Mutations in the gene encoding nuclear lamin A (LA) cause the premature aging disease Hutchinson-Gilford Progeria Syndrome. The most common of these mutations results in the expression of a mutant LA, with a 50-aa deletion within its C terminus. In this study, we demonstrate that this deletion leads to a stable farnesylation and carboxymethylation of the mutant LA (LA⌬50/progerin). These modifications cause an abnormal association of LA⌬50/ progerin with membranes during mitosis, which delays the onset and progression of cytokinesis. Furthermore, we demonstrate that the targeting of nuclear envelope/lamina components into daughter cell nuclei in early G 1 is impaired in cells expressing LA⌬50/ progerin. The mutant LA also appears to be responsible for defects in the retinoblastoma protein-mediated transition into S-phase, most likely by inhibiting the hyperphosphorylation of retinoblastoma protein by cyclin D1/cdk4. These results provide insights into the mechanisms responsible for premature aging and also shed light on the role of lamins in the normal process of human aging.cell division ͉ nuclear lamins ͉ nuclear structure ͉ progeria ͉ protein farnesylation

We have used two-dimensional 1H NMR spectroscopy to determine the solution structure of the DNA oligonucleotide d(5'-CGCTAGCG-3')2 complexed with the bis-intercalating dye 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis[4-(3-methyl -2,3- dihydrobenzo-1,3-thiazolyl-2-methylidene)qui nolinium] tetraiodide (TOTO). The determination of the structure was based on total relaxation matrix analysis of the NOESY cross-peak intensities using the program MARDIGRAS. Improved procedures to consider the experimental "noise" in NOESY spectra during these calculations have been employed. The NOE-derived distance restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for the TOTO complex from both A-form and B-form dsDNA starting structures. The root-mean-square (rms) deviation of the coordinates for the 40 structures of the complex was 1.45 A. The local DNA structure is distorted in the complex. The helix is unwound by 60 degrees and has an overall helical repeat of 12 base pairs, caused by bis-intercalation of TOTO. The poly(propylenamine) linker chain is located in the minor groove of dsDNA. Calculations indicate that the benzothiazole ring system is twisted relative to the quinoline in the uncomplexed TOTO molecule. The site selectivity of TOTO for the CTAG-CTAG site is explained by its ability to adapt to the base pair propeller twist of dsDNA to optimize stacking and the hydrophobic interaction between the thymidine methyl group and the benzothiazole ring. There is a 3000-fold fluorescence enhancement upon binding of TOTO to dsDNA. Rotation about the cyanine methine bonds is possible in free TOTO, allowing relaxation nonradiatively. When bound to dsDNA, the benzothiazole ring and the quinolinium ring are clamped by the nucleobases preventing this rotation, and the chromophore loses excitation energy by fluorescence instead.

