Biomedical subjects

Paul L Fox

Publications and source records attributed to Paul L Fox.

At least 19 recordsLinked to original sources

PubMed2022

Analysis of nested alternate open reading frames and their encoded proteins.

Transcriptional and post-transcriptional mechanisms diversify the proteome beyond gene number, while maintaining a sequence relationship between original and altered proteins. A new mechanism breaks this paradigm, generating novel proteins by translating alternative open reading frames (Alt-ORFs) within canonical host mRNAs. Uniquely, 'alt-proteins' lack sequence homology with host ORF-derived proteins. We show global amino acid frequencies, and consequent biochemical characteristics of Alt-ORFs nested within host ORFs (nAlt-ORFs), are genetically-driven, and predicted by summation of frequencies of hundreds of encompassing host codon-pairs. Analysis of 101 human nAlt-ORFs of length ≥150 codons confirms the theoretical predictions, revealing an extraordinarily high median isoelectric point (pI) of 11.68, due to anomalous charged amino acid levels. Also, nAlt-ORF proteins exhibit a >2-fold preference for reading frame 2 versus 3, predicted mitochondrial and nuclear localization, and elevated codon adaptation index indicative of natural selection. Our results provide a theoretical and conceptual framework for exploration of these largely unannotated, but potentially significant, alternative ORFs and their encoded proteins.

Journal Article↗

PubMed2007

Regulation of ceruloplasmin in human hepatic cells by redox active copper: identification of a novel AP-1 site in the ceruloplasmin gene.

Cp (ceruloplasmin), a copper containing plasma protein, mainly synthesized in the liver, is known to be functional between the interface of iron and copper metabolism. We have reported previously that Cp is regulated by cellular iron status, but the process of the regulation of Cp by copper still remains a subject for investigation. In the present paper, we show that PDTC (pyrrolidine dithiocarbamate), a thiol compound widely known to increase intracellular redox copper, regulates Cp expression in hepatic cells by a copper-dependent transcriptional mechanism. To find out the mechanism of induction, chimeric constructs of the Cp 5'-flanking region driving luciferase were transfected into human hepatic cells. Deletion and mutational analyses showed the requirement of a novel APRE [AP-1 (activator protein-1) responsive element] present about 3.7 kb upstream of the translation initiation site. The role of AP-1 was confirmed by electrophoretic mobility-shift analysis. Western blot and overexpression studies detected the AP-1 as a heterodimer of c-jun and c-fos proteins. The activation of AP-1 was found to be copper-dependent as a specific extracellular chelator bathocuproine disulfonic acid blocked PDTC-mediated induction of AP-1-DNA binding and increased reporter gene activity. Whereas, in a copper-free medium, PDTC failed to activate either AP-1 or Cp synthesis, supplementation of copper could reverse AP-1 activation and Cp synthesis. Our finding is not only the first demonstration of regulation of Cp by redox copper but may also explain previous findings of increased Cp expression in cancers like hepatocarcinoma, where the intracellular copper level is higher in a redox compromised environment.

Antioxidants↗

PubMed2007

L13a blocks 48S assembly: role of a general initiation factor in mRNA-specific translational control.

Transcript-specific translational control restricts macrophage inflammatory gene expression. The proinflammatory cytokine interferon-gamma induces phosphorylation of ribosomal protein L13a and translocation from the 60S ribosomal subunit to the interferon-gamma-activated inhibitor of translation (GAIT) complex. This complex binds the 3'UTR of ceruloplasmin mRNA and blocks its translation. Here, we elucidate the molecular mechanism underlying repression by L13a. Translation of the GAIT element-containing reporter mRNA is sensitive to L13a-mediated silencing when driven by internal ribosome entry sites (IRESs) that require initiation factor eIF4G, but is resistant to silencing when driven by eIF4F-independent IRESs, demonstrating a critical role for eIF4G. Interaction of L13a with eIF4G blocks 43S recruitment without suppressing eIF4F complex formation. eIF4G attack, e.g., by virus, stress, or caspases, is a well-known mechanism of global inhibition of protein synthesis. However, our studies reveal a unique mechanism in which targeting of eIF4G by mRNA-bound L13a elicits transcript-specific translational repression.

