Search PubMed⌕ Search

Biomedical subjects

V I Zannis

Publications and source records attributed to V I Zannis.

At least 37 records · Page 2Linked to original sources
PubMed1997

Activation of CAAT enhancer-binding protein delta (C/EBPdelta) by interleukin-1 negatively influences apolipoprotein C-III expression.

Tissue-specific transcription of the apolipoprotein C-III (apoC-III) gene is mainly regulated by synergistic interactions between the liver-enriched transcription factor HNF-4, which binds to the proximal promoter, and ubiquitous factors, which bind to the upstream enhancer region. Here we show that apoC-III expression in HepG2 cells is negatively regulated in response to interleukin-1 (IL-1), and this inhibition is mainly due to transcriptional repression. CAAT enhancer-binding protein delta (C/EBPdelta) was found to be the main mediator of IL-1-induced suppression. Analysis of the apoC-III promoter revealed two IL-1 response elements. The first is located in the proximal promoter region D and the second in the distal enhancer region I. Proximal element D is a high affinity binding site for C/EBPdelta, while the enhancer element I is not directly recognized by this transcription factor. Functional analysis of different combinations of homologous and heterologous promoter constructs revealed that indirect interaction of C/EBPdelta with site I, in the context of the full promoter, leads to repression. C/EBPdelta is activated by phosphorylation during IL-1-induced signal transduction pathway. This modification is important for both DNA binding activity and indirect transrepression of the apoC-III promoter.

Animals↗
PubMed1997

Interaction of nascent ApoE2, ApoE3, and ApoE4 isoforms expressed in mammalian cells with amyloid peptide beta (1-40). Relevance to Alzheimer's disease.

Population studies have established that one of the common isoforms of apolipoprotein E, the apoE4, is associated with higher incidence and earlier age of onset of late onset familial Alzheimer's disease (AD), whereas apoE2 may have the opposite effect. The apoE3 and apoE4 isoforms were shown to display different binding reactivities with amyloid beta peptide (Abeta) and tau protein in vitro. On the basis of these findings, it has been proposed that the apoE isoforms may modulate positively or negatively the formation of either the neurofibrillary tangles or the amyloid deposits in the brain of patients with AD. To study the interaction of Abeta with nascent apoE isoforms we have expressed their cDNAs in baby hamster kidney (BHK-21) cells using the Semliki Forest Virus expression system. Analysis of the secreted apoE by one- and two-dimensional gel electrophoresis and immunoblotting showed that the nascent apoE is heavily modified with carbohydrate chains containing sialic acid. A dimeric form of apoE is formed with apoE2 and apoE3 but not with apoE4 isoforms. Analysis of the interaction of nascent apoE2, apoE3, and apoE4 produced by BHK-21 cells with Abeta (1-40) under physiological conditions (pH 7.4, 37 degrees C) showed that the efficiency of the apoE monomer-Abeta complex formation follows the order apoE2 > apoE3 >> apoE4. In addition, the apoE2 dimer formed a complex with Abeta more efficiently than the apoE3 dimer. The isoform-specific differences in binding were temperature-dependent and are attenuated upon decrease of the temperature. The binding behavior of the monomeric apoE is different from that reported for plasma apoE3 and apoE4 or commercially available apoE3 and apoE4 preparations and similar to that described for apoE3 and apoE4 produced by human embryonic kidney (HEK-293) cells. It appears that the efficiency of binding between each of three main apoE isoforms and Abeta correlates inversely with the risk of developing late-onset familial AD and may indicate possible involvement of apoE in the binding and clearance of Abeta in vivo.

Alzheimer Disease↗
PubMed1997

Ultraspiracle, a Drosophila retinoic X receptor alpha homologue, can mobilize the human thyroid hormone receptor to transactivate a human promoter.