Polychlorinated biphenyls (PCBs) may undergo cytochrome P-450-catalyzed hydroxylations to form chlorinated dihydroxybiphenyl metabolites. When the hydroxyl groups are ortho or para to each other, oxidation to a quinone may be catalyzed by peroxidases present within the cell. In order to study the reactivity of PCB-derived quinones, selected chlorophenyl 1,2- and 1,4-benzoquinones were synthesized and characterized, including their reduction potentials against a saturated calomel electrode. Two quinones, 4-(4'-chlorophenyl)-1,2-, and 4-(3',4'-dichlorophenyl)-1,2-benzoquinone, were obtained via the oxidation of the corresponding dihydroxybiphenyls with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Six 1,4-benzoquinones were synthesized via the Meerwein arylation: 2-(2'-chlorophenyl)-1,4-, 2-(3'-chlorophenyl)-1,4-, 2-(4'-chlorophenyl)-1,4-, 2-(2',5'-dichlorophenyl)-1,4-, 2-(3',4'-dichlorophenyl)-1,4-, and 2-(3',5'-dichlorophenyl)-1,4-benzoquinone. As a model study, the rate of reactivity of 2-(4'-chlorophenyl)-1,4-benzoquinone toward the nitrogen nucleophiles glycine, L-arginine, L-histidine- and L-lysine was determined under pseudo-first-order conditions at pH 7.4. The rate constants ranged from 0.45 to 0.75 min-1 M-1. Higher rates were obtained under conditions of higher pH. Two reaction products were identified as the 5- and 6-ring addition products in the ratio of 1:4. In contrast, the reaction of 2-(4'-chlorophenyl)-1,4-benzoquinone with the sulfur nucleophiles glutathione or N-acetyl-L-cysteine was instantaneous. The major product of the reaction of glutathione with 2-(4'-chlorophenyl)-1,4-benzoquinone was also the 6-ring addition product. The hydroquinone thioether could be enzymatically reoxidized to the quinone thioether. Also, the influence of atmospheric oxygen and superoxide dismutase on the rates of the following horseradish peroxidase/H2O2-catalyzed oxidations was investigated: 3,4-dichloro-2',5'-dihydroxybiphenyl to 2-(3',4'-dichlorophenyl)-1,4-benzoquinone and 3,4-dichloro-3',4'-dihydroxybiphenyl to 4-(3',4'-dichlorophenyl)-1,2-benzoquinone. While the presence or absence of atmospheric oxygen did not alter the rates of the oxidation reactions, the presence of superoxide dismutase significantly increased the rates of both oxidation reactions, having the greater effect on the oxidation of the 1,4-hydroquinone. These data show that PCB-derived quinones react with both nitrogen and sulfur nucleophiles of the cell and may explain, in part, the toxic effects of individual PCBs and PCB formulations, such as glutathione depletion, oxidative stress, and cell death.

Hutchinson-Gilford progeria syndrome (HGPS), a rare disease that results in what appears to be premature aging, is caused by the production of a mutant form of prelamin A known as progerin. Progerin retains a farnesyl lipid anchor at its carboxyl terminus, a modification that is thought to be important in disease pathogenesis. Inhibition of protein farnesylation improves the hallmark nuclear shape abnormalities in HGPS cells and ameliorates disease phenotypes in mice harboring a knockin HGPS mutation (Lmna HG/+ ). The amelioration of disease, however, is incomplete, leading us to hypothesize that nonfarnesylated progerin also might be capable of eliciting disease. To test this hypothesis, we created knockin mice expressing nonfarnesylated progerin (Lmna nHG/+ ). Lmna nHG/+ mice developed the same disease phenotypes observed in Lmna HG/+ mice, although the phenotypes were milder, and mouse embryonic fibroblasts (MEFs) derived from these mice contained fewer misshapen nuclei. The steady-state levels of progerin in Lmna nHG/+ MEFs and tissues were lower, suggesting a possible explanation for the milder phenotypes. These data support the concept that inhibition of protein farnesylation in progeria could be therapeutically useful but also suggest that this approach may be limited, as progerin elicits disease phenotypes whether or not it is farnesylated.

Lamin A is formed from prelamin A by four post-translational processing steps-farnesylation, release of the last three amino acids of the protein, methylation of the farnesylcysteine and the endoproteolytic release of the C-terminal 15 amino acids (including the farnesylcysteine methyl ester). When the final processing step does not occur, a farnesylated and methylated prelamin A accumulates in cells, causing a severe progeroid disease, restrictive dermopathy (RD). Whether RD is caused by the retention of farnesyl lipid on prelamin A, or by the retention of the last 15 amino acids of the protein, is unknown. To address this issue, we created knock-in mice harboring a mutant Lmna allele (LmnanPLAO) that yields exclusively non-farnesylated prelamin A (and no lamin C). These mice had no evidence of progeria but succumbed to cardiomyopathy. We suspected that the non-farnesylated prelamin A in the tissues of these mice would be strikingly mislocalized to the nucleoplasm, but this was not the case; most was at the nuclear rim (indistinguishable from the lamin A in wild-type mice). The cardiomyopathy could not be ascribed to an absence of lamin C because mice expressing an otherwise identical knock-in allele yielding only wild-type prelamin A appeared normal. We conclude that lamin C synthesis is dispensable in mice and that the failure to convert prelamin A to mature lamin A causes cardiomyopathy (at least in the absence of lamin C). The latter finding is potentially relevant to the long-term use of protein farnesyltransferase inhibitors, which lead to an accumulation of non-farnesylated prelamin A.