Binding Sites↗

PubMed2006

Sox6 cell-autonomously stimulates erythroid cell survival, proliferation, and terminal maturation and is thereby an important enhancer of definitive erythropoiesis during mouse development.

Erythropoiesis, the essential process of hematopoietic stem cell development into erythrocytes, is controlled by lineage-specific transcription factors that determine cell fate and differentiation and by the hormone erythropoietin that stimulates cell survival and proliferation. Here we identify the Sry-related high-mobility-group (HMG) box transcription factor Sox6 as an important enhancer of definitive erythropoiesis. Sox6 is highly expressed in proerythroblasts and erythroblasts in the fetal liver, neonatal spleen, and bone marrow. Mouse fetuses and pups lacking Sox6 develop erythroid cells slowly and feature misshapen, short-lived erythrocytes. They compensate for anemia by elevating the serum level of erythropoietin and progressively enlarging their erythropoietic tissues. Erythroid-specific inactivation of Sox6 causes the same phenotype, demonstrating cell-autonomous roles for Sox6 in erythroid cells. Sox6 potentiates the ability of erythropoietin signaling to promote proerythroblast survival and has an effect additive to that of erythropoietin in stimulating proerythroblast and erythroblast proliferation. Sox6 also critically facilitates erythroblast and reticulocyte maturation, including hemoglobinization, cell condensation, and enucleation, and ensures erythrocyte cytoskeleton long-term stability. It does not control adult globin and erythrocyte cytoskeleton genes but acts by stabilizing filamentous actin (F-actin) levels. Sox6 thus enhances erythroid cell development at multiple levels and thereby ensures adequate production and quality of red blood cells.

Actins↗

PubMed2006

Caveolin-1 polarization in migrating endothelial cells is directed by substrate topology not chemoattractant gradient.

Polarization is a hallmark of migrating cells, and an asymmetric distribution of proteins is essential to the migration process. Caveolin-1 is highly polarized in migrating endothelial cells (EC). Several studies have shown caveolin-1 accumulation in the front of migrating EC while others report its accumulation in the EC rear. In this paper we address these conflicting results on polarized localization of caveolin-1. We find evidence for the hypothesis that different modes of locomotion lead to differences in protein polarization. In particular, we show that caveolin-1 is primarily localized in the rear of cells migrating on a planar substrate, but in the front of cells traversing a three-dimensional pore. We also show that a chemoattractant, present either as a gradient or ubiquitously in the medium, does not alter caveolin-1 localization in cells in either mode of locomotion. Thus we conclude that substrate topology, and not the presence of a chemoattractant, directs the polarization of caveolin-1 in motile ECs.

Animals↗

PubMed2006

Translational control of ceruloplasmin gene expression: beyond the IRE.

Translational control is a common regulatory mechanism for the expression of iron-related proteins. For example, three enzymes involved in erythrocyte development are regulated by three different control mechanisms: globin synthesis is modulated by heme-regulated translational inhibitor, erythroid 5-aminolevulinate synthase translation is inhibited by binding of the iron regulatory protein to the iron response element in the 5'-untranslated region (UTR); and 15-lipoxygenase is regulated by specific proteins binding to the 3'-UTR. Ceruloplasmin (Cp) is a multi-functional, copper protein made primarily by the liver and by activated macrophages. Cp has important roles in iron homeostasis and in inflammation. Its role in iron metabolism was originally proposed because of its ferroxidase activity and because of its ability to stimulate iron loading into apo-transferrin and iron efflux from liver. We have shown that Cp mRNA is induced by interferon (IFN)-gamma in U937 monocytic cells, but synthesis of Cp protein is halted by translational silencing. The silencing mechanism requires binding of a cytosolic inhibitor complex, IFN-Gamma-Activated Inhibitor of Translation (GAIT), to a specific GAIT element in the Cp 3'-UTR. Here, we describe our studies that define and characterize the GAIT element and elucidate the specific trans-acting proteins that bind the GAIT element. Our experiments describe a new mechanism of translational control of an iron-related protein and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation.

