Progeroid syndrome patients with ZMPSTE24 deficiency could benefit when treated with rapamycin and dimethylsulfoxide.
ABSTRACT: Patients with progeroid syndromes such as mandibuloacral dysplasia, type B (MADB) and restrictive dermopathy (RD) harbor mutations in zinc metalloproteinase (ZMPSTE24), an enzyme essential for posttranslational proteolysis of prelamin A to form mature lamin A. Dermal fibroblasts from these patients show increased nuclear dysmorphology and reduced proliferation; however, the efficacy of various pharmacological agents in reversing these cellular phenotypes remains unknown. In this study, fibroblasts from MADB patients exhibited marked nuclear abnormalities and reduced proliferation that improved upon treatment with rapamycin and dimethylsulfoxide but not with other agents, including farnesyl transferase inhibitors. Surprisingly, fibroblasts from an RD patient with a homozygous null mutation in ZMPSTE24, resulting in exclusive accumulation of prelamin A with no lamin A on immunoblotting of cellular lysate, exhibited few nuclear abnormalities and near-normal cellular proliferation. An unbiased proteomic analysis of the cellular lysate from RD fibroblasts revealed a lack of processing of vimentin, a cytoskeletal protein. Interestingly, the assembly of the vimentin microfibrils in MADB fibroblasts improved with rapamycin and dimethylsulfoxide. We conclude that rapamycin and dimethylsulfoxide are beneficial for improving nuclear morphology and cell proliferation of MADB fibroblasts. Data from a single RD patient's fibroblasts also suggest that prelamin A accumulation by itself might not be detrimental and requires additional alterations at the cellular level to manifest the phenotype.
Project description:Patients with progeroid syndromes like mandibuloacral dysplasia, type B (MADB) and restrictive dermopathy (RD), harbor mutations in zinc metalloproteinase (ZMPSTE24), an enzyme essential for post-translational proteolysis of prelamin A to form mature lamin A. Dermal fibroblasts from these patients show increased nuclear dysmorphology and reduced proliferation, however, efficacy of various pharmacological agents in reversing these cellular phenotypes remains unknown. In this study, fibroblasts from MADB patients exhibited marked nuclear abnormalities and reduced proliferation which improved upon treatment with rapamycin and dimethylsulfoxide but not with other agents including farnesyl transferase inhibitors. Surprisingly, fibroblasts from an RD patient with homozygous null mutation in ZMPSTE24, resulting in exclusive accumulation of prelamin A with no lamin A on immunoblotting of cellular lysate, exhibited few nuclear abnormalities and near normal cellular proliferation. An unbiased proteomic analysis of the cellular lysate from RD fibroblasts revealed lack of processing of vimentin, a cytoskeletal protein. Interestingly, the assembly of the vimentin microfibrils in MADB fibroblasts improved with rapamycin and dimethylsulfoxide. We conclude that rapamycin and dimethylsulfoxide are beneficial for improving nuclear morphology and cell proliferation of MADB fibroblasts. Data from RD fibroblasts suggest that prelamin A accumulation by itself may not be detrimental.
Project description:Restrictive dermopathy (RD) is a lethal human genetic disorder characterized by very tight, thin, easily eroded skin, rocker bottom feet, and joint contractures. This disease was recently reported to be associated with a single heterozygous mutation in ZMPSTE24 and hypothesized to be a digenic disorder (Navarro et al, Lamin A and ZMPSTE24 (FACE-1) defects cause nuclear disorganization and identify restrictive dermopathy as a lethal neonatal laminopathy. Hum Mol Genet 13:2493-2503, 2004). ZMPSTE24 encodes an enzyme necessary for the correct processing and maturation of lamin A, an intermediate filament component of the nuclear envelope. Here we present four unrelated patients with homozygous mutations in ZMPSTE24 and a fifth patient with compound heterozygous mutations in ZMPSTE24. Two of the three different mutations we found are novel, and all are single base insertions that result in messenger RNA frameshifts. As a consequence of the presumed lack of ZMPSTE24 activity, prelamin A, the unprocessed toxic form of lamin A, was detected in the nuclei of both cultured cells and tissue from RD patients, but not in control nuclei. Abnormally aggregated lamin A/C was also observed. These results indicate that RD is an autosomal recessive laminopathy caused by inactivating ZMPSTE24 mutations that result in defective processing and nuclear accumulation of prelamin A.
