Muscular Dystrophy Mutations Impair the Nuclear Envelope Emerin Self-assembly Properties

Abstract

More than 100 genetic mutations causing X-linked Emery–Dreifuss muscular dystrophy have been identified in the gene encoding the integral inner nuclear membrane protein emerin. Most mutations are nonsense or frameshift mutations that lead to the absence of emerin in cells. Only very few cases are due to missense or short in-frame deletions. Molecular mechanisms explaining the corresponding emerin variants’ loss of function are particularly difficult to identify because of the mostly intrinsically disordered state of the emerin nucleoplasmic region. We now demonstrate that this EmN region can be produced as a disordered monomer, as revealed by nuclear magnetic resonance, but rapidly self-assembles in vitro. Increases in concentration and temperature favor the formation of long curvilinear filaments with diameters of approximately 10 nm, as observed by electron microscopy. Assembly of these filaments can be followed by fluorescence through Thioflavin-T binding and by Fourier-transform Infrared spectrometry through formation of β-structures. Analysis of the assembly properties of five EmN variants reveals that del95–99 and Q133H impact filament assembly capacities. In cells, these variants are located at the nuclear envelope, but the corresponding quantities of emerin–emerin and emerin–lamin proximities are decreased compared to wild-type protein. Furthermore, variant P183H favors EmN aggregation in vitro, and variant P183T provokes emerin accumulation in cytoplasmic foci in cells. Substitution of residue Pro183 might systematically favor oligomerization, leading to emerin aggregation and mislocalization in cells. Our results suggest that emerin self-assembly is necessary for its proper function and that a loss of either the protein itself or its ability to self-assemble causes muscular dystrophy.

Emerin self‐assembly mechanism: role of the LEM domain

“…Figure 2A shows that long curvilinear filaments were detected for EmN, as reported earlier [25], and also for EmN132. Therefore, we incubated the different fragments at a fixed concentration of 600 lM during 1 week at 293 K. We then observed the samples using negative staining electron microscopy.…”

“…1A) is composed of a globular LEM domain, from residue 1 to residue 45 [28], and a large intrinsically disordered region, from residue 46 to residue 187 [25,29]. 1A) is composed of a globular LEM domain, from residue 1 to residue 45 [28], and a large intrinsically disordered region, from residue 46 to residue 187 [25,29].…”

“…Its N-terminal nucleoplasmic region (residues 1-221) contains all six mutations described above. We have found that fragment from residue 1 to residue 187 also self-associates in vitro and that residues 95-99 are important for self-association in vitro and in cells [25]. Fragment from residue 170 to residue 220 is sufficient to bind other emerin molecules homotypically [24].…”

“…These interactions are key events of the nucleocytoskeletal coupling machinery and critically contribute to nuclear structure. We and others showed that emerin self-associates in vitro and in cells and that defects in oligomerization were observed together with impaired lamin binding in the case of two mutants causing Emery-Dreifuss muscular dystrophy [24,25]. Recent X-ray crystallography studies have revealed that changes in SUN2 oligomeric states are associated with large structural rearrangements and regulate nesprin binding [20][21][22][23].…”

Immunohistochemistry on a panel of Emery–Dreifuss muscular dystrophy samples reveals nuclear envelope proteins as inconsistent markers for pathology

“…Thus, the relevance of this redistribution to EDMD pathology is unclear even in the patients where it was observed. One recent study suggested a link between emerin cytoplasmic accumulation and pathology in that emerin-p.P183T assembles into oligomers that perhaps cannot pass through the peripheral channels of the nuclear pore complexes [38]. Nonetheless, unlike this particular case, most reported emerin mutations result in a loss of protein.…”