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Biomedical subjects

Stephen G Young

Publications and source records attributed to Stephen G Young.

At least 37 records · Page 2Linked to original sources
PubMed2006

A farnesyltransferase inhibitor improves disease phenotypes in mice with a Hutchinson-Gilford progeria syndrome mutation.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the production of a truncated prelamin A, called progerin, which is farnesylated at its carboxyl terminus. Progerin is targeted to the nuclear envelope and causes misshapen nuclei. Protein farnesyltransferase inhibitors (FTI) mislocalize progerin away from the nuclear envelope and reduce the frequency of misshapen nuclei. To determine whether an FTI would ameliorate disease phenotypes in vivo, we created gene-targeted mice with an HGPS mutation (LmnaHG/+) and then examined the effect of an FTI on disease phenotypes. LmnaHG/+ mice exhibited phenotypes similar to those in human HGPS patients, including retarded growth, reduced amounts of adipose tissue, micrognathia, osteoporosis, and osteolytic lesions in bone. Osteolytic lesions in the ribs led to spontaneous bone fractures. Treatment with an FTI increased adipose tissue mass, improved body weight curves, reduced the number of rib fractures, and improved bone mineralization and bone cortical thickness. These studies suggest that FTIs could be useful for treating humans with HGPS.

Animals↗
PubMed2005

Compensatory increase in hepatic lipogenesis in mice with conditional intestine-specific Mttp deficiency.

Microsomal TG transfer protein (MTTP) is required for the assembly and secretion of TG (TG)-rich lipoproteins from both enterocytes and hepatocytes. Liver-specific deletion of Mttp produced a dramatic reduction in plasma very low density lipoprotein-TG and virtually eliminated apolipoprotein B100 (apoB100) secretion yet caused only modest reductions in plasma apoB48 and apoB48 secretion from primary hepatocytes. These observations prompted us to examine the phenotype following intestine-specific Mttp deletion because murine, like human enterocytes, secrete virtually exclusively apoB48. We generated mice with conditional Mttp deletion in villus enterocytes (Mttp-IKO), using a tamoxifen-inducible, intestine-specific Cre transgene. Villus enterocytes from chow-fed Mttp-IKO mice contained large cytoplasmic TG droplets and no chylomicron-sized particles within the secretory pathway. Chow-fed, Mttp-IKO mice manifested steatorrhea, growth arrest, and decreased cholesterol absorption, features that collectively recapitulate the phenotype associated with abetalipoproteinemia. Chylomicron secretion was reduced dramatically in vivo, in conjunction with an approximately 80% decrease in apoB48 secretion from primary enterocytes. Additionally, although plasma and hepatic cholesterol and TG content were decreased, Mttp-IKO mice demonstrated a paradoxical increase in both hepatic lipogenesis and very low density lipoprotein secretion. These findings establish distinctive features for MTTP involvement in intestinal chylomicron assembly and secretion and suggest that hepatic lipogenesis undergoes compensatory induction in the face of defective intestinal TG secretion.

Animals↗
PubMed2005

Prelamin A, Zmpste24, misshapen cell nuclei, and progeria--new evidence suggesting that protein farnesylation could be important for disease pathogenesis.

Prelamin A undergoes multistep processing to yield lamin A, a structural protein of the nuclear lamina. Prelamin A terminates with a CAAX motif, which triggers farnesylation of a C-terminal cysteine (the C of the CAAX motif), endoproteolytic release of the last three amino acids (the AAX), and methylation of the newly exposed farnesylcysteine residue. In addition, prelamin A is cleaved a second time, releasing 15 more residues from the C terminus (including the farnesylcysteine methyl ester), generating mature lamin A. This second cleavage step is carried out by an endoplasmic reticulum membrane protease, ZMPSTE24. Interest in the posttranslational processing of prelamin A has increased with the recognition that certain progeroid syndromes can be caused by mutations that lead to an accumulation of farnesyl-prelamin A. Recently, we showed that a key cellular phenotype of these progeroid disorders, misshapen cell nuclei, can be ameliorated by inhibitors of protein farnesylation, suggesting a potential strategy for treating these diseases. In this article, we review the posttranslational processing of prelamin A, describe several mouse models for progeroid syndromes, explain the mutations underlying several human progeroid syndromes, and summarize recent data showing that misshapen nuclei can be ameliorated by treating cells with protein farnesyltransferase inhibitors.