We have analyzed the functional domains of the Drosophila orphan receptor Ultraspiracle (usp), a homologue of the vertebrate retinoic X receptor alpha, as well as the ability of heterodimers between usp and the thyroid hormone receptor beta (T3Rbeta) to transactivate the human apolipoprotein A-II (apoA-II) promoter. DNA binding assays demonstrated that heterodimers of usp and the human T3Rbeta can bind to the hormone response element (HRE) of the regulatory element AIIJ (-734 to -716) of the human apoA-II promoter. Cotransfection experiments have shown that the combination of usp and T3Rbeta can transactivate the human apoA-II promoter in COS-1 cells 7-8-fold in the presence of thyroid hormone (T3). The observed transactivation was not affected by the deletion of the amino-terminal residues 1-85 of usp, which represent a putative transactivation domain, suggesting that the function of usp is to recruit T3Rbeta. Furthermore, a mutant usp, with impaired DNA binding properties, can form heterodimers with T3Rbeta in vitro but has reduced ability to transactivate the human apoA-II promoter. A minimal thymidine kinase (tk) promoter driven by four AIIJ regulatory elements is repressed to 20% of its original activity by T3Rbeta and the repression is relieved by usp/T3Rbeta heterodimers. Deletion analysis demonstrated that factors bound to the regulatory elements AIIJ, AIIAB, and AIIH participate in the usp/T3Rbeta-mediated transactivation of the human apoA-II promoter. Similarly to element AIIJ, element AIIAB binds usp/T3Rbeta heterodimers, whereas element AIIH binds a COS-1 nuclear activity that is supershifted with anti-hepatic nuclear factor 1 antibodies. The findings suggest that optimal transactivation of the apoA-II promoter by usp/T3Rbeta heterodimers requires complex interactions between these heterodimers and factors bound to other regulatory elements. The observed transcriptional activation through heterodimer formation between nuclear receptors from species as divergent in the evolutionary scale as insects and mammals indicates that the functional domains of these proteins have been highly conserved.

Animals↗
PubMed1997

The carboxyl-terminal hydrophobic residues of apolipoprotein A-I affect its rate of phospholipid binding and its association with high density lipoprotein.

We performed a series of mutations in the human apolipoprotein A-I (apoA-I) gene designed to alter specific amino acid residues and domains implicated in lecithin:cholesterol acyltransferase (LCAT) activation or lipid binding. We used the mutant apoA-I forms to establish nine stable cell lines, and developed strategies for the large scale production and purification of the mutated apoA-I proteins from conditioned media. HDL and dimyristoyl phosphatidylcholine binding assays using the variant apoA-I forms have shown that replacement of specific carboxyl-terminal hydrophobic residues Leu222, Phe225, and Phe229 with lysines, as well as replacement of Leu211, Leu214, Leu218, and Leu219 with valines, diminished the ability of apoA-I to bind to HDL and to lyse dimyristoyl phosphatidylcholine liposomes. The findings indicate that Leu222, and Phe225, Phe229 located in the putative random coil region, and Leu211, Leu214, Leu218, and Leu219 located in the putative helix 8, are important for lipid binding. In contrast, substitutions of alanines for specific charged residues in putative helices 7, 8, or 9 as well as various point mutations in other regions of apoA-I, did not affect the ability of the variant apoA-I forms to bind to HDL or to lyse dimyristoyl phosphatidylcholine liposomes. Cross-linking experiments confirmed that the carboxyl-terminal domain of apoA-I participates in the self-association of the protein, as demonstrated by the inability of the carboxyl-terminal deletion mutants delta185-243 and delta209-243 to form higher order aggregates in solution. Lecithin:cholesterol acyltransferase analysis, using reconstituted HDL particles prepared by the sodium cholate dialysis method, has shown that mutants (Pro165-->Ala,Gln172-->Glu) (Leu211-->Val,Leu214-->Val, Leu218-->Val,Leu219-->Val), Leu222-->Lys,Phe225-->Lys, Phe229-->Lys) and delta209-243 reduced LCAT activation (38-68%). Mutant (Glu191-->Ala,His193-->Ala,Lys195-->Ala) enhanced LCAT activation (131%), and mutant (Ala152-->Leu, Leu159-->Trp) exhibited normal LCAT activation as compared with the wild type proapoA-I and plasma apoA-I forms [corrected]. The apparent catalytic efficiency (Vmax(app)/Km(app)) of the apoA-I mutants ranged from 17.8 to 107.2% of the control and was the result of variations in both the Km and the Vmax in the different mutants. These findings indicate that putative helices 6 and 7, and the carboxyl-terminal helices 8 and 9 contribute to the optimum activation of lecithin:cholesterol acyltransferase. In addition to their use in the present study, the variant apoA-I forms generated will serve as valuable reagents for the identification of the domains and residues of apoA-I involved in binding the scavenger receptor BI, and facilitating cholesterol efflux from cells as well as aid in the structural analysis of apoA-I.

