Expression of disease-causing lamin A mutants impairs the formation of DNA repair foci

Abstract

Lamins are nuclear proteins that are components of a fibrous network underlying the inner nuclear membrane termed the nuclear lamina, and are also distributed throughout the interior of the nucleus (reviewed by Goldman et al., 2002). Lamins have been classified into two types based on biochemical properties and expression patterns. B-type lamins are expressed in most cells and are encoded by two separate genes, B1 and B2, whereas the A-type lamins have been detected primarily in differentiated cell types. The lamin A gene (LMNA) encodes lamin A and C transcripts, as well as germcell-specific lamin C2. Lamins belong to the intermediate filament family of proteins and have a short N-terminal head domain followed by an ␣-helical rod domain and a globular tail domain. The C-termini of lamins A, B1 and B2 bear a CAAX motif that is post-translationally modified and, in the case of lamin A, is subjected to a further maturation step by which prelamin A is proteolytically cleaved to give mature lamin A (reviewed by Stuurman et al., 1998).Mutations in human LMNA cause several debilitating diseases, collectively termed laminopathies, that affect skeletal and cardiac muscle, adipose, bone and neuronal tissues, and also cause premature ageing syndromes. The majority of mutations cause autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) (Bonne et al., 1999), whereas other mutations cause dilated cardiomyopathy with conduction system disease (DCM) (Fatkin et al., 1999), limb girdle muscular dystrophy , or familial partial lipodystrophy (FPLD) (Shackleton et al., 2000;Cao and Hegele, 2000). Mutations in LMNA have also been linked to the relatively rare autosomal recessive disorders CharcotMarie-Tooth disorder type 2 (De Sandre-Giovannoli et al., 2002) and mandibuloacral dysplasia (Novelli et al., 2002), and more recently to restrictive dermopathy (Navarro et al., 2004). Many of the above mutations are missense mutations that occur throughout the gene, although the FPLD mutations are clustered near the C-terminus. An interesting finding has been the linkage of mutations in LMNA to Hutchinson-Gilford progeria syndrome (HGPS) (Eriksson et al., 2003; De SandreGiovannoli et al., 2003) and to atypical Werner's syndrome . The most frequent mutation in HGPS is a nucleotide substitution (GGC to GGT) that does not cause an amino acid change (G608G) but activates a cryptic splice site that leads to a deletion of 50 amino acids near the C-terminus (residues 607-656), giving rise to a truncated protein termed lamin A del50 or progerin, which retains the C-terminus of prelamin A and is not proteolytically processed. A few missense mutations such as R471C and R527C that do not affect processing of the C-terminus of lamin A have also been linked to HGPS and atypical progerias (Eriksson et al., 2003;Cao and Hegele, 2003). Cells from patients expressing mutant lamins often exhibit a range of dominant-negative effects such as abnormal nuclear morphology, aberrant lamin assembly and altered gene regulation (reviewed by Wilson, 2000;Worman...

The R527H mutation in LMNA gene causes an increased sensitivity to ionizing radiation

“…This suggests that expression of lamin A mutants affect γ-H2AX redistribution into the nucleus in the presence of DNA damage. This result is in agreement with data described by Manju and coworkers, 30 who observed that cells expressing LMNA mutations causing progeria syndromes (Del50, R471C, R527C and L530P), form a lower percentage of γ-H2AX foci in response to mild doses of cisplatin or UV irradiation, in contrast to cells expressing wild-type GFP-lamin A or the LMNA disease-causing mutations H222P and R482L. Interestingly, a significant higher level of residual damage was observed in irradiated-MADA cells after 24 h from IR.…”

Identification of Markers for Cellular Stress and Senescence in Laminopathic Cells by Proteomic Analysis

“…1A, B). However, GFP-tagged lamin mutant Q294P was distributed in aggregates throughout the nucleus, and its expression resulted in aberrant nuclear morphology and disruption of the endogenous lamin A network, consistent with our earlier observation in other cell types (Manju et al, 2006). Importantly, we made the novel observation that endogenous lamin B1 was depleted in 88% of cells expressing lamin A mutant Q294P compared to <5% of cells expressing wild-type lamin A (Fig.…”

“…Cells were transfected with a green fluorescent protein (GFP)-tagged lamin A construct by electroporation and maintained in the medium for 24 h. The two constructs that were used were wild-type lamin A and Q294P lamin mutant that causes EMD and have been described earlier (Manju et al, 2006). GFPexpressing cells were sorted in a fluorescence activated cell sorter (FACS) in order to obtain >99% cells expressing the GFP-tagged lamin.…”

Lamin A precursor induces barrier-to-autointegration factor nuclear localization

“…GFP-BAF, 17 full-length FLAG-tagged rat prelamin A (LA-WT, pCI mammalian expression vector) and the mutated constructs LA-C661M, LA-L647R and LA-∆50 were transiently transfected into HEK293 cells using FuGene reagent (Roche). 9,12,23,44 Biochemical and immunofluorescence analyses were performed 24 hours after transfection. In human skin fibroblast cells the accumulation of different prelamin A intermediates were obtained using 25 µM mevinolin (Sigma), 10,12 or 20 µM 1-N-acetyl-S-farnesyl-Lcysteine methylester (AFCMe, Alexis).…”