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Biomedical subjects

Stephen G Young

Publications and source records attributed to Stephen G Young.

14 recordsLinked to original sources
PubMed2003

Targeting Ras signaling through inhibition of carboxyl methylation: an unexpected property of methotrexate.

The antifolate methotrexate is one of the most successful drugs in cancer chemotherapy. Although its efficacy is widely attributed to a decrease in nucleotide biosynthesis (1), methotrexate is known to increase homocysteine (2), a compound associated with an elevated risk of heart disease, Alzheimer's disease (3), and neural tube defects (4). A potential mechanism for the detrimental effects of homocysteine is cellular hypomethylation from an increase in S-adenosylhomocysteine (5), an inhibitor of methyltransferases including isoprenylcysteine carboxyl methyltransferase (Icmt). Among the substrates of Icmt is the monomeric G protein Ras, a critical component of many signaling pathways that regulate cell growth and differentiation. Because carboxyl methylation of Ras is important for proper plasma membrane localization and function (6), we investigated the role of Icmt in the antiproliferative effect of methotrexate. After methotrexate treatment of DKOB8 cells, Ras methylation is decreased by almost 90%. This hypomethylation is accompanied by a mislocalization of Ras to the cytosol and a 4-fold decrease in the activation of p44 mitogen-activated protein kinase and Akt. Additionally, cells lacking Icmt are highly resistant to methotrexate. Whereas cells expressing wild-type levels of Icmt are inhibited by methotrexate, stable expression of myristoylated H-Ras, which does not require carboxyl methylation for membrane attachment (7), confers resistance to methotrexate. These results suggest that inhibition of Icmt is a critical component of the antiproliferative effect of methotrexate, expanding our understanding of this widely used drug and identifying Icmt as a target for drug discovery.

Animals↗
PubMed2003

Eliminating atherogenesis in mice by switching off hepatic lipoprotein secretion.

BACKGROUND: LDL receptor-deficient "apolipoprotein (apo)-B100-only" mice (Ldlr-/-Apob100/100 have elevated LDL cholesterol levels on a chow diet and develop severe aortic atherosclerosis. We hypothesized that both the hypercholesterolemia and the susceptibility to atherosclerosis could be eliminated by switching off hepatic lipoprotein production. METHODS AND RESULTS: We bred Ldlr-/-Apob100/100 mice that were homozygous for a conditional allele for Mttp (the gene for microsomal triglyceride transfer protein) and the inducible Mx1-Cre transgene. In these animals, which we called "Reversa mice," the hypercholesterolemia could be reversed, without modifying the diet or initiating a hypolipidemic drug, by the transient induction of Cre expression in the liver. After Cre induction, hepatic Mttp expression was virtually eliminated (as judged by quantitative real-time PCR), hepatic lipoprotein secretion was abolished (as judged by electron microscopy), and LDLs were virtually eliminated from the plasma. Intestinal lipoprotein production was unaffected. In mice fed a chow diet, Cre induction reduced plasma cholesterol levels from 233.9+/-46.0 to 37.2+/-6.5 mg/dL. In mice fed a high-fat diet, cholesterol levels fell from 525.7+/-32.2 to 100.6+/-14.3 mg/dL. The elimination of hepatic lipoprotein production completely prevented both the development of atherosclerosis and the changes in gene expression that accompany atherogenesis. CONCLUSIONS: We developed mice in which hypercholesterolemia can be reversed with a genetic switch. These mice will be useful for understanding gene-expression changes that accompany the reversal of hypercholesterolemia and atherosclerosis.

Animals↗
PubMed2003

Blocking microsomal triglyceride transfer protein interferes with apoB secretion without causing retention or stress in the ER.

Microsomal triglyceride transfer protein (MTP) is an intraluminal protein in the endoplasmic reticulum (ER) that is essential for the assembly of apolipoprotein B (apoB)-containing lipoproteins. In this study, we examine how the livers of mice respond to two distinct methods of blocking MTP function: Cre-mediated disruption of the gene for MTP and chemical inhibition of MTP activity. Blocking MTP significantly reduced plasma levels of triglycerides, cholesterol, and apoB-containing lipoproteins in both wild-type C57BL/6 and LDL receptor-deficient mice. While treating LDL receptor-deficient mice with an MTP inhibitor for 7 days lowered plasma lipids to control levels, liver triglyceride levels were increased by only 4-fold. Plasma levels of apoB-100 and apoB-48 fell by >90% and 65%, respectively, but neither apoB isoform accumulated in hepatic microsomes. Surprisingly, loss of MTP expression was associated with a nearly complete absence of apoB-100 in hepatic microsomes. Levels of microsomal luminal chaperone proteins [e.g., protein disulfide isomerase, glucose-regulated protein 78 (GRP78), and GRP94] and cytosolic heat shock proteins (HSPs) (e.g., HSP60, HSC, HSP70, and HSP90) were unaffected by MTP inhibition. These findings show that the liver responds rapidly to inhibition of MTP by degrading apoB and preventing its accumulation in the ER. The rapid degradation of secretion-incompetent apoB in the ER may block the induction of proteins associated with unfolded protein and heat shock responses.