Apolipoprotein A-I↗
PubMed1997

Apolipoproteins of HDL can directly mediate binding to the scavenger receptor SR-BI, an HDL receptor that mediates selective lipid uptake.

The class B type I scavenger receptor, SR-BI, binds HDL, mediates selective uptake of HDL cholesteryl esters by cultured cells, and its expression is coordinately regulated with steroidogenesis in several endocrine tissues (adrenal, ovary, testes). SR-BI can also bind LDL and anionic phospholipids, which raised the possibility that HDL apolipoproteins might not participate directly in HDL binding. We have examined the ability of individual human HDL apolipoproteins (apoA-I, apoA-II, and apoC-III) reconstituted into phospholipid/unesterified cholesterol complexes to bind to murine SR-BI (mSR-BI) expressed in stably transfected cultured cells. All three apolipoprotein/phospholipid/unesterified cholesterol complexes specifically associated with mSR-BI expressing cells with high affinity and competed for the binding of HDL, while apolipoprotein-free complexes did not. Furthermore, lipid-free forms of these soluble apolipoproteins also competed for HDL and apolipoprotein/ phospholipid/cholesterol complex association with mSR-BI, but locust high density lipophorin and bovine serum albumin were not effective competitors.Thus, all three of the HDL apolipoproteins (apoA-I, apoA-II, and apoC-III) tested can directly mediate binding to mSR-BI, and this multiligand apolipoprotein receptor may be responsible for at least some of the multilipoprotein and apolipoprotein binding activity previously observed in cells and tissues.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1996

Isolation and characterization of a third isoform of human hepatocyte nuclear factor 4.

Hepatocyte nuclear factor 4 (HNF-4) is an essential positive regulator of a large number of liver-specific genes. We report here the isolation of three HNF-4 isoforms from a human liver cDNA library. hHNF-4A and hHNF-4B, differing by the insertion of 10 amino acids in the C-terminal region, have been previously identified in mouse, rat and human liver. The novel isoform, hHNF-4C, is identical to hHNF-4A and B in the regions encompassing the DNA-binding and dimerization domains, but contains a different C-terminal domain. Similar to the other isoforms, hHNF-4C is produced in a limited number of tissues and represents 2.6-13% of the total hHNF-4 mRNA population, depending on the cell type. The chromosomal origin of all three isoforms has been localized to human chromosome 20. hHNF-4C can form heterodimers with hHNF-4A and B in vitro, and exhibits similar transactivation potential as hHNF-4A or B in transient transfection assays, suggesting that the divergent C-terminal region is not part of the transactivation domain.

Amino Acid Sequence↗
PubMed1996

Binding specificity and modulation of the ApoA-I promoter activity by homo- and heterodimers of nuclear receptors.

Three proximal regulatory elements, AIB, AIC, and AID, of the apoA-I gene are necessary and sufficient for its hepatic expression in vivo and in vitro. DNA binding and competition assays showed that elements AIB and AID contain hormone response elements composed of imperfect direct repeats that support the binding of the hepatic nuclear factor-4, other nuclear orphan receptors, and the ligand-dependent nuclear receptors retinoic X receptor (RXRalpha), RXRalpha/RARalpha, and RXRalpha/T3Rbeta. Substitution mutations on repeats 1 and 2 in the hormone response sites of elements AIB and AID, respectively, abolished the binding of all nuclear receptors and reduced promoter activity to background levels, indicating the importance of both hormone response elements for the hepatic expression of the apoA-I gene. Cotransfection experiments in HepG2 cells with normal and mutated promoter constructs and plasmids expressing nuclear hormone receptors showed that RXRalpha homodimers transactivated the wild type promoter 150% of control, in the presence of 9-cis-retinoic acid (RA), whereas RXR alpha/T3R beta heterodimers repressed transcription to 60% of control, in the presence of T3. RXR alpha/RAR alpha and hepatic nuclear factor-4 did not affect the transcription, driven by the proximal apoA-I promoter. Potassium permanganate and dimethyl sulfate interference experiments showed that RXRalpha homodimers, RXRalpha/RARalpha, and RXRalpha/T3Rbeta heterodimers participate in protein-DNA interactions with 12, 13, and 11 out of the 14 nucleotides, respectively, that span repeats 1 and 2 and the spacer region separating them on the hormone response element of element AID. The binding of RXRalpha homodimers and RXRalpha/T3Rbeta heterodimers is associated with ligand-dependent activation by 9-cis-RA or repression by T3. Upon deletion or mutation of repeat 1, homodimeric binding of RXRalpha is lost whereas heterodimeric binding is retained. This heterodimeric binding to the mutated element AID is mediated solely by interactions with repeat 2 and one adjacent nucleotide and is confined to a heptameric core recognition motif. The interactions of the RXRalpha heterodimers with repeat 2 are associated with low levels of ligand-independent transcriptional activity. The findings suggest that the specific types of homo- and heterodimers of nuclear hormone receptors occupying the hormone response elements of apoA-I and the availability of the ligand may play an important role in the transcriptional regulation of the human apoA-I gene.

