A heterozygous p.S143P mutation in LMNA associates with proteasome dysfunction and enhanced autophagy-mediated degradation of mutant lamins A and C

Abstract

Lamins A and C are nuclear intermediate filament proteins that form a proteinaceous meshwork called lamina beneath the inner nuclear membrane. Mutations in the LMNA gene encoding lamins A and C cause a heterogenous group of inherited degenerative diseases known as laminopathies. Previous studies have revealed altered cell signaling pathways in lamin-mutant patient cells, but little is known about the fate of mutant lamins A and C within the cells. Here, we analyzed the turnover of lamins A and C in cells derived from a dilated cardiomyopathy patient with a heterozygous p.S143P mutation in LMNA. We found that transcriptional activation and mRNA levels of LMNA are increased in the primary patient fibroblasts, but the protein levels of lamins A and C remain equal in control and patient cells because of a meticulous interplay between autophagy and the ubiquitin-proteasome system (UPS). Both endogenous and ectopic expression of p.S143P lamins A and C cause significantly reduced activity of UPS and an accumulation of K48-ubiquitin chains in the nucleus. Furthermore, K48-ubiquitinated lamins A and C are degraded by compensatory enhanced autophagy, as shown by increased autophagosome formation and binding of lamins A and C to microtubule-associated protein 1A/1B-light chain 3. Finally, chaperone 4-PBA augmented protein degradation by restoring UPS activity as well as autophagy in the patient cells. In summary, our results suggest that the p.S143P-mutant lamins A and C have overloading and deleterious effects on protein degradation machinery and pharmacological interventions with compounds enhancing protein degradation may be beneficial for cell homeostasis.

Keywords: autophagy; degradation; disease mutations; lamin A/C (LMNA); ubiquitin (Ub); ubiquitin-proteasome degradation system.

FIGURE 1

Expression and degradation of lamins A and C are enhanced in LMNA mutant patient cells. (A) RT-qPCR analysis of LMNA expression in the patient fibroblasts carrying a heterozygous p.S143P LMNA mutation. Allele-specific primers detecting either wild-type (c.427T) or mutant (c.427C) alleles, or primers detecting both (LMNA) were used. N = 3 individual experiments. (B) LC-ESI-MS/MS analysis of peptide fragments covering both wild-type and mutant lamins A and C in the patient cells. The sequences specific to the p.143S and p.143P lamin A and C peptide fragments are shown on the top, and the corresponding peaks in the mass spectra are encircled. (C) RT-qPCR analysis of relative overall LMNA expression in the control and patient fibroblasts (N = 3). (D) Luciferase assay measuring activity of an upstream −1.3 kb LMNA promotor sequence in the control and patient fibroblasts (N = 3). (E) Western blot analysis shows protein levels of lamins A and C (LA/C) in the patient and control fibroblasts treated with 300 μg/ml cycloheximide (CHX) for given time points. Numerical values show levels of lamins A and C normalized to actin, which was used as a loading control. (F) Confocal microscopy images from control and patient cells stained for lamins A and C before or after 16-h treatment with 50 µM leptomycin B (LMB). Mid-plane confocal sections are shown. Scale bar 10 µm. (G) Mean fluorescence intensities (AU) were measured from confocal images and plotted (N = 300). Note upregulation of lamins A and C after 16-h treatment with 50 µM LMB in the patient fibroblasts. The whiskers show the mean values ±s.d. and boxplots show the 75th and 25th percentiles of the calculated intensities, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

Lamins A and C are ubiquitinated by K48-linked chains in LMNA mutant patient fibroblasts. (A) Representative confocal microscopy images from control and patient fibroblasts stained for lamins A and C and K48-linked ubiquitin chains. Scale bar 10 µm. (B) Pulldown with GST-tagged recombinant pan-tandem ubiquitin-binding entity (pan-TUBE) under denatured conditions. (C) Pulldown with lamin A antibody under denatured conditions. K48-ubiquitin levels were normalized to GAPDH, which was used as a loading control. (D-E) Proximity ligation assay (PLA) of lamins A and C and K48-ubiquitin in the control and patient fibroblasts as calculated from >300 individual cells. Cells with more than three PLA signals were considered positive. Data are expressed as mean ± s.e.m, *p < 0.05. (F) Proteasome activity of control and patient fibroblasts treated with or without 20 mM NH₄Cl for 24 h. N = 3 individual experiments. (G) Chymotrypsin-like activity of 20S proteasomes isolated from control and patient fibroblasts (N = 3). (H) Chymotrypsin-like activity of 20S proteasomes isolated from Hela cells expressing either FLAG-tagged WT-LA or p.S143P-LA and treated with or without 20 mM NH₄Cl for 24 h (N = 3). The whiskers show mean values ±s.d **p < 0.01, ***p < 0.001.

