Biomedical subjects

S J Demosky

Publications and source records attributed to S J Demosky.

8 recordsLinked to original sources

PubMed1992

Regulation of LDL receptor, apoB, and apoE protein and mRNA in Hep G2 cells.

The regulation of low-density lipoprotein (LDL) receptor activity, protein synthesis, and cellular mRNA content was evaluated in the human hepatoma cell line Hep G2. Incubation of the cells with LDL led to a complete downregulation of LDL receptor mRNA and LDL receptor protein synthesis. This LDL regulation of the LDL receptor and its mRNA was both time- and concentration-dependent. In contrast to protein synthesis and cellular mRNA concentrations of the LDL receptor, which were reduced to undetectable levels by prolonged incubation in the presence of LDL, LDL receptor activity was reduced to only 44% of preincubation levels. These findings support the presence of a second metabolic pathway for LDL uptake in human hepatocytic cells. The effect of LDL on cellular LDL receptor expression was specific for LDL because incubation in the presence of HDL did not affect any of these study end points. The potential coordinate regulation of the expression of the LDL receptor with its principal ligands, apolipoproteins (apo) B and E, was also investigated. In contrast to the LDL receptor mRNA downregulation with LDL incubation, cellular apoB and apoE mRNA concentrations were not affected by either LDL or HDL. Secretion of apoB, however, was significantly increased by incubating Hep G2 cells with LDL. These findings indicate that, in contrast to LDL receptor which is regulated at the mRNA level, the ligands for the LDL receptor are regulated either co- or post-translationally.

Apolipoproteins B↗

PubMed1991

Apolipoprotein B upstream suppressor site: identification of an element which can decrease apolipoprotein B transcription.

Elevated plasma levels of apolipoprotein B (apoB) may predispose to development of premature coronary atherosclerosis. We have identified the first well localized domain of the apoB gene which can effect negative regulation of its transcription. This region binds trans-activating factors present only in apoB producing cell lines. Mutagenesis of this region causes up-regulation of its transcriptional activity. We have termed this element apoB upstream suppressor site (aBUSS) and its trans-activators the apoB repressor proteins (ARP). aBUSS and ARP may play important roles in the transcriptional modulation of apoB.

Animals↗

PubMed1990

Both apolipoproteins B-48 and B-100 are synthesized and secreted by the human intestine.

Apolipoprotein B (apoB), an apolipoprotein associated with very low density lipoproteins and the atherogenic low density lipoproteins (LDL), directs the metabolism of lipoprotein particles in plasma by interacting with the LDL receptor. Utilizing human intestinal biopsy organ cultures, we have studied the synthesis of intestinal apoB in man. Intestinal organ cultures from normal adults (n = 6) were incubated in the presence of protease inhibitors in media supplemented with [35S]methionine. Media from these cultures were evaluated by sequential NaDodSO4 polyacrylamide gel electrophoresis, radioautography, and Western blot analyses, and intestinal biopsies were studied using immunohistochemistry. The relative abundance of apoB-100 and apoB-48 mRNA was assessed using reverse transcriptase-polymerase chain reaction followed by primer extension. Although apoB-48 was the principal isoprotein that was newly synthesized by intestinal organ cultures, apoB-100 was also synthesized and secreted by human intestinal organ cultures with 16 +/- 3% of the intestinal apoB mRNA coding for apoB-100. These results establish that apoB-100 is produced by the human intestine. The synthesis of the atherogenic apoB-100 by the intestine has pathophysiologic implications for the development of diet-induced atherosclerosis.

Adult↗

PubMed1989

Human apolipoprotein A-I. Post-translational modification by covalent phosphorylation.

In vitro phosphorylation of purified human plasma apolipoprotein A-I (apoA-I) by a recently characterized Ca2+/calmodulin-dependent kinase (Beg, Z. H., Stonik, J. A., and Brewer, H. B., Jr. (1987) J. Biol. Chem. 262, 13228-13240) was time-, Ca2+-, and calmodulin-dependent. Maximal phosphorylation of human apoA-I revealed a stoichiometry of approximately 1 mol of PO4/mol of apoA-I. Phosphorylation of apoA-I resulted in an increase of two negative charges and consequently a shift to a more acidic pI for each apoA-I isoform following isoelectrofocusing. Dephosphorylation of 32P-apoA-I with either phosphatase I or a Ca2+/calmodulin-dependent phosphatase was associated with a virtually complete loss of 1 mol of 32PO4/mol of apoA-I. Phosphoamino acid analysis of a purified 32P-peptide established that the phosphorylation occurred on a single serine residue. Automated Edman degradation of the purified 32P-peptide revealed a single amino acid sequence and indicated that phosphorylation occurred on the serine at residue 201 in the apoA-I sequence. ApoA-I was shown to be secreted as a phosphoapolipoprotein by HepG-2 cells as well as primary human hepatocytes. Analysis of HepG-2 cells established that intracellular apoA-I, like secreted apoA-I, is phosphorylated. Dephosphorylation of both secreted and intracellular 32P-apoA-I revealed the loss of radioactivity in the apoA-I protein bands. These data provide the initial description of a post-translational modification involving reversible phosphorylation of extracellular as well as intracellular apoA-I on a serine residue. These combined results suggest that synthesis and secretion of apoA-I as a phosphoapolipoprotein in HepG-2 cells as well as primary human hepatocytes may play an important role in lipoprotein assembly, intracellular transport as well as processing, and lipoprotein secretion.

Adenosine Triphosphate↗

PubMed1988

Apolipoprotein B synthesized by Hep G2 cells undergoes fatty acid acylation.