Amino Acid Sequence↗
PubMed1995

Complex interactions between SP1 bound to multiple distal regulatory sites and HNF-4 bound to the proximal promoter lead to transcriptional activation of liver-specific human APOCIII gene.

Footprinting analysis of the human apoCIII promoter identified a set of four proximal (A-D) and six distal (E-J) regulatory elements between nucleotides -792 and -25 [Ogami, K., et al. (1990) J. Biol. Chem. 265, 9808-9815]. The distal regulatory elements of the apoCIII gene increase by 10-fold the strength of the homologous as well as of heterologous proximal promoters. Required for such transcriptional enhancement is the presence of an intact hormone response element (HRE) on the proximal promoter which binds a variety of nuclear hormone receptors. To understand the mechanism of this transcriptional activation, we identified the nature and the importance of the factors which bind to the upstream regulatory elements of the apoCIII promoter by DNA binding, competition, supershift, and transient transfection assays. These analyses showed that the upstream apoCIII promoter contains multiple binding sites for the ubiquitous transcription factor SP1, which recognizes the regulatory elements F, H, and I. The regulatory element G represents a specialized HRE which is recognized by the orphan receptors ARP-1 and EAR-3 but not by HNF-4. A single activity designated CIII J1 binds to the regulatory element J. The same or a similar activity binds as a minor component to the regulatory elements F and I where SP1 is the predominant binding activity. Finally, a minor activity designated CIII 15 binds to the regulatory element I.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein C-III↗
PubMed1994

Transcriptional regulation of the apolipoprotein A-IV gene involves synergism between a proximal orphan receptor response element and a distant enhancer located in the upstream promoter region of the apolipoprotein C-III gene.

Apolipoprotein A-IV expression is limited to intestinal and hepatic cells, suggesting a tissue specific transcriptional regulation of its gene. To investigate the mechanism controlling apo A-IV transcription we have analysed its promoter region by in vitro DNA binding and transient transfection experiments. DNase I footprinting analysis of the proximal promoter with rat liver nuclear extracts revealed four protected regions: AIVA (-32 to -22), AIVB (-84 to -42), AIVC (-148 to -92) and AIVD (-274 to -250). Element AIVC which is necessary for maximal promoter activity, binds HNF-4, Arp-1 and Ear-3 with similar affinity in a mutually exclusive manner. HNF-4 transactivated chimeric constructs containing intact AIVC site in the context of either the apo A-IV promoter or the heterologous thymidine kinase minimal promoter, while Arp-1 and Ear-3 repressed this activation. Increasing amounts of HNF-4 alleviated Arp-1 or Ear-3 mediated repression, suggesting that the observed opposing effects is a result of direct competition of these factors for the same recognition site. In transient transfection assays the apo A-IV promoter region (-700 to +10) had a very low activity in cells of hepatic (HepG2) and intestinal (CaCo2) origin. This activity was increased 13 to 18-fold when the upstream elements of the distantly linked apo C-III gene were fused to the proximal promoter. Results obtained with different 5' and 3' deletion constructs indicated that the cis-acting elements F to J between the nucleotides -500 and -890 of the apo C-III promoter were absolutely necessary to drive maximal enhancement in HepG2 and CaCo2 cells. The apo C-III upstream elements enhanced the activity of the minimal AdML promoter or the apo A-IV site C mutant less efficiently than the intact apo A-IV or AdML promoter constructs containing single HNF-4 sites. The findings suggest that the enhancer effect is mediated by synergistic interactions between the trans-acting factors which recognize the apo C-III regulatory elements and HNF-4 which binds to the proximal apo A-IV promoter.

