Neddylation modification stabilizes LC3B by antagonizing its ubiquitin-mediated degradation and promoting autophagy in skin

Abstract

The Atg8-family proteins, including LC3B (microtubule-associated protein 1 light chain 3 beta), are pivotal for key steps in the autophagy process. Proper regulation of LC3B homeostasis is essential for its function. Although LC3B is modulated by various posttranslational modifications (PTMs), the impact of these modifications on LC3B protein homeostasis remains unclear. Neddylation, a recently identified ubiquitin-like modification, plays diverse biological roles. Here, we identify LC3B as a specific target for neddylation. This modification weakens LC3B's interaction with the ubiquitin E3 ligases VHL and BIRC6, thereby reducing LC3B ubiquitination. Depletion of ubiquitin-conjugating enzyme E2M (UBE2M), the primary E2 enzyme in the neddylation pathway, destabilizes LC3B and suppresses autophagy activity. Heterozygous Ube2m knockout (Ube2m^+/-) mice exhibit pronounced aging-like phenotypes, with reduced LC3B expression and impaired autophagy in skin tissues. Our findings demonstrate that LC3B neddylation is vital for maintaining its stability and regulating autophagy flux, offering a potential therapeutic avenue to mitigate aging-related processes.

Keywords: LC3B; UBE2M; autophagy; neddylation; skin aging.

Fig. 1.

UBE2M time-dependently decreases in skin during aging. (A) and (B) Expression of UBE2M and NEDD8 in the skin tissue from 3, 9, and 14-mo-old mice by western blot, and quantification of the indicated bands using image J. (C) Immunohistochemical staining of UBE2M and NEDD8 and quantification of the skin tissues from 3, 9, and 14-mo-old mice. Semiquantitative analysis of the epidermis was performed. (Scale bar, 50 μm.) (D) Western blot analysis of LC3, p62, and Beclin1 in skin tissues from 3, 9, and 14-mo-old mice. (E) The overall presence of hair on the 3-mo-old WT and Ube2m^+/− mice. (F) Representative dermoscopy images for the back skin of 3-mo-old WT and Ube2m^+/− mice (the red triangle denotes hyperpigmentation, and the red arrows indicate white linear areas). (G) Histological staining of skin tissue with H&E from the back of WT and Ube2m^+/− mice. Enlarged images in the box were present on the Right side. The thickness of the epidermis was quantified from 12 different fields from three mice for each group. (Scale bar, 20 μm.) The data are presented as the means ± SD. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, according to one-way ANOVA with post hoc Tukey (A–C, and D) and unpaired Student’s t test (G). n = 3.

Fig. 2.

UBE2M deficiency disrupts autophagy-related (ATG) proteins and causes senescence. (A) Western blot analysis of ATG protein expression in the skin of Ube2m^+/− and WT mice, alongside statistical analysis of the indicated proteins. (B) Western blot analysis of the senescent marker proteins p21 and p16 in Ube2m^+/− and WT mice skin, alongside statistical analysis of the indicated proteins. (C) Representative immunofluorescence images of Ki67+ cells (green) in the back skin of WT and Ube2m^+/− mice. DAPI (blue) was utilized for nuclei labeling. Scale bar, 50 μm, and statistical analysis of Ki67+ cells was conducted. (D) and (E) Expression of LC3B in NHEK and HaCaT cells with UBE2M knockdown examined by western blot and quantification analysis. (F) and (G) The transcription and protein expression of SASP genes in HaCaT cells with UBE2M knockdown examined by qPCR and western blot. siRNA sequence targeting UBE2M was employed. The protein expression of SASP was quantified using Image J. Data are presented as the means ± SD of triplicate samples, and the P-value was calculated using an unpaired Student’s t test; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 3.