Animals↗
PubMed2005

Disruption of the phosphatidylserine decarboxylase gene in mice causes embryonic lethality and mitochondrial defects.

Most of the phosphatidylethanolamine (PE) in mammalian cells is synthesized by two pathways, the CDP-ethanolamine pathway and the phosphatidylserine (PS) decarboxylation pathway, the final steps of which operate at spatially distinct sites, the endoplasmic reticulum and mitochondria, respectively. We investigated the importance of the mitochondrial pathway for PE synthesis in mice by generating mice lacking PS decarboxylase activity. Disruption of Pisd in mice resulted in lethality between days 8 and 10 of embryonic development. Electron microscopy of Pisd-/- embryos revealed large numbers of aberrantly shaped mitochondria. In addition, fluorescence confocal microscopy of Pisd-/- embryonic fibroblasts showed fragmented mitochondria. PS decarboxylase activity and mRNA levels in Pisd+/- tissues were approximately one-half of those in wild-type mice. However, heterozygous mice appeared normal, exhibited normal vitality, and the phospholipid composition of livers, testes, brains, and of mitochondria isolated from livers, was the same as in wild-type littermates. The amount and activity of a key enzyme of the CDP-ethanolamine pathway for PE synthesis, CTP:phosphoethanolamine cytidylyltransferase, were increased by 35-40 and 100%, respectively, in tissues of Pisd+/- mice, as judged by immunoblotting; PE synthesis from [3H]ethanolamine was correspondingly increased in hepatocytes. We conclude that the CDP-ethanolamine pathway in mice cannot substitute for a lack of PS decarboxylase during development. Moreover, elimination of PE production in mitochondria causes fragmented, misshapen mitochondria, an abnormality that likely contributes to the embryonic lethality.

Animals↗
PubMed2005

Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans with progeroid syndromes.

Defects in the biogenesis of lamin A from its farnesylated precursor, prelamin A, lead to the accumulation of prelamin A at the nuclear envelope, cause misshapen nuclei, and result in progeroid syndromes. A deficiency in ZMPSTE24, a protease involved in prelamin A processing, leads to prelamin A accumulation, an absence of mature lamin A, misshapen nuclei, and a lethal perinatal progeroid syndrome: restrictive dermopathy (RD). Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutant prelamin A that cannot be processed to lamin A. The hallmark cellular abnormality in RD and HGPS is misshapen nuclei. We hypothesized that the farnesylation of prelamin A is important for its targeting to the nuclear envelope in RD and HGPS and that blocking farnesylation would ameliorate the nuclear shape abnormalities. Indeed, when RD fibroblasts were treated with a farnesyltransferase inhibitor (FTI), prelamin A was partially mislocalized away from the nuclear envelope, and the frequency of nuclear shape abnormalities was reduced (P < 0.0001). A FTI also mislocalized prelamin A and improved nuclear shape in Zmpste24-deficient mouse embryonic fibroblasts (P < 0.0001) and improved nuclear shape in human HGPS fibroblasts (P < 0.0001). Most remarkably, a FTI significantly improved nuclear shape in two fibroblast cell lines from atypical progeria patients with lamin A missense mutations in the absence of prelamin A accumulation (P = 0.0003 and P < 0.0001). These findings establish a paradigm for ameliorating the most obvious cellular pathology in lamin-related progeroid syndromes and suggest a potential strategy for treating these diseases.

Alkyl and Aryl Transferases↗
PubMed2005

Differential membrane localization of ERas and Rheb, two Ras-related proteins involved in the phosphatidylinositol 3-kinase/mTOR pathway.