Animals↗
PubMed1994

Purification and characterization of nuclear factors binding to the negative regulatory element D of human apolipoprotein A-II promoter: a negative regulatory effect is reversed by GABP, an Ets-related protein.

We have previously shown that transcription of the human apolipoprotein A-II (apoA-II) gene is controlled by a complex set of regulatory elements [Cardot et al. (1993) Biochemistry 32, 9080-9093]. We have also identified previously described, as well as new activities which bind to these elements and influence to varying degrees the transcription of the apoA-II gene. DNA binding and competition assays indicated that element D binds three new activities, designated AIID1, AIID2, and AIID4, as well as an activity related to C/EBP. Activities AIID1, AIID2, and AIID4 were purified and characterized further in order to determine their function on the transcriptional regulation of human apoA-II gene. SDS-PAGE analysis as well as photoaffinity cross-linking of the affinity-purified AIID2 showed that it consists of three proteins with molecular masses ranging between 54 and 63 kDa. The amino acid sequence of tryptic peptides obtained from AIID2 protein bands revealed that it is homologous to GABP, an Ets-related protein. Similar analysis showed that affinity-purified AIID4 has an apparent molecular mass of 130 kDa. AIID1 activity was purified partially; in addition to binding to the apoA-II promoter, AIID1 also binds to the regulatory element C of apoCIII and may play a role in the transcriptional regulation of both genes. Methylation interference of G residues and permanganate modification of T residues indicated that the binding sites of AIID2 and AIID4 were contiguous on element D. However, the binding site of AIID1 overlaps with the binding sites of both AIID2 and AIID4. This suggests that the binding of AIID1 and AIID2 or of AIID1 and AIID4 may be mutually exclusive, whereas AIID2 and AIID4 may bind simultaneously. Transcription from a minimal promoter containing elements AB, C, and D of apoA-II increased 1.5- to 1.6-fold when element D is deleted, as well as by promoter mutations which eliminated the binding of both AIID1 and/or AIID4 to element D, but permitted the binding of AIID2/GABP. The findings suggest that element D has a negative regulatory role on apoA-II gene transcription when it is occupied by protein AIID1 and/or AIID4. This negative effect is reversed when element D is occupied only by the regulatory factor AIID2/GABP.

Amino Acid Sequence↗
PubMed1994

Intracellular early and late modifications of human apolipoprotein A-II. Effect of glutamine- +1 to leucine substitution.

It has been shown previously that apoA-II undergoes several intracellular modifications in HepG2 cells (Hussain & Zannis, 1990). In the present study, we have generated permanent cell lines in mouse C127 cells which express the normal apoA-II gene and a mutated form in which Gln+1 was substituted with Leu (Leu+1). This modification was designed to prevent cyclization of the N-terminal glutamine of apoA-II and thus identify the isoproteins which are precursors and products of the N-terminal cyclization reaction. The C127-expression cells were also utilized to study the cellular compartments where the apoA-II modifications occur as well as the importance of the modifications for apoA-II trafficking and secretion. We have found that apoA-II (Gln+1) synthesized by C127 and HepG2 cells had similar isoproteins. In both cell types, unmodified pro-apoA-II, designated isoprotein 3, had a similar isoelectric point as the cell-free translation product of apoA-II mRNA, suggesting that isoprotein 3 results from cleavage of the signal peptide. Isoprotein 3 represents an unmodified apoA-II isoprotein and undergoes an early modification into a more acidic isoprotein 1, which differs from isoprotein 3 by two negative charges. Brefeldin A treatment of the cells did not prevent the formation of isoprotein 1, suggesting that this modification occurs in a pre-Golgi compartment. Neuraminidase treatment of secreted apoA-II isoproteins did not affect isoprotein 1, indicating that it is not sialylated isoprotein. Isoprotein 1 undergoes further modifications which are consistent with cleavage of the propeptide, N-terminal cyclization and sialylation most likely resulting from O-glycosylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-II↗
PubMed1993

An indirect negative autoregulatory mechanism involved in hepatocyte nuclear factor-1 gene expression.