Project description:The human zinc metalloprotease ZMPSTE24 is an integral membrane protein crucial for the final step in the biogenesis of the nuclear scaffold protein lamin A, encoded by LMNA After farnesylation and carboxyl methylation of its C-terminal CAAX motif, the lamin A precursor (prelamin A) undergoes proteolytic removal of its modified C-terminal 15 amino acids by ZMPSTE24. Mutations in LMNA or ZMPSTE24 that impede this prelamin A cleavage step cause the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS), and the related progeroid disorders mandibuloacral dysplasia type B (MAD-B) and restrictive dermopathy (RD). Here, we report the development of a 'humanized yeast system' to assay ZMPSTE24-dependent cleavage of prelamin A and examine the eight known disease-associated ZMPSTE24 missense mutations. All mutations show diminished prelamin A processing and fall into three classes, with defects in activity, protein stability or both. Notably, some ZMPSTE24 mutants can be rescued by deleting the E3 ubiquitin ligase Doa10, involved in endoplasmic reticulum (ER)-associated degradation of misfolded membrane proteins, or by treatment with the proteasome inhibitor bortezomib. This finding may have important therapeutic implications for some patients. We also show that ZMPSTE24-mediated prelamin A cleavage can be uncoupled from the recently discovered role of ZMPSTE24 in clearance of ER membrane translocon-clogged substrates. Together with the crystal structure of ZMPSTE24, this humanized yeast system can guide structure-function studies to uncover mechanisms of prelamin A cleavage, translocon unclogging, and membrane protein folding and stability.
Project description:A variety of missense mutations in LMNA (the gene for lamin C and prelamin A) cause familial partial lipodystrophy (FPLD), a disease associated with reduced adipose tissue, particularly in the limbs. Several studies have reported that fibroblasts from FPLD subjects have an accumulation of prelamin A. Those findings were intriguing but also perplexing because many of the LMNA missense mutations associated with lipodystrophy are located in sequences distant from the sequences required for the farnesylation of prelamin A and ZMPSTE24-mediated conversion of prelamin A to mature lamin A. Here, we revisited the issue of prelamin A accumulation in the setting of FPLD mutations. We used western blots with lamin A/C antibodies and prelamin A-specific monoclonal antibodies to assess prelamin A levels in wild-type fibroblasts and fibroblasts carrying LMNA mutations associated with lipodystrophy (R482W, I299V, C591F, T528M). None of the mutant fibroblasts exhibited an accumulation of prelamin A. Also, the amount of prelamin A accumulation in response to lopinavir (an inhibitor of ZMPSTE24) was similar in wild-type and mutant fibroblasts. Thus, the LMNA lipodystrophy mutations that we examined did not lead to prelamin A accumulation, nor did they render those cells more susceptible to prelamin A accumulation when ZMPSTE24 was inhibited by lopinavir.