3' Untranslated Regions↗

PubMed2005

Regulation of macrophage ceruloplasmin gene expression: one paradigm of 3'-UTR-mediated translational control.

Ceruloplasmin (Cp) is a copper protein with important functions in iron homeostasis and in inflammation. Cp mRNA expression is induced by interferon (IFN)-g in U937 monocytic cells, but synthesis of Cp protein is halted after about 12 h by transcript-specific translational silencing. The silencing mechanism requires binding of a 4-component cytosolic inhibitor complex, IFN-gamma-activated inhibitor of translation (GAIT), to a defined structural element (GAIT element) in the Cp 3'-UTR. Translational silencing of Cp mRNA requires the essential proteins of mRNA circularization, suggesting that the translational inhibition requires end-to-end mRNA closure. These studies describe a new mechanism of translational control, and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation.

3' Untranslated Regions↗

PubMed2005

Protein kinase Cdelta-dependent phosphorylation of syndecan-4 regulates cell migration.

Endothelial cell (EC) migration is a complex process requiring exquisitely coordinated focal adhesion assembly and disassembly. Protein kinase C (PKC) is known to regulate focal adhesion formation. Because lysophosphatidylcholine (lysoPC), a major lipid constituent of oxidized low-density lipoprotein, can activate PKC and inhibit EC migration, we explored the signaling cascade responsible for this inhibition. LysoPC increased PKCdelta activity, measured by in vitro kinase activity assay, and increased PKCdelta phosphorylation. Decreasing PKCdelta activation, using pharmacological inhibitors or antisense oligonucleotides, diminished the antimigratory effect of lysoPC. LysoPC-induced PKCdelta activation was followed by increased phosphorylation of the transmembrane proteoglycan, syndecan-4, and decreased binding of PKCalpha to syndecan-4, with a concomitant decrease in PKCalpha activity. A reciprocal relationship was noted between the interaction of PKCalpha and alpha-actinin with syndecan-4. These changes were temporally related to the observed changes in cell morphology and the inhibition of migration of ECs incubated with lysoPC. The data suggested that generalized activation of PKCdelta by lysoPC initiated a cascade of events, including phosphorylation of syndecan-4, displacement and decreased activity of PKCalpha, binding of alpha-actinin to syndecan-4, and disruption of the time- and site-specific regulation of focal adhesion complex assembly and disassembly required for normal cell migration.

Actinin↗

PubMed2005

Equality of the sexes: found in translation.

Male fruit flies (and humans) have half the number of X chromosomes that females have, yet the expression of X-linked proteins in males is the same because of a sex-specific compensatory mechanism. In this issue of Cell, report that a unique two-stage translation silencing mechanism thwarts synthesis of a male-specific transcription activator in female flies, ensuring that gene dosage compensation occurs only in males.

Animals↗

PubMed2005

Unexpected role of ceruloplasmin in intestinal iron absorption.

Ferroxidases are essential for normal iron homeostasis in most organisms. The paralogous vertebrate ferroxidases ceruloplasmin (Cp) and hephaestin (Heph) are considered to have nonidentical functions in iron transport: plasma Cp drives iron transport from tissue stores while intestinal Heph facilitates iron absorption from the intestinal lumen. To clarify the function of Cp, we acutely bled Cp-/- mice to stress iron homeostasis pathways. Red cell hemoglobin recovery was defective in stressed Cp-/- mice, consistent with low iron availability. Contrary to expectations, iron was freely released from spleen and liver stores in Cp-/- mice, but intestinal iron absorption was markedly impaired. Phlebotomy of wild-type mice caused a striking shift of Cp from the duodenal epithelium to the underlying lamina propria, suggesting a critical function of Cp in basolateral iron transport. Regulated relocalization of intestinal Cp may represent a fail-safe mechanism in which Cp shares with Heph responsibility for iron absorption under stress.