LC3B is a target for neddylation. (A) LC3B was modified upon the overexpression of UBE2M in HaCaT cell lines detected by western blot. (B) The expression of LC3B and Beclin1 upon a variety of E2 proteins overexpression in 293 T cells. (C) NAE inhibitor MLN4924 blocked the UBE2M-mediated modified form of LC3B in HaCaT cell lines. The cells were administered MLN4924 (0.3 μM, 12 h) before harvesting. Quantification was employed as indicated. n = 3. (D) LC3B physically associates with UBE2M and NEDD8. Flag-LC3B was transfected into 293 T cells and treated with 0.3 μM MLN4924 for 12 h. Cell lysis was followed by immunoprecipitation and quantification analysis. (E) Endogenous LC3B associated with NEDD8 via coimmunoprecipitation with LC3B antibody. Mouse IgG was utilized as a control. (F) Colocalization analysis of LC3B with UBE2M and NEDD8. HaCaT cells were transfected with UBE2M constructs, and then costained with LC3B (Red) and NEDD8 (Green), UBE2M (Green). Boxed areas were amplified to indicate their colocalization (yellow). Nuclei were stained with DAPI (blue). (Scale bar, 10 μm.) (G) Nuclear colocalization of LC3B and NEDD8. HaCaT cells with UBE2M overexpression were prepermeabilized to remove the cytoplasmic inclusions, followed by LC3B (red) and NEDD8 (green) immunofluorescence staining. Nuclei were stained with DAPI (blue). (Scale bar, 10 μm.) (H) Both NLS-Flag-LC3B and NES-Flag-LC3B constructs weaken their interactions with NEDD8. 293 T cells were transfected with these LC3B constructs for 48 h, followed by anti-Flag immunoprecipitation and quantification analysis as indicated. n = 3. (I) Both NLS-Flag-LC3B and NES-Flag-LC3B constructs decrease the PE conjugation of LC3B. Anti-Flag immunoprecipitation and anti-LC3B immunoblot were employed. The red triangle denotes PE conjugation of LC3B (LC3B II), and the red asterisk denotes neddylated LC3B. (J) The LC3B^G120A mutation abolishes the conjugation of NEDD8 to LC3B. 293 T cells were transfected with Flag-tagged WT or mutant LC3B for 48 h, followed by anti-Flag immunoprecipitation. The red box indicates the neddylated LC3B. (K) The interaction of LC3B and NEDD8 in 293 T cells with siRNA-mediated knockdown of ATG5, ATG3, and ATG7. Anti-Flag immunoprecipitation and quantification analysis were employed as indicated. n = 3. (L) PLA analysis of the interaction between FLAG-LC3B (WT or mutations) and NEDD8. HaCaT cells were transfected with Flag-tagged LC3B WT and its mutants. PLA was conducted with antibodies against NEDD8 and Flag. Nuclei were stained with DAPI (blue). PLA puncta (red) for each condition were imaged by confocal microscopy. (Scale bar, 10 μm.) The quantification of PLA was plotted, n = 8 to 15. The data are presented as the means ± SD. ns, not significant, **P < 0.01, ***P < 0.001, and ****P < 0.0001, according to unpaired Student’s t test (C–E, and J) and one-way ANOVA with post hoc Tukey (H, K, and L).

Fig. 4.

CUL2-RBX1 and CUL4B–RBX1 are required E3 ligases for LC3B neddylation. (A) The interaction of LC3B and NEDD8 with and without RBX1 knockdown using siRNA. Anti-Flag immunoprecipitation and the correspondent quantification analysis were employed as indicated. (B) The knockdown efficiency of relative cullins characterized by qRT-PCR. The indicated cullins were knocked down using siRNA in 293 T cells with UBE2M overexpression. (C) The neddylation of LC3B under various cullins knockdown conditions. The quantification of the indicated bands was calculated using image J. The P value was calculated comparing each group with NC. (D) and (E) The physical interaction of NEDD8 and Flag-LC3B in 293 T cells with and without CUL2 or CUL4B overexpression. Cell lysates were immunoprecipitated using Flag antibodies, alongside quantification analysis. The data are presented as the means ± SD. ns, not significant, *P < 0.05, **P < 0.01, according to unpaired Student’s t test (A, B, D, and E) and one-way ANOVA with post hoc Tukey (C). n = 3.

Fig. 5.

LC3B K42 is the primary neddylation site. (A) Identification of putative neddylation sites in LC3B via Flag-tagged LC3B mutations immunoprecipitation and NEDD8 immunoblotting in 293 T cells alongside NEDD8 overexpression. (B) The pulldown assay of NEDD8 with mCherry-tagged LC3B mutations in 293 T cells by endogenous NEDD8 immunoprecipitation and mCherry immunoblotting. Mouse IgG was utilized as a control. (C) PLA analysis of neddylation of LC3B WT and mutations in HaCaT cells with Flag-tagged LC3B and its mutations expression (Top panel). (Scale bar, 10 μm.) PLA foci in individual samples were quantified (Bottom panel). Data are presented as means ± SD, and the P-value for PLA quantification was calculated via one-way ANOVA with post hoc Tukey (ns, not significant, ****P < 0.0001). (D) Mapping the K42 on the tertiary structure of LC3B predicted using AlphaFold. (E) Z-dock software was utilized for covalent docking. The Lys42 residue on LC3B and Gly76 on NEDD8 were binding sites. (F) Sequence alignment of human LC3B with those of four other species suggests the conservation of Lys42. Human LC3 K42 and the related amino acids across other species are indicated by a red box.