Project description:Several progeroid disorders are caused by deficiency in the endoprotease ZMPSTE24 which leads to accumulation of prelamin A at the nuclear envelope. ZMPSTE24 cleaves prelamin A twice: at the third carboxyl-terminal amino acid following farnesylation of a -CSIM motif; and 15 residues upstream to produce mature lamin A. The carboxyl-terminal cleavage can also be performed by RAS-converting enzyme 1 (RCE1) but little is known about the importance of this cleavage for the ability of prelamin A to cause disease. Here, we found that knockout of RCE1 delayed senescence and increased proliferation of ZMPSTE24-deficient fibroblasts from a patient with non-classical Hutchinson-Gilford progeria syndrome (HGPS), but did not influence proliferation of classical LMNA-mutant HGPS cells. Knockout of Rce1 in Zmpste24-deficient mice at postnatal week 4-5 increased body weight and doubled the median survival time. The absence of Rce1 in Zmpste24-deficient fibroblasts did not influence nuclear shape but reduced an interaction between prelamin A and AKT which activated AKT-mTOR signaling and was required for the increased proliferation. Prelamin A levels increased in Rce1-deficient cells due to a slower turnover rate but its localization at the nuclear rim was unaffected. These results strengthen the idea that the presence of misshapen nuclei does not prevent phenotype improvement and suggest that targeting RCE1 might be useful for treating the rare progeroid disorders associated with ZMPSTE24 deficiency.
Project description:Hutchinson-Gilford progeria syndrome (HGPS) is a devastating premature aging disease resulting from a mutation in the LMNA gene, which encodes nuclear lamins A and C. Lamin A is synthesized as a precursor (prelamin A) with a C-terminal CaaX motif that undergoes farnesylation, endoproteolytic cleavage, and carboxylmethylation. Prelamin A is subsequently internally cleaved by the zinc metalloprotease Ste24 (Zmpste24) protease, which removes the 15 C-terminal amino acids, including the CaaX modifications, to yield mature lamin A. HGPS results from a dominant mutant form of prelamin A (progerin) that has an internal deletion of 50 aa near the C terminus that includes the Zmpste24 cleavage site and blocks removal of the CaaX-modified C terminus. Fibroblasts from HGPS patients have aberrant nuclei with irregular shapes, which we hypothesize result from the abnormal persistence of the farnesyl and/or carboxylmethyl CaaX modifications on progerin. If this hypothesis is correct, inhibition of CaaX modification by mutation or pharmacological treatment should alleviate the nuclear morphology defect. Consistent with our hypothesis, we find that expression in HeLa cells of GFP-progerin or an uncleavable form of prelamin A with a Zmpste24 cleavage site mutation induces the formation of abnormal nuclei similar to those in HGPS fibroblasts. Strikingly, inhibition of farnesylation pharmacologically with the farnesyl transferase inhibitor rac-R115777 or mutationally by alteration of the CaaX motif dramatically reverses the abnormal nuclear morphology. These results suggest that farnesyl transferase inhibitors represent a possible therapeutic option for individuals with HGPS and/or other laminopathies due to Zmpste24 processing defects.
Project description:The zinc metalloprotease ZMPSTE24 plays a critical role in nuclear lamin biology by cleaving the prenylated and carboxylmethylated 15-amino acid tail from the C-terminus of prelamin A to yield mature lamin A. A defect in this proteolytic event, caused by a mutation in the lamin A gene (LMNA) that eliminates the ZMPSTE24 cleavage site, underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Likewise, mutations in the ZMPSTE24 gene that result in decreased enzyme function cause a spectrum of diseases that share certain features of premature aging. Twenty human ZMPSTE24 alleles have been identified that are associated with three disease categories of increasing severity: mandibuloacral dysplasia type B (MAD-B), severe progeria (atypical 'HGPS') and restrictive dermopathy (RD). To determine whether a correlation exists between decreasing ZMPSTE24 protease activity and increasing disease severity, we expressed mutant alleles of ZMPSTE24 in yeast and optimized in vivo yeast mating assays to directly compare the activity of alleles associated with each disease category. We also measured the activity of yeast crude membranes containing the ZMPSTE24 mutant proteins in vitro. We determined that, in general, the residual activity of ZMPSTE24 patient alleles correlates with disease severity. Complete loss-of-function alleles are associated with RD, whereas retention of partial, measureable activity results in MAD-B or severe progeria. Importantly, our assays can discriminate small differences in activity among the mutants, confirming that the methods presented here will be useful for characterizing any new ZMPSTE24 mutations that are discovered.
