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.

MeCP2-E1 isoform is a dynamically expressed, weakly DNA-bound protein with different protein and DNA interactions compared to MeCP2-E2

“…To allow possible N-terminal in vitro modifications in a mammalian cell system, the NTDs of MeCP2-E1 and MeCP2-E2 were expressed in HEK293T cells and were subsequently purified using immunoprecipitation of recombinant fusion protein, followed by mass spectrometry and peptide analysis. Our MS analysis of PTM for MeCP2-E1 was reported previously (6), and showed no peptides with N-terminal methionine (NM), indicating complete NM excision (NME) at the first residue (P1) position. Acetylation of the initial alanine residue (P’1) after NME was observed (Fig.…”

“…Differentiation was performed using a sequential treatment of Retinoic Acid (Sigma-Aldrich) and BDNF (Alomone Labs) as previously described (19). For transfection experiments cells were seeded and one day later were transfected with MeCP2-E1 and MeCP2-E2 C-terminal GFP fusion expression vectors (6) using Lipofectamine 3000 (Invitrogen) according to manufacturer instructions. Cycloheximide treatments were performed 48 h after transfection.…”

“…The remaining sequence is identical for both isoforms, and encompasses the methyl binding domain (MBD), intervening domain (ID), transcriptional repression domain (TRD) and C-terminal domain (CTD) (5). MeCP2-E1 is likely the ancestral form of the protein, as orthologues are present across vertebrate evolution, whereas orthologous sequences of the exon 2 coding region have only been found in mammalian genomes (6).…”

MeCP2 AT-Hook1 mutations in patients with intellectual disability and/or schizophrenia disrupt DNA binding and chromatin compaction in vitro

“…The two isoforms are identical for the remainder of the protein, including the methyl‐CpG‐binding domain (MBD) and transcriptional repressor domain (TRD), however the MECP2_E1 transcript is the more highly expressed in brain (Mnatzakanian et al., ; Kriaucionis & Bird, 2004). Recently, we have reviewed N‐terminal post‐translational modifications, translation efficiency and half‐life of the MECP2_E1 isoform in detail (Sheikh, de Paz, Akhtar, Ausió, & Vincent, ).…”

MeCP2 and Chromatin Compartmentalization

“…Finally, we address how these properties regulate and mediate the ability of MeCP2 to orchestrate chromatin compartmentalization and higher order genome architecture.Cells 2020, 9, 878 2 of 31 the predominant isoform in brain and has an earlier expression onset than MeCP2 e2 [6]. The two isoforms are commonly considered as functionally equivalent, yet recent evidence shows that MeCP2 e1 plays a role in neuronal maturation [7] and is more relevant for RTT [8][9][10]. In view of the fact that MeCP2 e2 isoform was the first to be known and a much larger body of literature pertains to this isoform, we will, throughout, use amino acid coordinates from MeCP2 e2 isoform.Both variants include two functionally characterized domains: the methyl-CpG binding domain (MBD) and the transcriptional repression domain (TRD).…”