Zmpste24 is an integral membrane metalloproteinase of the endoplasmic reticulum. Biochemical studies of tissues from Zmpste24-deficient mice (Zmpste24 ؊/؊ ) have indicated a role for Zmpste24 in the processing of CAAX-type prenylated proteins. Here, we report the pathologic consequences of Zmpste24 deficiency in mice. Zmpste24 ؊/؊ mice gain weight slowly, appear malnourished, and exhibit progressive hair loss. The most striking pathologic phenotype is multiple spontaneous bone fractures-akin to those occurring in mouse models of osteogenesis imperfecta. Cortical and trabecular bone volumes are significantly reduced in Zmpste24 ؊/؊ mice. Zmpste24 ؊/؊ mice also manifested muscle weakness in the lower and upper extremities, resembling mice lacking the farnesylated CAAX protein prelamin A. Prelamin A processing was defective both in fibroblasts lacking Zmpste24 and in fibroblasts lacking the CAAX carboxyl methyltransferase Icmt but was normal in fibroblasts lacking the CAAX endoprotease Rce1. Muscle weakness in Zmpste24 ؊/؊ mice can be reasonably ascribed to defective processing of prelamin A, but the brittle bone phenotype suggests a broader role for Zmpste24 in mammalian biology.metalloproteinase ͉ knockout mice ͉ brittle bones ͉ CAAX motif T he mammalian zinc metalloproteinase Zmpste24 has attracted attention because it shares a high degree of sequence identity with Ste24p, a Saccharomyces cerevisiae enzyme required for the maturation of the farnesylated mating pheromone a-factor (1-3). Ste24p plays two distinct roles in a-factor biogenesis (2, 4). First, it acts as a CAAX endoprotease, clipping off the C-terminal three amino acids from the protein (i.e., the ϪAAX of the CAAX motif) (3). Release of the ϪAAX from a-factor can also be mediated by Rce1p, the CAAX endoprotease involved in Ras processing (3). The removal of the ϪAAX exposes a carboxyl-terminal farnesylcysteine, which is methylated by Ste14p (5). Second, Ste24p clips the amino-terminal extension of a-factor, rendering it susceptible to a final endoproteolytic cleavage by Axl1p or Ste23p (6). Aside from a-factor, no other substrates for Ste24p have been identified, but other substrates likely exist because genetic screens in yeast have demonstrated that STE24 mutations can reverse the topological orientation of membrane proteins (7) and can affect the viability of yeast with mutations in genes encoding actin cytoskeleton proteins (8).Zmpste24 faithfully carries out both of Ste24p's processing steps in a-factor biogenesis and thus is a bona fide Ste24p ortholog (2, 9). Although it would be tempting to speculate that Zmpste24 processes an ''a-factor-like'' peptide in mammals, no a-factor ortholog has yet been identified. We have previously speculated that prelamin A (a precursor to lamin A, a component of the nuclear lamina) might be a Zmpste24 substrate (2, 6) because prelamin A (like yeast a-factor) is a farnesylated CAAX protein that undergoes more than one proteolytic processing step (10). After the removal of the C-terminal ϪAAX, an additional 15 res...

