Fig 2: PARKIN down regulation and impairment of mitophagy in DS fibroblasts.a, b Whole-cell extracts from 2 N and DS cells were analyzed by western blot for PARKIN and PINK1. Each quantitative data was normalized with ACTIN. (n ≥ 3). c 2 N and DS cells were assessed for PARKIN and PINK1 mRNA by quantitative real-time PCR. mRNA levels were normalized to ACTIN mRNA, used as internal control. Data display the fold-changes of PARKIN, PINK1 and SQSTM1 (p62) mRNAs relative to control cells (n ≥ 3). d Mitochondrion/cytosol fractionation was performed to assay the presence of PARKIN and LC3-II within the mitochondrial fraction. VDAC1 and COXIV were used as markers of purity for mitochondrial fraction. βIII-TUBULIN was used as a marker of the cytosolic fraction. e Quantitative data of LC3-II in the mitochondrial fraction, normalized with VDAC1 (n = 5). f Mitochondrial fractionation was performed to assess the accumulation of p62, LC3-II, NDP52 and OPTINEURIN (OPTN) within the mitochondria in untreated and treated fibroblasts with a lysosomal-inhibiting agent, Bafilomycin A (Bafil., 10 nM) for 6 h. Each protein was normalized with VDAC1 with quantitation in (g). (n ≥ 3) h 2 N and DS cells were treated with Concanamycin A (ConA, 50 nM) for 2 h to block lysosome function. The cells were fixed and immunostained for LC3 and TOM20 in order to evaluate mitophagy. White arrows point to the co-localization of puncta into the high magnification image of the boxed area. Scale bar 10 μm. The graph shows the percentage of LC3/TOM20 colocalization calculated by JACoP plugin of ImageJ. Quantitation based on a minimum of 50 cells per conditions from three independent experiments. i GSEA analysis of transcriptomic changes comparing DS fibroblasts to 2 N fibroblasts, using a list of transcriptional factors that have been described to be involved in mitophagy46,47 (for list of genes, see Supp. Table 1). As described in Fig. 1d, the vertical bars represent genes ranked horizontally by moderated t-statistics; Pink, blue and grey shaded rectangles upregulated, downregulated and genes with no change respectively. The plot shows significant downregulation of TFs involved in mitophagy in DS compared to 2 N fibroblasts (p < 0.001; FDR < 0.001). j Volcano plots for whole transcriptomes of differentially expressed genes between DS and 2 N fibroblasts, with TFs involved in mitophagy, used in (I) for GSEA analysis highlighted with labels. Statistical analysis was performed using Student’s T-test or one-way ANOVA with Tukey’s multiple comparisons test. (**p < 0.01; ***p < 0.001). Immunoblots reported are from one representative experiment

Fig 3: Functional domains in the targeted autophagy receptors and library design. (A) OPTN, p62 and NDP52 are multifunctional proteins that contain domains mediating multimerization (blue) and cargo recognition (green). In p62, the ZZ domain (orange) binds arginylated (Nt-R) substrates (Zhang et al., 2018). The SKICH domain (magenta) of NDP52 has been shown to bind to intracellular membranes (Von Muhlinen et al., 2012). The four amino acid linear LC3 interaction region (LIR) is shown in bold in its broader amino acid context. (B) Surface representation of the LIR binding site of LC3B in complex with a pS177 OPTN peptide (cyan). Region 1 (orange) and region 2 (purple) are shown. Hydrophobic binding pocket 1 (HP1) coordinates position 0 (Trp, Phe or Tyr) residues and HP 2 coordinates position +3 residues (Leu, Val or Ile) in canonical LIR peptides. (C) LC3B (pdb: 3VTU) and GATE-16 (pdb: 1EO6) library design. Surface exposed residues (spheres) in region 1 (orange) and region 2 (purple) that mediate binding to LIR motifs have been randomized and are shown below in the context of their aligned primary amino acid sequence. Amino acid positions of the boundaries of regions 1 and 2 are shown.

Fig 4: Engineered LC3B and GATE-16 variants co-immunoprecipitate endogenous OPTN, p62 or NDP52. (A–C) Co-immunoprecipitation (Co-IP) of (A) OPTN, (B) NDP52, or (C) p62 binding variants and corresponding wt controls expressed as SGFP2 fusion proteins in stable inducible HeLa cells. Western blots were probed with indicated antibodies. (D–F) Mass spectrometry of Co-IPs using anti-GFP beads from stable HeLa cells expressing inducibly SGFP2 fusion proteins of LC3Bwt, GATE-16wt, OPTN.LC3Bv or p62.GATE-16v. In case of NDP52.LC3Bv, HeLa cells were transiently transfected with SGFP2 fusion constructs either expressing LC3Bwt or NDP52.LC3Bv. Detected proteins (gray) were plotted as log2 enrichment vs log10 intensity. Endogenous OPTN (blue), p62 (orange), NDP52 (green) and other LIR containing proteins (black) are shown as indicated.

Fig 5: NDP52 binds selectively to Ub 6AcK.a Selected interactors of acetylated Ub variants with a distinct binding pattern. Red indicates enrichment, whereas blue indicates lack of enrichment. b Confirmation of interactions by western blot analysis. 20% of the elution fraction of the affinity enrichment were subjected to western blot analysis with antibodies specific for the indicated proteins. Input represents 1% of the HEK293T cell extract used for affinity enrichment. c Verification of interactions by in vitro binding assays using recombinant NDP52 and UCHL3 and the bait molecules indicated. Binding reactions were analyzed by SDS-PAGE followed by Coomassie blue staining (PD). 10% of the proteins used in the binding assay are shown in the lower panel (Input). Asterisk indicates the running position of an UCHL3-Ub adduct that was recently described80. d In vitro binding assay using Strep-tagged versions of nonmodified Ub (wt), Ub acetylated at K6 (6AcK), M1-linked Ub dimers (diUb), and empty beads as affinity matrices and recombinant full-length NDP52 (fl) or a truncation variant of NDP52 devoid of the C-terminal Ub-binding zinc finger (ΔZF2). Binding reactions were analyzed by SDS-PAGE followed by Coomassie blue staining (PD). 10% of the proteins used in the binding assay are shown in the lower panel (Input). A schematic of full-length NDP52 and NDP52 ΔZF2 is shown at the top. e In vitro binding assays with GST fusion proteins of full-length NDP52 (fl) and NDP52 ΔZF2 with the Ub variants indicated. GST was used as control. Proteins bound to the different GST proteins were analyzed by western blot using an antibody directed against Ub. b–e The experiments shown are representative of two independent experiments. Source data are provided as a Source Data file.