Animals↗
PubMed2003

BayGenomics: a resource of insertional mutations in mouse embryonic stem cells.

The BayGenomics gene-trap resource (http://baygenomics.ucsf.edu) provides researchers with access to thousands of mouse embryonic stem (ES) cell lines harboring characterized insertional mutations in both known and novel genes. Each cell line contains an insertional mutation in a specific gene. The identity of the gene that has been interrupted can be determined from a DNA sequence tag. Approximately 75% of our cell lines contain insertional mutations in known mouse genes or genes that share strong sequence similarities with genes that have been identified in other organisms. These cell lines readily transmit the mutation to the germline of mice and many mutant lines of mice have already been generated from this resource. BayGenomics provides facile access to our entire database, including sequence tags for each mutant ES cell line, through the World Wide Web. Investigators can browse our resource, search for specific entries, download any portion of our database and BLAST sequences of interest against our entire set of cell line sequence tags. They can then obtain the mutant ES cell line for the purpose of generating knockout mice.

Animals↗
PubMed2002

Immunochemical evidence that human apoB differs when expressed in rodent versus human cells.

LDL from human apolipoprotein B-100 (apoB-100) transgenic (HuBTg+/+) mice contains more triglyceride than LDL from normolipidemic subjects. To obtain novel monoclonal antibody (MAb) probes of apoB conformation, we generated hybridomas from HuBTg+/+ that had been immunized with LDL isolated from human plasma. One apoE-specific and four anti-apoB-100-specific hybridomas were identified. Two MAbs, 2E1 and 3D11, recognized an epitope in the amino-terminal 689 residues of apoB in native apoB-containing lipoproteins (LpBs) from human plasma or from the supernatant of human hepatoma HepG2 cells, but did not react with LpB from HuBTg+/+ mice or LpB secreted by human apoB-100-transfected rat McArdle 7777 hepatoma cells. 2E1 reacted weakly and 3D11 reacted strongly with apoB from HuBTg+/+ mice after SDS-PAGE. The lack of expression of the 2E1 and 3D11 epitopes on native LpB from HuBTg+/+ mice did not solely reflect the abnormal lipid composition of murine LpB. Both epitopes were detected in all human plasma samples tested and in all human plasma LpB classes. Therefore, human apoB expressed by rodent hepatocytes or hepatoma cells appears to adopt a different conformation or undergoes different posttranslational modification than apoB expressed in human hepatocytes or hepatoma cells.

Animals↗
PubMed2002

Hematologic effects of inactivating the Ras processing enzyme Rce1.

Posttranslational processing of Ras proteins has attracted considerable interest as a potential target for anticancer drug discovery. Rce1 encodes an endoprotease that facilitates membrane targeting of Ras and other prenylated proteins by releasing the carboxyl-terminal 3 amino acids (ie, the -AAX of the CAAX motif). Homozygous Rce1 mutant embryos (Rce1(-/-)) die late in gestation. To characterize the role of Rce1 in hematopoiesis, we performed adoptive transfers and investigated cells from the recipients. Rce1(-/-) fetal liver cells rescued lethally irradiated recipients and manifested normal long-term repopulating potential in competitive repopulation assays. The recipients of Rce1(-/-) cells developed modest elevations in mature myeloid cells (neutrophils + monocytes), but remained well. Bone marrow cells from mice that received transplants of Rce1(-/-) activated extracellular signal-related kinase (ERK) normally in response to granulocyte-macrophage colony-stimulating factor. These data suggest that pharmacologic inhibitors of Rce1 will have minimal effects on normal hematopoietic cells.

Adoptive Transfer↗
PubMed2002

Defining the importance of phosphatidylserine synthase 2 in mice.

Phosphatidylserine synthase 1 (Pss1) and phosphatidylserine synthase 2 (Pss2) produce phosphatidylserine by exchanging serine for the head groups of other phospholipids. Pss1 and Pss2 are structurally similar (approximately 32% amino acid identity) but differ in their substrate specificities, with Pss1 using phosphatidylcholine for the serine exchange reaction and Pss2 using phosphatidylethanolamine. Whether Pss1 and Pss2 are both required for mammalian growth and development is not known, and no data exist on the relative contributions of the two enzymes to serine exchange activities in different tissues. To address those issues and also to define the cell type-specific expression of Pss2, we generated Pss2-deficient mice in which a beta-galactosidase marker is expressed from Pss2 regulatory sequences. Histologic studies of Pss2-deficient mice revealed very high levels of beta-galactosidase expression in Sertoli cells of the testis and high levels of expression in brown fat, neurons, and myometrium. The ability of testis extracts from Pss2-deficient mice to catalyze serine exchange was reduced by more than 95%; reductions of approximately 90% were noted in the brain and liver. However, we found no perturbations in the phospholipid content of any of these tissues. As judged by Northern blots, the expression of Pss1 was not up-regulated in Pss2-deficient cells and tissues. Testis weight was reduced in Pss2-deficient mice, and some of the male mice were infertile. We conclude that Pss2 is responsible for the majority of serine exchange activity in in vitro assays, but a deficiency in this enzyme does not cause perturbations in phospholipid content or severe developmental abnormalities.