Hutchinson-Gilford progeria syndrome (HGPS), a progeroid syndrome in children, is caused by mutations in LMNA (the gene for prelamin A and lamin C) that result in the deletion of 50 aa within prelamin A. In normal cells, prelamin A is a ''CAAX protein'' that is farnesylated and then processed further to generate mature lamin A, which is a structural protein of the nuclear lamina. The mutant prelamin A in HGPS, which is commonly called progerin, retains the CAAX motif that triggers farnesylation, but the 50-aa deletion prevents the subsequent processing to mature lamin A. The presence of progerin adversely affects the integrity of the nuclear lamina, resulting in misshapen nuclei and nuclear blebs. We hypothesized that interfering with protein farnesylation would block the targeting of progerin to the nuclear envelope, and we further hypothesized that the mislocalization of progerin away from the nuclear envelope would improve the nuclear blebbing phenotype. To approach this hypothesis, we created a gene-targeted mouse model of HGPS, generated genetically identical primary mouse embryonic fibroblasts, and we then examined the effect of a farnesyltransferase inhibitor on nuclear blebbing. The farnesyltransferase inhibitor mislocalized progerin away from the nuclear envelope to the nucleoplasm, as determined by immunofluoresence microscopy, and resulted in a striking improvement in nuclear blebbing (P < 0.0001 by 2 statistic). These studies suggest a possible treatment strategy for HGPS.aging ͉ lamin A͞C ͉ laminopathy H utchinson-Gilford progeria syndrome (HGPS) is a progeroid syndrome characterized by a host of aging-like phenotypes, including a wizened appearance of the skin, osteoporosis, alopecia, and premature atherosclerosis (1). Children with HGPS die at the mean age of 13, generally from myocardial infarctions or strokes (1). This disease is caused by the accumulation of a mutant form of prelamin A that cannot be processed to mature lamin A (1). In normal cells, wild-type prelamin A is virtually undetectable because it is fully converted to mature lamin A, a structural protein of the nuclear lamina (2, 3). The nuclear lamina is an intermediate filament meshwork adjacent to the inner nuclear membrane that provides structural support for the nucleus (2, 3).Prelamin A contains a nuclear localization signal and terminates with a CAAX motif (2), in which C is a cysteine, A residues are usually aliphatic amino acids, and X can be one of many different residues. CAAX motifs are also found on lamin B1, lamin B2, the Ras family of proteins, and many other cellular proteins. The CAAX motif triggers three sequential enzymatic posttranslational modifications, beginning with protein prenylation. In the case of prelamin A, the first processing step is carried out by protein farnesyltransferase (FTase) and involves the addition of a 15-carbon farnesyl lipid to the thiol group of the cysteine within the CAAX motif. Second, the last 3 aa of the protein (i.e., ϪAAX) are removed by a prenylprotein-specific endoprotease. For p...

Progerias are rare genetic diseases characterized by premature aging. Several progeroid disorders are caused by mutations that lead to the accumulation of a lipid-modified (farnesylated) form of prelamin A, a protein that contributes to the structural scaffolding for the cell nucleus. In progeria, the accumulation of farnesyl-prelamin A disrupts this scaffolding, leading to misshapen nuclei. Previous studies have shown that farnesyltransferase inhibitors (FTIs) reverse this cellular abnormality. We tested the efficacy of an FTI (ABT-100) in Zmpste24-deficient mice, a mouse model of progeria. The FTI-treated mice exhibited improved body weight, grip strength, bone integrity, and percent survival at 20 weeks of age. These results suggest that FTIs may have beneficial effects in humans with progeria.

Zmpste24 is a metalloproteinase required for the processing of prelamin A to lamin A, a structural component of the nuclear lamina. Zmpste24 deficiency results in the accumulation of prelamin A within cells, a complete loss of mature lamin A, and misshapen nuclear envelopes. Zmpste24-deficient (Zmpste24 ؊/؊ ) mice exhibit retarded growth, alopecia, micrognathia, dental abnormalities, osteolytic lesions in bones, and osteoporosis, which are phenotypes shared with Hutchinson-Gilford progeria syndrome, a human disease caused by the synthesis of a mutant prelamin A that cannot undergo processing to lamin A. Zmpste24 ؊/؊ mice also develop muscle weakness. We hypothesized that prelamin A might be toxic and that its accumulation in Zmpste24 ؊/؊ mice is responsible for all of the disease phenotypes. We further hypothesized that Zmpste24 ؊/؊ mice with half-normal levels of prelamin A (Zmpste24 ؊/؊ mice with one Lmna knockout allele) would be subjected to less toxicity and be protected from disease. Thus, we bred and analyzed Zmpste24 ؊/؊ Lmna ؉/؊ mice. As expected, prelamin A levels in Zmpste24 ؊/؊ Lmna ؉/؊ cells were significantly reduced. Zmpste24 ؊/؊ Lmna ؉/؊ mice were entirely normal, lacking all disease phenotypes, and misshapen nuclei were less frequent in Zmpste24 ؊/؊ Lmna ؉/؊ cells than in Zmpste24 ؊/؊ cells. These data suggest that prelamin A is toxic and that reducing its levels by as little as 50% provides striking protection from disease.Hutchinson-Gilford progeria syndrome ͉ prelamin A

The segmental premature aging disease, Hutchinson-Gilford Progeria (HGPS) is caused by a truncated and farnesylated form of Lamin A. In a mouse model for HGPS, a similar Lamin A variant causes the proliferative arrest and death of post-natal but not embryonic fibroblasts. Arrest is due to an inability to produce a functional extracellular matrix (ECM), as growth on normal ECM rescues proliferation. The defects are associated with inhibition of canonical Wnt signaling, due to reduced nuclear localization and transcriptional activity of Lef1, but not Tcf4, in both mouse and human progeric cells. Defective Wnt signaling, affecting ECM synthesis, maybe critical to the etiology of HGPS as mice exhibit skeletal defects and apoptosis in major blood vessels proximal to the heart. These results establish a functional link between the nuclear envelope/lamina and the cell surface/ECM and may provide insights into the role of Wnt signaling and the ECM in aging.