Apolipoprotein B is the principal protein associated with cholesterol transport in the blood and has been proposed to play a central role in human atherogenesis. The unique hydrophobic nature of this large (512 kDa), glycosylated apolipoprotein differs from that of the other apolipoproteins. Since another apolipoprotein, apolipoprotein A-I, has been recently shown to have covalently bound fatty acids, potential fatty acid acylation of apolipoprotein B was investigated. The human hepatoma cell line, Hep G2, synthesizes apoB-100 and secretes the apolipoprotein into the culture medium. After a 24-hr incubation with [14C]palmitate and [14C]stearate, the label was incorporated into apoB-100 when assessed by a sodium dodecyl sulfate polyacrylamide gel electrophoresis, autoradiography, immunoblot analysis, and immunoprecipitation. Hydroxylamine treatment, which hydrolyzes ester and thioester bonds, removed the radiolabel. ApoB-100 isolated from Hep G2 cells by ultracentrifugation and preparative sodium dodecyl sulfate gel electrophoresis was hydrolyzed and analyzed by gas-liquid chromatography-mass spectrometry. In contrast to circulating apoB in low density lipoproteins, both palmitate and stearate were present in newly synthesized apoB-100. These results establish that newly synthesized apoB-100 undergoes covalent acylation with palmitate and stearate. The acylation of apoB may play an important role in lipoprotein particle secretion. In addition, derangements in apoB fatty acid acylation may lead to dyslipoproteinemia.

Acylation↗

PubMed1986

The expressed human hepatic receptor for low-density lipoproteins differs from the fibroblast low-density lipoprotein receptor.

The role of the cellular receptor for the low-density lipoproteins (LDL) in cholesterol transport was initially defined through the study of nonhepatic cells in vitro. Since the liver is central in plasma lipoprotein metabolism, an investigation of hepatic lipoprotein receptors is important for understanding normal lipoprotein transport. Utilizing human hepatic and fibroblast membranes, the characteristics of receptors for LDL from hepatic and nonhepatic tissues were directly compared. Human hepatic membranes reversibly bound LDL within 5 min. Although both fibroblast and hepatic membranes saturably bound LDL at 37 degrees C, the fibroblast LDL receptor affinity (Kd = 2.5 X 10(-8) M) and number (5.5 X 10(12) sites/mg membrane protein) were greater than the hepatic receptor affinity (Kd = 10.8 X 10(-8) M) and number (0.5 X 10(12) sites/mg membrane protein). In contrast to the fibroblast LDL receptor which was unable to bind LDL in the presence of EDTA, the hepatic LDL receptor binding of LDL was only partially blocked by EDTA. The binding of LDL to its hepatic receptor is highly temperature-dependent, and studies utilizing both radiolabeled LDL and colloidal gold-labeled LDL indicate that little, if any, binding of LDL hepatic membranes occur at 0-4 degrees C. The hepatic membrane receptor(s) (Mr approximately equal to 270 000 and 330 000) differ from that of the fibroblast LDL receptor (Mr approximately equal to 130 000) and these proteins are present in hepatic membranes from a patient lacking the fibroblast LDL receptor. These data indicate that an expressed hepatic LDL receptor has unique properties different from those of the fibroblast LDL receptor and that the expressed protein(s) is genetically distinct from the fibroblast receptor.

Biological Transport, Active↗

PubMed1985

Distinct hepatic receptors for low density lipoprotein and apolipoprotein E in humans.

Since the liver is a central organ for lipid and lipoprotein synthesis and catabolism, hepatic receptors for specific apolipoproteins on plasma lipoproteins would be expected to modulate lipid and lipoprotein metabolism. The role of hepatic receptors for low density lipoproteins and apolipoprotein E-containing lipoproteins was evaluated in patients with complementary disorders in lipoprotein metabolism: abetalipoproteinemia and homozygous familial hypercholesterolemia. In addition, hepatic membranes from a patient with familial hypercholesterolemia were studied and compared before and after portacaval shunt surgery. The results establish that the human liver has receptors for apolipoproteins B and E. Furthermore, in the human, hepatic receptors for low density lipoproteins and apolipoprotein E are genetically distinct and can undergo independent control.

Abetalipoproteinemia↗

PubMedDate not supplied

Characterization of a human hepatic receptor for high density lipoproteins.

Characterization of the membrane receptor for the low density lipoproteins (LDL) has led to insights into cellular receptor physiology as well as mammalian lipid transport. Result with LDL have stimulated the search for specific receptors for other plasma lipoproteins. Receptors for high density lipoproteins (HDL) have been identified in human fibroblasts and smooth muscle cells. Specificity for this receptor has been difficult to define since normal HDL contains several apolipoproteins, and particles containing apolipoproteins B and E have been shown to compete for HDL binding. In the present study, we demonstrate that HDL isolated from a patient devoid of apolipoprotein E was bound specifically by human hepatic membranes. This binding reached saturation within 2 hours and was EDTA-resistant. Assuming a single receptor model, we found that 2.9 x 10(15) receptors/mg membrane protein bound with an affinity KD = 3.5 x 10(-7) M at 0 to 4 degrees C and KD = 1.9 x 10(-7) M at 37 degrees C. The binding was effectively competed with intact HDL3, with HDL3 that had undergone selective arginine and lysine residue modification, and with antibodies to apolipoproteins A-I and A-II. However, LDL, asialofetuin, and HDL3 which had undergone tyrosine modification by nitration, and anti-apolipoprotein B did not compete with apo A-I HDL binding. In contrast to LDL binding, the human hepatoma cell line, HEPG2, increased HDL binding with cholesterol loading that was specific for HDL3. Thus, hepatic tissue can modulate its recognition of HDL. Finally, hepatic membranes from a patient lacking normal hepatic LDL receptors bound apo A-I HDL normally. These data indicate that a saturable, specific regulatable receptor for apo E-free HDL is present in human liver.

Apolipoprotein A-I↗