA) or empty plasmid control (Vector Co) for 24 h, incubated with Bafilomycin A1 (BafA1) (200 nM) for 4 h, immunoprecipitated (IP) with anti-HA-beads, and analyzed by immunoblotting. C: reciprocal IP of (B) using HeLa cells transfected with enhanced green fluorescent protein (EGFP)-LC3B and hABIN-1-HA and IP with anti-GFP-beads. D: HeLa cells transfected with Vector Co, plasmids encoding hABIN-1-HA full length (FL), or mutants defective in the LIR1-, LIR2-, LIR1 + 2-, or ubiquitin-binding domain in ABIN and NEMO (UBAN)-domain and IP with anti-HA antibody. β-Actin served as the loading control for the input. Immunoprecipitated ABIN-1-HA or EGFP-LC3B served as loading controls for the respective IPs. E: glutathione-S-transferase (GST)-pull-down assay of ABIN-1 or the indicated mutants. Purified GST, GST4x-Ubiquitin (Ub), GST-LC3A, or GST-LC3B purified from Escherichia coli were immobilized on GST beads and incubated with bacterial purified recombinant maltose binding protein (MBP)-tagged ABIN-1 protein. ABIN-1 fused to MBP was detected by Western blotting. Ponceau S staining was used to visualize GST-fusion proteins; n = 3 replicates. NBD, NEMO-binding domain.

Figure 2.

A20 binding and inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1) colocalizes with LC3B and lysosomal-associated membrane protein 1 (LAMP-1) and translocate to damaged mitochondria. A and B: representative confocal images of HeLa cells overexpressing monomeric enhanced green fluorescent protein (mEGFP)-hABIN-1 (green). A: cells cotransfected with mCherry-LC3B (red) for 24 h and treated with Torin-1 (1 μM) or dimethylsulfoxide (DMSO) (1 μM) and Bafilomycin A1 (BafA1, 300 nM) for 4 h. B: cells stimulated with Torin-1 and immunostained for endogenous LAMP-1 (red). Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar = 10 μm. C: mCherry-Parkin expressing HeLa cells treated with oligomycin/antimycin A (OA) (10 µM, 4 h) or DMSO (10 µM, 4 h) and Western blotted to analyze whole cell lysate, mitochondrial-, and cytosolic-fractions. Translocase of outer membrane (TOM)20 and cytochrome c oxidase subunit II (COXII) served as mitochondrial markers; n = 3 replicates. Co, empty plasmid control; DOX, doxycycline hyclate.

Figure 3.

A20 binding and inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1) is processed during autophagy. A: Western blot analysis of ABIN-1, p62, and 1A/1B light chain 3B (LC3B) expression in HeLa cells treated with rapamycin (100 nM) and/or Bafilomycin A1 (BafA1) (4 and 8 h, 200 nM; 24 h, 10 nM) for the indicated time points. Quantification of ABIN-1 (B), p62 (C), and LC3B-II (D). The protein levels were normalized to β-actin. Data represent means ± standard deviation (SD); n = 3 replicates; (*P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 4.

A20 binding and inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1)-dependent degradation of mitophagy substrates. A: HeLa-mCherry-Parkin [wild type (WT) control] and CRISPR/Cas9-mediated ABIN-1 KO-HeLa-mCherry-Parkin cells (ABIN-1 KO) stimulated with oligomycin and antimycin A (OA, 1 µM, 24 h) and analyzed by Western blotting. Quantification of mitofusin-2 (MFN2) (B), voltage-dependent anion-selective channel 1 (VDAC1) (C), translocase of outer membrane (TOM)20 (D), and cytochrome c oxidase subunit II (COXII) (E). The protein levels were normalized to β-actin. Data represent means ± standard deviation (SD); n = 3 replicates; *P < 0.05.

Figure 5.

Absence of A20 binding and inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1) partially inhibits mitophagy. A: representative data for mt-mKEIMA-Parkin-expressing HeLa cells transfected with siRNA control (siRNA Co) and siRNA-ABIN-1 for 48 h. Cells were treated with dimethylsulfoxide (DMSO) (10 μM), oligomycin and antimycin A (OA, 10 μM) or OA (10 μM) together with BafA1 (200 nM) for 6 h and analyzed by fluorescence-activated cell sorting for lysosomal positive mt-mKEIMA, representing cells undergoing mitophagy. B: quantification of (A). C: Western blot analysis of mt-mKEIMA-Parkin-expressing HeLa cells treated with siRNA Co or siRNA-ABIN-1 after 48 h. β-Actin served as the loading control. Data represent means ± standard deviation (SD); n = 5 replicates; *P < 0.05 and ***P < 0.001); n.s. = not significant.

Figure 6.

A model of A20 binding and inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1)-mediated modulation of mitophagy. Autophagy induces the 1A/1B light chain 3 (LC3)-interacting region (LIR) 1 and 2 motifs-dependent binding of ABIN-1 to LC3B-II on the forming phagophore membrane and subsequent colocalization with LC3B-positive autophagosomes and lysosomal associated membrane protein 1 (LAMP-1)-containing autophagolysosomes. Furthermore, autophagy induces translocation of ABIN-1 to damaged mitochondria where it mediates degradation of mitochondrial outer membrane proteins mitofusin-2 (MFN2), translocase of outer membrane (TOM20), and voltage-dependent anion-selective channel 1 (VDAC1), together with its own processing via. the autophagic pathway, resulting in the selective degradation of damaged mitochondria by mitophagy.