Animals↗

PubMed2004

Noncanonical function of glutamyl-prolyl-tRNA synthetase: gene-specific silencing of translation.

Aminoacyl tRNA synthetases (ARS) catalyze the ligation of amino acids to cognate tRNAs. Chordate ARSs have evolved distinctive features absent from ancestral forms, including compartmentalization in a multisynthetase complex (MSC), noncatalytic peptide appendages, and ancillary functions unrelated to aminoacylation. Here, we show that glutamyl-prolyl-tRNA synthetase (GluProRS), a bifunctional ARS of the MSC, has a regulated, noncanonical activity that blocks synthesis of a specific protein. GluProRS was identified as a component of the interferon (IFN)-gamma-activated inhibitor of translation (GAIT) complex by RNA affinity chromatography using the ceruloplasmin (Cp) GAIT element as ligand. In response to IFN-gamma, GluProRS is phosphorylated and released from the MSC, binds the Cp 3'-untranslated region in an mRNP containing three additional proteins, and silences Cp mRNA translation. Thus, GluProRS has divergent functions in protein synthesis: in the MSC, its aminoacylation activity supports global translation, but translocation of GluProRS to an inflammation-responsive mRNP causes gene-specific translational silencing.

Amino Acyl-tRNA Synthetases↗

PubMed2004

Oxidative stress, caveolae and caveolin-1.

Oxidative stress underlies a range of pathophysiological conditions. Reactive oxygen species are also generated intracellularly to serve as second messengers and some are linked to caveolae/raft signalling systems. The effect of oxidative stress on caveolin-1 expression, post-translational modifications, membrane trafficking and function are described.

Animals↗

PubMed2004

Role of reactive oxygen species in inhibition of endothelial cell migration by oxidized low-density lipoprotein.

OBJECTIVE: Endothelial cell migration is inhibited by oxidized low-density lipoprotein (oxLDL) and lysophosphatidylcholine (lysoPC). The purpose of this study was to explore the mechanism of this inhibition, specifically the role of reactive oxygen species. METHODS: The ability of oxLDL, lysoPC, and known superoxide generators to stimulate endothelial cell production of reactive oxygen species and inhibit endothelial cell migration under the same conditions was assessed. Reactive oxygen species production was assessed with dichlorofluorescein. Migration was studied with a razor scrape assay and measured after 24 hours. In addition, the ability of various antioxidants, added before initiation of the scrape assay, to restore endothelial cell migration in oxLDL was determined. RESULTS: OxLDL and lysoPC, at concentrations that stimulated reactive oxygen species production, also inhibited endothelial cell migration. Other agents that generated superoxide also inhibited endothelial cell migration, but hydrogen peroxide did not. Of a variety of antioxidants assessed for their ability to preserve endothelial cell migration in the presence of oxLDL, only superoxide dismutase and reduced nicotinamide adenine dinucleotide (phosphate) oxidase inhibitors (diphenyleneiodonium, quinacrine, hydralazine) preserved endothelial cell migration. CONCLUSIONS: These data suggest that oxLDL inhibits endothelial cell migration through a superoxide-dependent mechanism and that reduced nicotinamide adenine dinucleotide (phosphate) oxidase is the cellular source of the superoxide. CLINICAL RELEVANCE: OxLDL inhibits endothelial cell migration, and may impair healing of arterial injuries. The mechanism of oxidized LDL inhibition is not known. Our in vitro studies show that the inhibitory properties are related to production of reactive oxygen species. Superoxide dismutase or inhibitors of reduced nicotinamide adenine dinucleotide phosphate oxidase can preserve endothelial migration in the presence of oxLDL. This might improve the healing of endothelial injuries at sites of arterial repair or angioplasty, especially in lipid-laden arterial walls.

Animals↗

PubMed2004

Alpha-tocopherol preserves endothelial cell migration in the presence of cell-oxidized low-density lipoprotein by inhibiting changes in cell membrane fluidity.