Project description:Several progeroid disorders, including Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (ZMPSTE24 deficiency), arise when a farnesylated and methylated form of prelamin A accumulates at the nuclear envelope. Here, we found that a hypomorphic allele of isoprenylcysteine carboxyl methyltransferase (ICMT) increased body weight, normalized grip strength, and prevented bone fractures and death in Zmpste24-deficient mice. The reduced ICMT activity caused prelamin A mislocalization within the nucleus and triggered prelamin A-dependent activation of AKT-mammalian target of rapamycin (mTOR) signaling, which abolished the premature senescence of Zmpste24-deficient fibroblasts. ICMT inhibition increased AKT-mTOR signaling and proliferation and delayed senescence in human HGPS fibroblasts but did not reduce the levels of misshapen nuclei in mouse and human cells. Thus, targeting ICMT might be useful for treating prelamin A-associated progeroid disorders.
Project description:Lamin A is part of a complex structural meshwork located beneath the nuclear envelope and is involved in both structural support and the regulation of gene expression. Lamin A is initially expressed as prelamin A, which contains an extended carboxyl terminus that undergoes a series of post-translational modifications and subsequent cleavage by the endopeptidase ZMPSTE24 to generate lamin A. To facilitate investigations of the role of this cleavage in normal and disease states, we developed a monoclonal antibody (PL-1C7) that specifically recognizes prelamin A at the intact ZMPSTE24 cleavage site, ensuring prelamin A detection exclusively. Importantly, PL-1C7 can be used to determine prelamin A localization and accumulation in cells where lamin A is highly expressed without the use of exogenous fusion proteins. Our results show that unlike mature lamin A, prelamin A accumulates as discrete and localized foci at the nuclear periphery. Furthermore, whereas treatment with farnesylation inhibitors of cells overexpressing a GFP-prelamin A fusion protein results in the formation of large nucleoplasmic clumps, these aggregates are not observed upon similar treatment of cells expressing endogenous prelamin A or in cells lacking ZMPSTE24 expression and/or activity. Finally, we show that specific laminopathy-associated mutations exhibit both positive and negative effects on prelamin A accumulation, indicating that these mutations affect prelamin A processing efficiency in different manners.
Project description:The nuclear lamins A, B, and C are intermediate filament proteins that form a nuclear scaffold adjacent to the inner nuclear membrane in higher eukaryotes, providing structural support for the nucleus. In the past two decades it has become evident that the final step in the biogenesis of the mature lamin A from its precursor prelamin A by the zinc metalloprotease ZMPSTE24 plays a critical role in human health. Defects in prelamin A processing by ZMPSTE24 result in premature aging disorders including Hutchinson Gilford Progeria Syndrome (HGPS) and related progeroid diseases. Additional evidence suggests that defects in prelamin A processing, due to diminished ZMPSTE24 expression or activity, may also drive normal physiological aging. Because of the important connection between prelamin A processing and human aging, there is increasing interest in how ZMPSTE24 specifically recognizes and cleaves its substrate prelamin A, encoded by LMNA. Here, we describe two humanized yeast systems we have recently developed to examine ZMPSTE24 processing of prelamin A. These systems differ from one another slightly. Version 1.0 is optimized to analyze ZMPSTE24 mutations, including disease alleles that may affect the function or stability of the protease. Using this system, we previously showed that some ZMPSTE24 disease alleles that affect stability can be rescued by the proteasome inhibitor bortezomib, which may have therapeutic implications. Version 2.0 is designed to analyze LMNA mutations at or near the ZMPSTE24 processing site to assess whether they permit or impede prelamin A processing. Together these systems offer powerful methodology to study ZMPSTE24 disease alleles and to dissect the specific residues and features of the lamin A tail that are required for recognition and cleavage by the ZMPSTE24 protease.