FIGURE 3

Lamins A and C are increasingly committed to autophagy in LMNA mutant patient fibroblasts. (A) Confocal microscopy images from the patient and control fibroblasts stained for autophagy-related proteins Atg5 and Atg7. Scale bar 10 µm. (B) Calculated mean fluorescence intensity values from Atg5 and Atg7 stainings. The whiskers show mean values ± s.d and boxplots show the 75th and 25th percentiles (N = 300), *** p < 0.001. (C) Confocal microscopy images from the patient and control fibroblasts treated with or without 20 mM NH₄Cl for 24h and stained for Atg8/LC3-I/II and SQSTM1/p62. Note the accumulation of LC3-I/II or p62 after NH₄Cl treatment especially in the patient cells. Scale bar 10 µm. (D) Calculated mean fluorescence intensity values from LC3-I/II staining. The whiskers show mean values ± s.d and boxplots show the 75th and 25th percentiles (N = 300), *** p < 0.001 (compared to untreated control cells). (E) Western blot analysis from control and patient fibroblasts treated with 20 mM NH₄Cl for 24 h. Pooled LC3-I/II and p62 levels were normalized to GAPDH, which was used as a loading control. Note the increase of LC3-II after NH₄Cl treatment. (F) Calculated mean fluorescence intensity values from p62 staining. (N = 300), *** p < 0.001 (G–H) Proximity ligation assay (PLA) detecting association of lamins A and C with LC3-I/II in the control and patient fibroblasts. The percentage of cells with PLA signals was determined from >300 cells and the cells with more than three PLA signals were considered positive. Data is expressed as mean ± s.e.m, * p < 0.05, ** p < 0.01, *** p < 0.001.

FIGURE 4

Crosstalk between autophagy and UPS is impaired in LMNA mutant patient cells. (A) LysoTracker Red DND-99 staining from untreated and MG132-treated control and patient cells. (B) Calculated mean fluorescence intensity values show more LysoTracker positive lysosomes in the patient cells and their number is further increased after 24-h treatment with 1 µM MG132. (C) Western blot analysis from control and patient cells treated with 1 µM MG132 or 20 mM NH₄Cl for 24 h. Protein levels of lamins A and C (LA/C) were normalized to GAPDH, which was used as a loading control. All the proteins were detected on the same membrane. (D) Confocal microscopy images from control and patient cells treated with MG132 and stained for lamins A and C and K48-ubiquitin. Insets show cytosolic structures that co-stain with lamins A and C and K48 antibodies. Scale bar 10 µm. (E) Chymotrypsin-like activity as measured in control and patient cells treated with 5 mM 4-PBA for 24 h (N = 3 individual experiments) **p < 0.01. (F) Western blot analysis of control and patient cells treated with or without 5 mM 4-PBA. Autophagy was inhibited with 20 mM NH₄Cl and GAPDH was used as a loading control. (G) Calculated mean fluorescence intensity values (AU) from control and patient fibroblasts treated with or without 5 mM 4-PBA for 24 h and stained for lamins A and C (N = 300). The whiskers show mean values ±s.d and boxplots show the 75th and 25th percentiles, *p < 0.05, ***p < 0.001.

FIGURE 5

Illustrative picture of degradation of K48-ubiquitinated lamins A and C. In normal cells, wild-type lamins A and C are partially ubiquitinated with K48-linked chains that lead to UPS-mediated degradation of lamins A and C. In the patient cells, lamins A and C are increasingly K48-ubiquitinated, presumably due to increased turnover and production, as well as proteasomal dysfunction. This leads to saturation of UPS and an accumulation of K48-linked ubiquitin chains within the nucleus. Saturation and dysfunction of UPS further lead to compensatory degradation of K48-lamins A and C through autophagy, created with BioRender.com.