Lamin A and lamin C, both products of Lmna, are key components of the nuclear lamina. In the mouse, a deficiency in both lamin A and lamin C leads to slow growth, muscle weakness, and death by 6 weeks of age. Fibroblasts deficient in lamins A and C contain misshapen and structurally weakened nuclei, and emerin is mislocalized away from the nuclear envelope. The physiologic rationale for the existence of the 2 different Lmna products lamin A and lamin C is unclear, although several reports have suggested that lamin A may have particularly important functions, for example in the targeting of emerin and lamin C to the nuclear envelope. Here we report the development of lamin C-only mice (Lmna LCO/LCO ), which produce lamin C but no lamin A or prelamin A (the precursor to lamin A). Lmna LCO/LCO mice were entirely healthy, and Lmna LCO/LCO cells displayed normal emerin targeting and exhibited only very minimal alterations in nuclear shape and nuclear deformability. Thus, at least in the mouse, prelamin A and lamin A appear to be dispensable. Nevertheless, an accumulation of farnesyl-prelamin A (as occurs with a deficiency in the prelamin A processing enzyme Zmpste24) caused dramatically misshapen nuclei and progeria-like disease phenotypes. The apparent dispensability of prelamin A suggested that lamin A-related progeroid syndromes might be treated with impunity by reducing prelamin A synthesis. Remarkably, the presence of a single Lmna LCO allele eliminated the nuclear shape abnormalities and progeria-like disease phenotypes in Zmpste24 -/-mice. Moreover, treating Zmpste24 -/-cells with a prelamin A-specific antisense oligonucleotide reduced prelamin A levels and significantly reduced the frequency of misshapen nuclei. These studies suggest a new therapeutic strategy for treating progeria and other lamin A diseases.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by a LMNA mutation that leads to the synthesis of a mutant prelamin A that is farnesylated but cannot be further processed to mature lamin A. A more severe progeroid disorder, restrictive dermopathy (RD), is caused by the loss of the prelamin A-processing enzyme, ZMPSTE24. The absence of ZMPSTE24 prevents the endoproteolytic processing of farnesyl-prelamin A to mature lamin A and leads to the accumulation of farnesyl-prelamin A. In both HGPS and RD, the farnesyl-prelamin A is targeted to the nuclear envelope, where it interferes with the integrity of the nuclear envelope and causes misshapen cell nuclei. Recent studies have shown that the frequency of misshapen nuclei can be reduced by treating cells with a farnesyltransferase inhibitor (FTI). Also, administering an FTI to mouse models of HGPS and RD ameliorates the phenotypes of progeria. These studies have prompted interest in testing the efficacy of FTIs in children with HGPS.

Precision, long-term stability, linearity and accuracy of the x-ray peripheral quantitative computerized tomographic (pQCT) bone scanner XCT 3000 (Norland-Stratec Medical Sys.) were evaluated using the European Forearm Phantom (EFP). In vivo measurements were assessed using a standardized procedure at the distal femur and the distal tibia. In the patient-scan mode, the spatial resolution of the system was 1.04 +/- 0.05 lp/mm as measured at the 10% level of the modulation transfer function (MTF). The contrast-detail diagram (CDD) yielded a minimal difference in attenuation coefficient (AC) of 0.07 cm(-1) at an object size of 0.5 mm. The effective dose for humans was calculated to be less than 1.5 microSv per scan. Short-term precision in vivo was expressed as root mean square standard deviation of paired measurements of 20 healthy volunteers (RMSSD = 0.5%). At the distal femur total volumetric density (ToD) and total cross-sectional area (ToA) were found to be less sensitive to positioning errors than at the distal tibia. Structural parameters like the polar cross-sectional moment of inertia (CSMIp) or the polar cross-sectional moment of resistance (CSMRp) showed a good short-term precision at the distal femur (RMSSD = 1.2 and 1.4%). The relation between the two skeletal sites with respect to CSMIp or CSMRp showed a high coefficient of determination (r2 = 0.77 and 0.74).