A -type lamins are intermediate filaments and major components of the nuclear lamina, a filamentous network underlying the inner nuclear membrane that provides structural and mechanical stability for the nucleus in nearly all differentiated cells. A-type lamins interact with heterochromatin and transcriptional regulators, highlighting their important role in chromatin organization, gene expression, and DNA repair.1 The 2 main A-type lamin isoforms, lamin-A and lamin-C, arise from alternative splicing of the LMNA gene. The precursor of lamin-A, prelamin-A, undergoes a complex post-translational maturation comprising a step of C-terminal farnesylation followed by carboxymethylation and a proteolytic cleavage by the metalloprotease ZMPSTE24, resulting in the carboxymethylated C-terminal removal of the protein, including its farnesyl group, and in the release of mature lamin-A. 1LMNA mutations cause inherited diseases commonly named laminopathies, including muscular dystrophies, cardiomyopathies, progeroid phenotypes, and lipodystrophic syndromes.2 Among them, the Dunnigan-type familial partial lipodystrophy (FPLD2; OMIM #151660) is mainly attributable to LMNA p.R482 heterozygous substitutions. 3,4 This syndrome is characterized by a gradual atrophy of subcutaneous adipose tissue in the extremities, gluteal, and truncal areas © 2013 American Heart Association, Inc. heterozygous substitutions, and the effects of p.R482W-prelamin-A overexpression in human coronary artery endothelial cells. In 68% of FPLD2 patients, early atherosclerosis was attested by clinical cardiovascular events, occurring before the age of 45 in most cases. In transduced endothelial cells, exogenous wild-type-prelamin-A was correctly processed and localized, whereas p.R482W-prelamin-A accumulated abnormally at the nuclear envelope. Patients' fibroblasts also showed a predominant nuclear envelope distribution with a decreased rate of prelamin-A maturation. Only p.R482W-prelamin-A induced endothelial dysfunction, with decreased production of NO, increased endothelial adhesion of peripheral blood mononuclear cells, and cellular senescence. p.R482W-prelamin-A also induced oxidative stress, DNA damages, and inflammation. These alterations were prevented by treatment of endothelial cells with pravastatin, which inhibits prelamin-A farnesylation, or with antioxidants. In addition, pravastatin allowed the correct relocalization of p.R482W-prelamin-A within the endothelial cell nucleus. These data suggest that farnesylated p.R482W-prelamin-A accumulation at the nuclear envelope is a toxic event, leading to cellular oxidative stress and endothelial dysfunction. Conclusions-LMNA p.R482 mutations, responsible for FPLD2, exert a direct proatherogenic effect in endothelial cells, which could contribute to patients' early atherosclerosis. (Arterioscler Thromb Vasc Biol. 2013;33:2162-2171.)
The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-alpha-phosphatidyl ethanolamine (DC-Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent-labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene delivery in dividing and S-phase growth-arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. Strikingly, mu peptide associates with nuclei and nucleoli of cells within less than 15 mins incubation of LMD with cells, which suggests that mu peptide has an NLS function. These NLS properties were confirmed by cloning of a mu-beta-galactosidase fusion protein that localises in the nuclei of cells after cytosolic translation. In dividing cells both LMD and LND deliver pDNA(Cy3) to nuclei within 30-45 min incubation with cells. By contrast, pDNA is detected only in the cytoplasm in growth-arrested cells over the period of time investigated, and not in the nuclei. LD systems prepared from DC-Chol/DOPE cationic liposomes and pDNA(Cy3) behave similarly to LMD systems, which suggests that mu peptide is unable to influence trafficking events in this current LMD formulation, in spite of its strong NLS capacity. We further describe the effect of polyethyleneglycol (PEG) on cellular uptake. "Stealth" systems obtained by post-coating LMD particles with fluorescent-labelled PEG molecules (0.5, 5 and 10 mol % fluorescein-PEG(5000)-N-hydroxysuccinimide) were prepared and shown to be internalised rapidly (mins) by cells, without detectable transgene expression. This result indicates that PEG blocks intracellular trafficking of pDNA.
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