Animals↗
PubMed2002

Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect.

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.

Age Factors↗
PubMed2002

Prenylcysteine lyase deficiency in mice results in the accumulation of farnesylcysteine and geranylgeranylcysteine in brain and liver.

In in vitro experiments, prenylcysteine lyase (Pcly) cleaves the thioether bond of prenylcysteines to yield free cysteine and the aldehyde of the isoprenoid lipid. However, the importance of this enzyme has not yet been fully defined at the biochemical or physiologic level. In this study, we show that Pcly is expressed at high levels in mouse liver, kidney, heart, and brain. To test whether Pcly deficiency would cause prenylcysteines to accumulate in tissues and result in pathologic consequences, we produced Pcly-deficient cell lines and Pcly-deficient mice (Pcly-/-). Pcly activity levels were markedly reduced in Pcly-/- cells and tissues. Pcly-/- fibroblasts were more sensitive than wild-type fibroblasts to growth inhibition when prenylcysteines were added to the cell culture medium. To determine if the reduced Pcly enzyme activity levels led to an accumulation of prenylcysteines within cells, mass spectrometry was used to measure farnesylcysteine and geranylgeranylcysteine levels in the tissues of Pcly-/- mice and wild-type controls. These studies revealed a striking accumulation of both farnesylcysteine and geranylgeranylcysteine in the brain and liver of Pcly-/- mice. This accumulation did not appear to be accompanied by significant pathologic consequences. Pcly-/- mice were healthy and fertile, and surveys of more than 30 tissues did not uncover any abnormalities. We conclude that prenylcysteine lyase does play a physiologic role in cleaving prenylcysteines in mammals, but the absence of this activity does not lead to major pathologic consequences.

Amino Acid Sequence↗
PubMed2002

Absence of the CAAX endoprotease Rce1: effects on cell growth and transformation.

After isoprenylation, the Ras proteins and other CAAX proteins undergo two additional enzymatic modifications-endoproteolytic release of the last three amino acids of the protein by the protease Rce1 and methylation of the carboxyl-terminal isoprenylcysteine by the methyltransferase Icmt. This postisoprenylation processing is thought to be important for the association of Ras proteins with membranes. Blocking postisoprenylation processing, by inhibiting Rce1, has been suggested as a potential approach for retarding cell growth and blocking cellular transformation. The objective of this study was to develop a cell culture system for addressing these issues. We generated mice with a conditional Rce1 allele (Rce1(flox)) and produced Rce1(flox/flox) fibroblasts. Cre-mediated excision of Rce1 (thereby producing Rce1(Delta/Delta) fibroblasts) eliminated Ras endoproteolytic processing and methylation and caused a partial mislocalization of truncated K-Ras and H-Ras fusion proteins within cells. Rce1(Delta/Delta) fibroblasts grew more slowly than Rce1(flox/flox) fibroblasts. The excision of Rce1 also reduced Ras-induced transformation, as judged by the growth of colonies in soft agar. The excision of Rce1 from a Rce1(flox/flox) skin carcinoma cell line also significantly retarded the growth of cells, and this effect was exaggerated by cotreatment of the cells with a farnesyltransferase inhibitor. These studies support the idea that interference with postisoprenylation processing retards cell growth, limits Ras-induced transformation, and sensitizes tumor cells to a farnesyltransferase inhibitor.

Alkyl and Aryl Transferases↗
PubMed2001

Blocking the secretion of hepatic very low density lipoproteins renders the liver more susceptible to toxin-induced injury.

Recently, we generated mice lacking microsomal triglyceride transfer protein (MTP) in the liver (Mttp(Delta/Delta)) and demonstrated that very low density lipoprotein secretion from hepatocytes was almost completely blocked. The blockade in lipoprotein production was accompanied by mild to moderate hepatic steatosis, but the mice appeared healthy. Although hepatic MTP deficiency appeared to be innocuous, we hypothesized that a blockade in very low density lipoprotein secretion and the accompanying steatosis might increase the sensitivity of Mttp(Delta/Delta) livers to additional hepatic insults. To address this issue, we compared the susceptibility of Mttp(Delta/Delta) mice and Mttp(flox/flox) controls to hepatic injury from Escherichia coli lipopolysaccharides, concanavalin A, and Pseudomonas aeruginosa exotoxin A. At baseline, neither the Mttp(Delta/Delta) nor the Mttp(flox/flox) mice had elevated serum transaminases or histologic evidence of hepatic inflammation. After the administration of the toxins, however, the Mttp(Delta/Delta) mice manifested higher levels of transaminases and, unlike the Mttp(flox/flox) mice, developed histologic evidence of hepatic inflammation. The toxic challenge induced tumor necrosis factor-alpha to a similar extent in Mttp(Delta/Delta) and Mttp(flox/flox) mice, but other parameters of injury (e.g. chemokine transcript levels and lipid peroxides) were disproportionately increased in the Mttp(Delta/Delta) mice. Our results suggest that blocking lipoprotein secretion in the liver may increase the susceptibility of the liver to certain toxic challenges.

ADP Ribose Transferases↗