OBJECTIVE: Endothelial cell (EC) migration is essential for healing areas of arterial injury and angioplasty sites. Iron or copper-oxidized low-density lipoprotein (oxLDL(Cu)) inhibits EC migration in vitro, but the effect of physiologically relevant monocyte/macrophage-oxidized LDL (oxLDL(cell)) is unknown. We postulated that oxLDL(cell) would inhibit EC migration and that this inhibition would be reversed by antioxidants. METHODS: The effect of oxLDL(Cu) and oxLDL(cell) on EC migration was studied by using a razor scrape assay, and migration was assessed after 24 hours. In addition, ECs were incubated with various antioxidants, including butylated hydroxytoluene (BHT), probucol, or alpha-tocopherol, for 1 hour prior to initiation of the scrape assay and application of oxLDL. RESULTS: Both oxLDL(Cu) and oxLDL(cell) inhibited migration. The antioxidants did not alter the antimigratory activity of oxLDL(Cu), but alpha-tocopherol preserved EC migration in the presence of oxLDL(cell). The lack of effect of BHT or probucol suggested that the effect of alpha-tocopherol resided not in its antioxidant activity but in its membrane-stabilizing properties. To test this theory, the effect of oxLDL and alpha-tocopherol on relative cell membrane fluidity was assessed by fluorescence recovery after photobleaching. Both oxLDL(Cu) and oxLDL(cell) increased relative membrane fluidity. Preincubation with alpha-tocopherol inhibited the increase in membrane fluidity of ECs incubated in oxLDL(cell) but not in oxLDL(Cu). CONCLUSIONS: These studies show that alpha-tocopherol preserves EC migration in oxLDL(cell) and hastens restoration of the endothelial monolayer after injury by inhibiting changes in membrane integrity caused by oxLDL. CLINICAL RELEVANCE: Recent studies find that vitamin E is not efficacious in the secondary prevention of cardiovascular events, perhaps because vitamin E does not efficiently block oxidation pathways known to be operative in atherosclerotic arteries. "Non-antioxidant" properties of vitamin E, however, could be important in the primary prevention of atherosclerosis and its complications. Our in vitro studies show that alpha-tocopherol can preserve endothelial migration in the presence of cell-oxidized LDL. This effect might improve the healing of endothelial injuries at sites of arterial repair or angioplasties, especially in lipid-laden arterial walls.

Animals↗

PubMed2004

Polarization of plasma membrane microviscosity during endothelial cell migration.

Cell movement is characterized by anterior-posterior polarization of multiple cell structures. We show here that the plasma membrane is polarized in moving endothelial cells (EC); in particular, plasma membrane microviscosity (PMM) is increased at the cell leading edge. Our studies indicate that cholesterol has an important role in generation of this microviscosity gradient. In vitro studies using synthetic lipid vesicles show that membrane microviscosity has a substantial and biphasic influence on actin dynamics; a small amount of cholesterol increases actin-mediated vesicle deformation, whereas a large amount completely inhibits deformation. Experiments in migrating ECs confirm the important role of PMM on actin dynamics. Angiogenic growth factor-stimulated cells exhibit substantially increased membrane microviscosity at the cell front but, unexpectedly, show decreased rates of actin polymerization. Our results suggest that increased PMM in lamellipodia may permit more productive actin filament and meshwork formation, resulting in enhanced rates of cell movement.

Actin Cytoskeleton↗

PubMed2004

Apolipoprotein A-I is a selective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjects with cardiovascular disease.

