Fig 1: The ubiquitin-binding activity of NDP52 is indispensable for NF-κB suppression. (A) Domain structure of Wild-type (WT) human NDP52 isoform 3 and its ΔUBZ mutant. V248A is associated with Crohn’s disease (31), and the D439R substitution causes the defect in ubiquitin-binding (32). SKICH, SKIP carboxyl homology; LIR, LC3-interacting region; GALBI, galectin-8 binding; and UBZ, ubiquitin-binding zinc finger. (B) The UBZ domain of NDP52 functions as the ubiquitin-binding site. In vitro MBP pulldown experiments using linear (M1)-, K48-, or K63-linked tetraubiquitins and MBP-fused lacZ, NDP52-WT, and D439R mutant were performed, and the bound ubiquitin chain was detected by immunoblotting. The precipitated MBP-fusion proteins were visualized by Coomassie Brilliant Blue (CBB) staining. (C) Low ubiquitin selectivity of NDP52. HA-tagged single Lys mutants of ubiquitin were expressed in HEK293T cells, and the lysates were pulled down by MBP-NDP52-WT or -D439R mutant. The precipitated ubiquitin was detected by an anti-HA antibody, and the precipitated MBP-fusion proteins were detected by CBB staining. (D) Linear polyubiquitin-binding of NDP52. LUBAC-generated linear polyubiquitin was pulled down by the MBP-NDP52-WT or -D439R mutant in vitro. The precipitated ubiquitin was detected by an anti-linear ubiquitin antibody, and the precipitated MBP-fused proteins were detected by CBB staining. (E) NDP52-WT, but not the D439R mutant, suppresses NF-κB activity. Effects of increasing amounts (0.025 μg, 0.075 μg, and 0.25 μg/well) of WT and D439R mutant of FLAG-NDP52 were examined, in the presence of either 10 ng/ml TNF-α or the co-expression of LUBAC subunits in HEK293T cells, by the NF-κB luciferase assay. (F) NDP52 does not inhibit the linear ubiquitination activity of LUBAC. HA-tagged LUBAC subunits (1.0 μg HA-HOIP, 0.2 μg HOIL-1L-HA, and 0.2 μg HA-SHARPIN/well) and increasing amounts (0.1 μg, 0.3 μg, and 1.0 μg/well) of FLAG-NDP52-WT, -D439R, and -ΔUBZ were co-expressed in HEK293T cells, and cell lysates were immunoblotted with the indicated antibodies. (G) NDP52 suppresses the canonical NF-κB pathway. The C-terminally tetraubiquitin-fused NEMO (NEMO-Ub4), WT and mutants of NDP52, and/or NIK were co-transfected with the NF-κB luciferase reporter, as indicated, and luciferase activity was measured at 24 h-post transfection. (E, G) Data are shown as Means ± SD (n = 3) by Huber-White Sandwich estimators for variance-covariance structures corrected with Bonferroni method. *P < 0.05, **P < 0.01, ****P < 0.0001, NS, not significant.
Fig 2: The C-terminal region of NDP52 is required for degradation of TRIF. a Schematic diagram of human NDP52 and its domain-deleted mutants. b HEK293T cells were transfected with Flag-TRIF together with HA-NDP52 or HA-tagged NDP52 mutant for 24 h. IP with anti-Flag agarose was carried out with clarified cell lysates, followed by IB with antibodies to HA epitope and Flag epitope. c HEK293T cells were transfected with Flag-TRIF together with HA-NDP52 or HA-tagged NDP52 mutant for 24 h. Cell lysates were analyzed by IB with antibodies to Flag epitope, HA epitope and β-actin. d, e HEK293T cells were transfected with Flag-TRIF and pNF-κB-Luc (d) or p561-luc (e) together with HA-NDP52, HA-tagged NDP52 mutant or empty vector (Mock) for 24 h. Then luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). *P < 0.01, for comparison with the value of Mock. All results are representative of three independent experiments
Fig 3: Model of autophagy activation via TBK1 at Ad endosome penetration sites.Ad entry induces rupture of the endosomal membrane exposing intracellular glycans which are detected by Gal8 (I). This step creates a recruitment and activation hub for TBK1 to the Ad penetration site. Autophagy receptors (NDP52, p62) are recruited independently of TBK1 (II). TBK1 recruitment results in high local concentrations at the penetration site probably leading to its activation by auto-phosphorylation. Once activated, TBK1 promotes autophagy activation as well as phosphorylates autophagy receptors increasing their affinity to LC3 (III). Autophagy activation generates autophagosomal membranes with LC3 and autophagy receptor link damaged membranes into the forming autophagosome to induce degradation of the damaged membrane (including associated pathogens, i.e. Ad M1) to restore homeostasis (IV). In contrast, Ad WT stalls autophagosome formation and/or maturation and avoids degradation by escaping into the cytosol.
Fig 4: Autophagy receptors defend against seeded tau aggregation. a–d, confocal images of 293T cells (a and b) and SH-SY5Y cells (c and d) expressing P301S tau treated with DyLight-labeled aggregated P301S tau (red) for 3 h, followed by 24-h growth and immunostaining with anti-NDP52, anti-p62, and anti-ubiquitin (green) (a and b) or treated with aggregated recombinant P301S tau for 3 h, followed by 48-h growth and immunostaining with AT100 (green) and anti-NDP52, anti-p62, and anti-ubiquitin (red) (c and d). Nuclei were visualized with DAPI (blue). Arrows indicate co-localization of tau seed–containing vesicles with NDP52 and seeded tau aggregates with p62. Scale bars, 10 μm. e, representative Western blots with HT7 of the total lysate and sarkosyl-soluble and sarkosyl-insoluble fractions of HeLa cells expressing P301S tau and treated with NDP52, p62, and optineurin siRNAs, followed by treatment with aggregated P301S tau. GAPDH was used as a loading control. f, densitometric analysis of HT7 blots of the sarkosyl-insoluble fractions from e. The results are the means ± S.E. n = 4; **, p < 0.01 (ANOVA).
Fig 5: NDP52 and TAX1BP1 are neither involved in the replication nor the release of MOPV or LASV infectious particles. (A) HeLa cells were transfected with the indicated siRNA 72 h before analysis of silencing efficiency by Western blotting (n = 4 independent experiments). (B–D) the same cells as in (A) were then infected with LASV or MOPV with an MOI of 0.1 for 1 h before being maintained for three days at 37 °C. Viral RNA was then extracted from the cells and supernatants for quantification by RTqPCR. Infectious particles from the supernatants were also titrated on Vero cells. The error bars represent the standard error of the means from four independent experiments. * indicates p < 0.05, as determined by the Mann–Whitney test.
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