Fig. 6.

Neddylation modification of LC3B promotes its stability against proteasome-dependent proteolysis. (A) The HA-Ub modification of LC3B is reduced upon UBE2M overexpression in 293 T cells detected by immunoprecipitation with Flag-LC3B (Top panel). The relative quantification of Ub modification was performed (Bottom panel). (B) The HA-Ub modification of the LC3B K42R mutation is increased compared to LC3B WT detected by immunoprecipitation with Flag-LC3B (Top panel). The relative quantification of Ub modification was performed (Bottom panel). (C) Co-IP analysis of the relationship between Flag-LC3B or Flag-LC3B K42R with VHL and BIRC6, respectively. The relative quantification was conducted by normalizing to the respective Flag bands in IP samples. (D) The HA-Ub modification of LC3B K42R is reduced upon BIRC6 knockdown in 293 T cells by CoIP analysis. (E) The HA-Ub modification of the LC3B K42R is increased under MG132 (20 μM, 12 h) treatment. The relative level of HA-Ub modification was quantified. (F) 293 T cells were transfected with plasmids encoding Flag-LC3B K42R and siRNA targeting BIRC6. Cells were incubated with 20 μM MG132 for 12 h before lysis and western blot. The relative quantification of Flag modification was performed. (G) The changes of LC3B levels were analyzed in UBE2M-knockdown 293 T cells with bafilomycin A1 (Baf A1, 40 nM, 4 h) or MG132 (20 μM, 12 h) treatment. Data are presented as means ± SD, and the P-value was calculated using unpaired Student’s t test (A–F), and one-way ANOVA with post hoc Tukey (G). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. n = 3.

Fig. 7.

Neddylation deficiency of LC3B results in autophagy-dysfunction. (A) Endogenous LC3B and p62 were detected in HaCaT cells with and without knockdown of UBE2M, in presence of starvation for 6 h or (and) 40 nM bafilomycin A1 for 4 h. The relative density of protein bands was quantified. (B) Western blot detection of total cell lysates from 293 T cells (wild type, stably overexpressing UBE2M, or treated with MLN4924) overexpressing Halo-LC3B or Halo-mGFP. After treatment with TMR-conjugated ligand and starvation, the cells were collected. Quantification of Halo band intensities was present. Data are presented as the means ± SD of triplicate samples, and the P-value was calculated using one-way ANOVA with post hoc Tukey and unpaired Student’s t test (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001).

Fig. 8.

Reduction of LC3B neddylation contributes to the keratinocyte cellular senescence. (A) The effects of Flag-LC3B WT and K42R on senescence-related proteins expression were detected in NHEK cells with UBE2M knockdown. Western blot and the correspondent quantification analysis were employed as indicated. (B) Ki67 staining to indicate the cell proliferation in NHEK with Flag-LC3B WT and K42R mutation expression upon UBE2M knockdown. Nuclei were stained with DAPI (blue). (Scale bar, 50 μm.) The ratio of Ki67-positive NHEK cells was quantified. (C) The expression of SASP-associated proteins and p62 affected by LC3 overexpression were detected in primary mouse epidermal keratinocytes isolated from the skin of Ube2m^+/− mice and WT mice. (D) The expression of p21 and p16 expression in HaCaT cells with ATG5 and UBE2M knockdown and Flag-LC3B overexpression as indicated, and the protein expression of p21 and p16 was quantified. (E) LC3B undergoes neddylation through a covalent linkage with NEDD8 facilitated by the E2 enzyme UBE2M and the E3 enzymes CUL2-RBX1 or CUL4B–RBX1. The neddylation of LC3B counteracts its ubiquitination and the subsequent protein degradation, thereby mitigating the skin aging associated with autophagy protein deficiencies. Data are presented as the means ± SD of triplicate samples, and the P-value was calculated using one-way ANOVA with post hoc Tukey (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001).