In recent studies we demonstrated that systemic levels of protein-bound nitrotyrosine (NO(2)Tyr) and myeloperoxidase (MPO), a protein that catalyzes generation of nitrating oxidants, serve as independent predictors of atherosclerotic risk, burden, and incident cardiac events. We now show both that apolipoprotein A-I (apoA-I), the primary protein constituent of HDL, is a selective target for MPO-catalyzed nitration and chlorination in vivo and that MPO-catalyzed oxidation of HDL and apoA-I results in selective inhibition in ABCA1-dependent cholesterol efflux from macrophages. Dramatic selective enrichment in NO(2)Tyr and chlorotyrosine (ClTyr) content within apoA-I recovered from serum and human atherosclerotic lesions is noted, and analysis of serum from sequential subjects demonstrates that the NO(2)Tyr and ClTyr contents of apoA-I are markedly higher in individuals with cardiovascular disease (CVD). Analysis of circulating HDL further reveals that higher NO(2)Tyr and ClTyr contents of the lipoprotein are each significantly associated with diminished ABCA1-dependent cholesterol efflux capacity of the lipoprotein. MPO as a likely mechanism for oxidative modification of apoA-I in vivo is apparently facilitated by MPO binding to apoA-I, as revealed by cross-immunoprecipitation studies in plasma, recovery of MPO within HDL-like particles isolated from human atheroma, and identification of a probable contact site between the apoA-I moiety of HDL and MPO. To our knowledge, the present results provide the first direct evidence for apoA-I as a selective target for MPO-catalyzed oxidative modification in human atheroma. They also suggest a potential mechanism for MPO-dependent generation of a proatherogenic dysfunctional form of HDL in vivo.

ATP Binding Cassette Transporter 1↗

PubMed2003

Regulated release of L13a from the 60S ribosomal subunit as a mechanism of transcript-specific translational control.

Transcript-specific translational control is generally directed by binding of trans-acting proteins to structural elements in the untranslated region (UTR) of the target mRNA. Here, we elucidate a translational silencing mechanism involving regulated release of an integral ribosomal protein and subsequent binding to its target mRNA. Human ribosomal protein L13a was identified as a candidate interferon-Gamma-Activated Inhibitor of Translation (GAIT) of ceruloplasmin (Cp) mRNA by a genetic screen for Cp 3'-UTR binding proteins. In vitro activity of L13a was shown by inhibition of target mRNA translation by recombinant protein. In response to interferon-gamma in vivo, the entire cellular pool of L13a was phosphorylated and released from the 60S ribosomal subunit. Released L13a specifically bound the 3'-UTR GAIT element of Cp mRNA and silenced translation. We propose a model in which the ribosome functions not only as a protein synthesis machine, but also as a depot for regulatory proteins that modulate translation.

3' Untranslated Regions↗

PubMed2003

Role of ceruloplasmin in macrophage iron efflux during hypoxia.

The reticuloendothelial system has a central role in erythropoiesis and iron homeostasis. An important function of reticuloendothelial macrophages is phagocytosis of senescent red blood cells. The iron liberated from heme is recycled for delivery to erythrocyte precursors for a new round of hemoglobin synthesis. The molecular mechanism by which recycled iron is released from macrophages remains unresolved. We have investigated the mechanism of macrophage iron efflux, focusing on the role of ceruloplasmin (Cp), a copper protein with a potent ferroxidase activity that converts Fe2+ to Fe3+ in the presence of molecular oxygen. As shown by others, Cp markedly increased iron binding to apotransferrin at acidic pH; however, the physiological significance of this finding is uncertain because little stimulation was observed at neutral pH. Introduction of a hypoxic atmosphere resulted in marked Cp-stimulated binding of iron to apotransferrin at physiological pH. The role of Cp in cellular iron release was examined in U937 monocytic cells induced to differentiate to the macrophage lineage. Cp added at its normal plasma concentration increased the rate of 55Fe release from U937 cells by about 250%. The stimulation was absolutely dependent on the presence of apotransferrin and hypoxia. Cp-stimulated iron release was confirmed in mouse peritoneal macrophages. Stimulation of iron release required an intracellular "labile iron pool" that was rapidly depleted in the presence of Cp and apotransferrin. Ferroxidase-mediated loading of iron into apotransferrin was critical for iron release because ferroxidase-deficient Cp was inactive and because holotransferrin could not substitute for apotransferrin. The extracellular iron concentration was critical as shown by inhibition of iron release by exogenous free iron, and marked enhancement of release by an iron chelator. Together these data show that Cp stimulates iron release from macrophages under hypoxic conditions by a ferroxidase-dependent mechanism, possibly involving generation of a negative iron gradient.

Animals↗