MeCP2_E1 N-terminal modifications affect its degradation rate and are disrupted by the Ala2Val Rett mutation

Abstract

Methyl CpG-binding protein 2 (MeCP2), the mutated protein in Rett syndrome (RTT), is a crucial chromatin-modifying and gene-regulatory protein that has two main isoforms (MeCP2_E1 and MeCP2_ E2) due to the alternative splicing and switching between translation start codons in exons one and two. Functionally, these two isoforms appear to be virtually identical; however, evidence suggests that only MeCP2_E1 is relevant to RTT, including a single RTT missense mutation in exon 1, Ala2Val. Here, we show that N-terminal co- and post-translational modifications differ for MeCP2_E1 and MeCP2_E1-Ala2Val, which result in different protein degradation rates in vitro. We report complete N-methionine excision (NME) for MeCP2_E1 and evidence of excision of multiple alanine residues from the N-terminal polyalanine stretch. For MeCP2_E1-Ala2Val, we observed only partial NME and N-acetylation (NA) of either methionine or valine. The localization of MeCP2_E1 and co-localization with chromatin appear to be unaffected by the Ala2Val mutation. However, a higher proteasomal degradation rate was observed for MeCP2_E1-Ala2Val compared with that for wild type MeCP2_E1. Thus, the etiopathology of Ala2Val is likely due to a reduced bio-availability of MeCP2 because of the faster degradation rate of the unmodified defective protein. Our data on the effects of the Ala2Val mutation on N-terminal modifications of MeCP2 may be applicable to Ala2Val mutations in other disease genes for which no etiopathological mechanism has been established.

Figure 1

MeCP2 N-terminal domain (NTD) sequence of wild type MeCP2_E1, Ala2Val (A2V) and E2, and the effect of NTD mutation MeCP2_E1 A2V on the translocation and localization of the protein. (A) Amino acid sequence of the MeCP2 NTD; residue counted as in (7) of the mutant MeCP2_E1-A2V and wild type MeCP2_E1/E2. (B) XY Image stacks of neuronally differentiated SK-N-SH (human neuroblastoma) cells that transiently expressed the full-length MeCP2 _E1 and mutant (A2V), representing the co-localization at chromocenters (DAPI; column 1) and the recombinant GFP-tagged protein (column 2) and the merge of 1 and 2 (column 3). (C) Mean of the Pearson correlation coefficient (PCC) values (MeCP2-WT n = 11 and MeCP2-A2V n = 15 cells; ±SD shown) of the wild type and mutant. (D) Scatter plot of blue and green pixel intensities, showing the co-localization of DNA (DAPI) and recombinant MeCP2 protein (GFP), respectively.

Figure 2

Mass spectrometry sequencing of the N-terminal of the MeCP2 protein (in vitro). N-terminal peptide coverage alignment chart and high-resolution mass spectra, showing N-methionine excision (NME) and N-acetylation (NA) of the N-terminus of (A) MeCP2_E1; (B) MeCP2_E1-p.A2V. NA (+42 Da) of N-terminus amino acid is shown highlighted in yellow.

Figure 3

Cell viability and cycloheximide (CHX) chase assays. (A) Representative confocal image of SK-N-SH cells treated with 10 µg/mL cycloheximide (merged images, 10X4 objective, 512X512 resolution). Blue (Hoechst 33342) for total cell staining, Red (propidium iodide) for dead nuclei staining and green channel for WT MeCP2_E1-GFP. (B) Graphical representation of averaged total cells of CHX-treated cells for 0, 24 and 48 h; (C) Average percent dead cells (blue/red merged) (D) Average percent transfected (WT-MeCP2_E1-GFP) cells (from B-C n = 6, ±error bars = SD). (E) Western blot (WB) analysis of CHX treated SH-SY5Y cells harvested after 0 (lane 1), 24 (lane 2) and 48 h (lane 3), using anti-GFP and anti-actin antibodies; (F) WB analysis of the CHX treated SK-N-SH cells harvested after 0 (lane 1), 24 (lane 2) and 48 h (lane 3), using anti-GFP and anti-tubulin antibodies. Replicate WBs are shown in Supplementary Material, Figure S4.

Figure 4

Live cell Real-Time dynamics, degradation rates and half-life of the MeCP2 protein. (A) FRAP assays, showing the recovery at different time points. (B) Fluorescent recovery of MeCP2 wild type and p.A2V. Averaged recovered intensities were plotted as a function of time from 299 image stacks (512 × 512) (SEM bar shown on every 10th value; n = 5). (C) Bleach chase of MeCP2_E1 WT and A2V. Real-time protein recovery slope of bleached, Pv(t), and unbleached P(t) cell with the function of time (7 h). (D) Protein degradation rates or removal rates (α) of the same cells shown in C. (E) Protein half-life and degradation rates calculated by taking the slope of the difference between the Pv(t) and P(t) protein fluorescence on a semi-logarithmic plot using a linear regression ($\mathrm{l}\mathrm{n}(P\left(t\right)-Pv\left(t\right))$(28).