Two Ras-related proteins, ERas and Rheb, which are involved in the phosphatidylinositol 3-kinase pathway, display high GTP affinity and have atypical CAAX motifs. The factors governing the intracellular localization of ERas and Rheb are incompletely understood. In the current study, we show by confocal microscopy that ERas is localized to the plasma membrane, whereas Rheb is confined to the endomembranes. Membrane localization of the two proteins was abolished by mutation of the cysteine of the CAAX motif. Membrane targeting was also abolished by a farnesyltransferase inhibitor but not by a geranylgeranyltransferase inhibitor. In mouse fibroblasts deficient in either Rce1 (Ras converting enzyme 1) or Icmt (isoprenylcysteine carboxyl methyltransferase), ERas was mislocalized mainly to the Golgi apparatus, whereas Rheb showed diffuse localization. Mutation of cysteines in the hypervariable region of ERas prevented the plasma membrane localization of ERas, very strongly suggesting that palmitoylation of the cysteines is essential for membrane targeting. The hypervariable region of Rheb does not contain cysteines or polybasic residues, and when it was replaced with the hypervariable region of H-Ras, Rheb displayed plasma membrane localization. These data indicate that ERas shares the same posttranslational modifications with H-Ras and N-Ras and is localized at the plasma membrane. Rheb also shares the same membrane-targeting pathway but because of the absence of palmitoylation is located on endomembranes.

Alkyl and Aryl Transferases↗
PubMed2005

Transgene insertion on mouse chromosome 6 impairs function of the uterine cervix and causes failure of parturition.

The molecular mechanisms controlling the initiation of parturition remain largely undefined. We report a new animal model in which parturition does not occur. A line of mice expressing a human apolipoprotein B (APOB) gene fail to deliver their young if the transgene is present in homozygous (Tg/Tg), but not in heterozygous (Tg/Wt), form. Cloning and mapping of the transgene insertion locus indicate that 10 copies of the 80-kilobase APOB genomic fragment inserted into mouse chromosome 6 result in a small, 390-base pair deletion of mouse genomic DNA. Nine other lines expressing the transgene have normal labor, suggesting that transgene insertion in this mutant line disrupted a mouse gene crucial for successful parturition. The pathophysiology of parturition failure in these animals was defined using physiological, endocrinological, and morphological techniques. Results indicate that luteolysis occurs in Tg/Tg mice but is delayed by 1 day. Delivery did not occur in mutant mice at term after spontaneous luteolysis or even after removal of progesterone action by ovariectomy or antiprogestin treatment. Biomechanical and functional studies of the uterus and cervix revealed that the primary cause of failed parturition in Tg/Tg mice was not inadequate uterine contractions of labor but, rather, a rigid, inelastic cervix at term that was abnormally rich in neutrophils and tissue monocytes. Characterization of the transgene insertional mutant, Tg/Tg, indicates that progesterone withdrawal is insufficient to complete parturition in the presence of inadequate cervical ripening at term.

20-alpha-Dihydroprogesterone↗
PubMed2005

Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation.

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 chi2 statistic). These studies suggest a possible treatment strategy for HGPS.

Alkyl and Aryl Transferases↗
PubMed2005

A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells.

Many key regulatory proteins, including members of the Ras family of GTPases, are modified at their C terminus by a process termed prenylation. This processing is initiated by the addition of an isoprenoid lipid, and the proteins are further modified by a proteolytic event and methylation of the C-terminal prenylcysteine. Although the biological consequences of prenylation have been characterized extensively, the contributions of prenylcysteine methylation to the functions of the modified proteins are not well understood. This reaction is catalyzed by the enzyme isoprenylcysteine carboxyl methyltransferase (Icmt). Recent genetic disruption studies have provided strong evidence that blocking Icmt activity has profound consequences on oncogenic transformation. Here, we report the identification of a selective small-molecule inhibitor of Icmt, 2-[5-(3-methylphenyl)-1-octyl-1H-indol-3-yl]acetamide (cysmethynil). Cysmethynil treatment results in inhibition of cell growth in an Icmt-dependent fashion, demonstrating mechanism-based activity of the compound. Treatment of cancer cells with cysmethynil results in mislocalization of Ras and impaired epidermal growth factor signaling. In a human colon cancer cell line, cysmethynil treatment blocks anchorage-independent growth, and this effect is reversed by overexpression of Icmt. These findings provide a compelling rationale for development of Icmt inhibitors as another approach to anticancer drug development.

Acetamides↗
PubMed2005

N-myristoyltransferase 1 is essential in early mouse development.

N-Myristoyltransferase (NMT) transfers myristate to an amino-terminal glycine of many eukaryotic proteins. In yeast, worms, and flies, this enzyme is essential for viability of the organism. Humans and mice possess two distinct but structurally similar enzymes, NMT1 and NMT2. These two enzymes have similar peptide specificities, but no one has examined the functional importance of the enzymes in vivo. To address this issue, we performed both genetic and biochemical studies. Northern blots with RNA from adult mice and in situ hybridization studies of day 13.5 embryos revealed widespread expression of both Nmt1 and Nmt2. To determine whether the two enzymes are functionally redundant, we generated Nmt1-deficient mice carrying a beta-galactosidase marker gene. beta-Galactosidase staining of tissues from heterozygous Nmt1-deficient (Nmt1+/-) mice and embryos confirmed widespread expression of Nmt1. Intercrosses of Nmt1+/- mice yielded no viable homozygotes (Nmt1-/-), and heterozygotes were born at a less than predicted frequency. Nmt1-/- embryos died between embryonic days 3.5 and 7.5. Northern blots revealed lower levels of Nmt2 expression in early development than at later time points, a potential explanation for the demise of Nmt1-/- embryos. To explore this concept, we generated Nmt1-/- embryonic stem (ES) cells. The Nmt2 mRNA could be detected in Nmt1-/- ES cells, but the total NMT activity levels were reduced by approximately 95%, suggesting that Nmt2 contributes little to total enzyme activity levels in these early embryo cells. The Nmt1-/- ES cells were functionally abnormal; they yielded small embryoid bodies in in vitro differentiation experiments and did not contribute normally to organogenesis in chimeric mice. We conclude that Nmt1 is not essential for the viability of mammalian cells but is required for development, likely because it is the principal N-myristoyltransferase in early embryogenesis.

Acyltransferases↗
PubMed2005

Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases.

The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal- AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the -AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1- and Icmt-deficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins.

Actins↗
PubMed2005

High-level lipoprotein [a] expression in transgenic mice: evidence for oxidized phospholipids in lipoprotein [a] but not in low density lipoproteins.

Efforts to elucidate the role of lipoprotein [a] (Lp[a]) in atherogenesis have been hampered by the lack of an animal model with high plasma Lp[a] levels. We produced two lines of transgenic mice expressing apolipoprotein [a] (apo[a]) in the liver and crossed them with mice expressing human apolipoprotein B-100 (apoB-100), generating two lines of Lp[a] mice. One had Lp[a] levels of approximately 700 mg/dl, well above the 30 mg/dl threshold associated with increased risk of atherosclerosis in humans; the other had levels of approximately 35 mg/dl. Most of the LDL in mice with high-level apo[a] expression was covalently bound to apo[a], but most of the LDL in the low-expressing line was free. Using an enzyme-linked sandwich assay with monoclonal antibody EO6, we found high levels of oxidized phospholipids in Lp[a] from high-expressing mice but not in LDL from low-expressing mice or in LDL from human apoB-100 transgenic mice (P <0.00001), even though all mice had similar plasma levels of human apoB-100. The increase in oxidized lipids specific to Lp[a] in high-level apo[a]-expressing mice suggests a mechanism by which increased circulating levels of Lp[a] could contribute to atherogenesis.

Animals↗
PubMed2005

Assembly of lipoprotein particles containing apolipoprotein-B: structural model for the nascent lipoprotein particle.

Apolipoprotein B (apoB) is the major protein component of large lipoprotein particles that transport lipids and cholesterol. We have developed a detailed model of the first 1000 residues of apoB using standard sequence alignment programs (ClustalW and MACAW) and the MODELLER6 package for three-dimensional homology modeling. The validity of the apoB model was supported by conservation of disulfide bonds, location of all proline residues in turns and loops, and conservation of the hydrophobic faces of the two C-terminal amphipathic beta-sheets, betaA (residues 600-763) and betaB (residues 780-1000). This model suggests a lipid-pocket mechanism for initiation of lipoprotein particle assembly. In a previous model we suggested that microsomal triglyceride transfer protein might play a structural role in completion of the lipid pocket. We no longer think this likely, but instead propose a hairpin-bridge mechanism for lipid pocket completion. Salt-bridges between four tandem charged residues (717-720) in the turn of the hairpin-bridge and four tandem complementary residues (997-1000) at the C-terminus of the model lock the bridge in the closed position, enabling the deposition of an asymmetric bilayer within the lipid pocket.

Amino Acid Sequence↗
PubMed2004

Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice.

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.

Alleles↗
PubMed2004

Lamin B1 is required for mouse development and nuclear integrity.

Lamins are key structural components of the nuclear lamina, an intermediate filament meshwork that lies beneath the inner nuclear membrane. Lamins play a role in nuclear architecture, DNA replication, and gene expression. Mutations affecting A-type lamins have been associated with a variety of human diseases, including muscular dystrophy, cardiomyopathy, lipodystrophy, and progeria, but mutations in B-type lamins have never been identified in humans or in experimental animals. To investigate the in vivo function of lamin B1, the major B-type lamin, we generated mice with an insertional mutation in Lmnb1. The mutation resulted in the synthesis of a mutant lamin B1 protein lacking several key functional domains, including a portion of the rod domain, the nuclear localization signal, and the CAAX motif (the carboxyl-terminal signal for farnesylation). Homozygous Lmnb1 mutant mice survived embryonic development but died at birth with defects in lung and bone. Fibroblasts from mutant embryos grew under standard cell-culture conditions but displayed grossly misshapen nuclei, impaired differentiation, increased polyploidy, and premature senescence. Thus, the lamin B1 mutant mice provide evidence for a broad and nonredundant function of lamin B1 in mammalian development. These mutant mice and cell lines derived from them will be useful models for studying the role of the nuclear lamina in various cellular processes.

Animals↗
PubMed2004

CD1d function is regulated by microsomal triglyceride transfer protein.

CD1d is a major histocompatibility complex (MHC) class I-related molecule that functions in glycolipid antigen presentation to distinct subsets of T cells that express natural killer receptors and an invariant T-cell receptor-alpha chain (invariant NKT cells). The acquisition of glycolipid antigens by CD1d occurs, in part, in endosomes through the function of resident lipid transfer proteins, namely saposins. Here we show that microsomal triglyceride transfer protein (MTP), a protein that resides in the endoplasmic reticulum of hepatocytes and intestinal epithelial cells (IECs) and is essential for lipidation of apolipoprotein B, associates with CD1d in hepatocytes. Hepatocytes from animals in which Mttp (the gene encoding MTP) has been conditionally deleted, and IECs in which Mttp gene products have been silenced, are unable to activate invariant NKT cells. Conditional deletion of the Mttp gene in hepatocytes is associated with a redistribution of CD1d expression, and Mttp-deleted mice are resistant to immunopathologies associated with invariant NKT cell-mediated hepatitis and colitis. These studies indicate that the CD1d-regulating function of MTP in the endoplasmic reticulum is complementary to that of the saposins in endosomes in vivo.

Abetalipoproteinemia↗
PubMed2004

Macrophage-targeted overexpression of urokinase causes accelerated atherosclerosis, coronary artery occlusions, and premature death.

BACKGROUND: Human atherosclerotic lesions contain elevated levels of urokinase plasminogen activator (uPA), expressed predominantly by macrophages. METHODS AND RESULTS: To test the hypothesis that macrophage-expressed uPA contributes to the progression and complications of atherosclerosis, we generated transgenic mice with macrophage-targeted overexpression of uPA. The uPA transgene was bred into the apolipoprotein E-null background, and transgenic mice and nontransgenic littermate controls were fed an atherogenic diet. uPA-transgenic mice had significantly elevated uPA activity in the atherosclerotic artery wall, of a magnitude similar to elevations reported in atherosclerotic human arteries. Compared with littermate controls, uPA-transgenic mice had accelerated atherosclerosis, dilated aortic roots, occlusive proximal coronary artery disease, myocardial infarcts, and early mortality. CONCLUSIONS: These data support the hypothesis that overexpression of uPA by artery wall macrophages is atherogenic and suggest that uPA inhibitors might be therapeutically useful.

Animals↗