Recent studies have revealed that hepatocyte nuclear factor 4 (HNF-4) is an essential positive regulator of another liver enriched transcription factor HNF-1, defining a transcriptional hierarchy between the two factors operating in hepatocytes. To assess the possible autoregulation of the HNF-1 gene we have examined the effect of HNF-1 on its own transcription. In transient transfection assays, HNF-1 strongly down-regulated transcription driven by its own promoter in HepG2 cells. In addition HNF-1 also repressed the activity of HNF-4 dependent ApoCIII and ApoAI promoters. The same effect was observed using vHNF-1, a distinct but highly related protein to HNF-1. Both HNF-1 and vHNF-1 downregulated HNF-4 activated transcription from intact and chimeric promoter constructs carrying various HNF-4 binding sites implying that they act by impeding HNF-4 binding or activity. DNA binding and cell free transcription experiments however failed to demonstrate any direct or indirect interaction of HNF-1 and vHNF-1 with the above regulatory regions. Both factors repressed HNF-4 induced transcription of the ApoCIII and HNF-1 genes in HeLa cells, arguing against the requirement of a hepatocyte specific function. These findings define an indirect negative autoregulatory mechanism involved in HNF-1 gene expression, which in turn may affect HNF-4 dependent transcription of other liver specific genes.

Animals↗
PubMed1993

Factors participating in the liver-specific expression of the human apolipoprotein A-II gene and their significance for transcription.

We have shown previously that the hepatic and intestinal transcription of the human apolipoprotein A-II (apoA-II) gene in cell cultures is controlled by a complex set of regulatory elements A-N [Chambaz, et al. (1991) J. Biol. Chem. 266, 11676-11685; Cardot, et al. (1991) J. Biol. Chem. 266, 24460-24470]. In the present communication, we have assessed the functional importance of each of the regulatory elements. In addition, we have used DNA binding and competition assays and protein fractionation to identify the hepatic nuclear activities which are involved in the regulation of the human apoA-II gene. Such activities may be of general importance for the regulation of liver-specific genes. The DNA binding and competition analysis showed that the regulatory elements M, D, and F bind new activities which have not been identified in apolipoprotein or other liver-specific promoters. These activities have been designated AIIM1 and AIIM2 for element M, AIID1 and AIID2 for element D, and AIIF2 for element F. The activity AIIM2 is present in liver, but absent in CaCo-2 cells. A set of regulatory elements binds activities which resemble liver-enriched or ubiquitous factors previously shown to play important roles in the regulation of their target genes. Thus, element I binds to activities related to NF1, and elements L, C, D, G, AB, and F bind to C/EBP alpha as well as other heat-stable activities. The affinity of the bacterially expressed C/EBP alpha for the various apoA-II regulatory regions follows the order: AIIL approximately AIIC > AIID > AIIF > AIIG > AIIAB. Protein fractionation showed that element J binds at least three hepatic nuclear activities and is also recognized by members of the nuclear receptor family, HNF4, EAR2, EAR3, and ARP1. Another liver-enriched factor, HNF1, was shown previously to bind to element H. Despite the importance of HNF1, HNF4, NF1, and C/EBP alpha in the regulation of numerous other target genes, deletion of the HNF1, NF1, and HNF4 and several C/EBP binding sites did not drastically affect the hepatic transcription of the apoA-II gene. Rather, the hepatic and intestinal transcription is affected severely by deletion of elements A, B, K, L, and N. In addition, the intestinal transcription is affected by deletion of elements C, J, and M. The in vivo physiological importance of these elements will require analysis of their function in transgenic animals. This analysis establishes the organization of several nuclear activities on the human apoA-II promoter.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Site-directed mutagenesis and structure-function analysis of the human apolipoprotein A-I. Relation between lecithin-cholesterol acyltransferase activation and lipid binding.

We have mutagenized the human apoA-I gene and have generated cell lines which express normal and mutant apoA-I forms. Point mutations were introduced which changed Gln-1, Gln-2 to Arg,Arg, Pro99 to His, and Pro121 to His. In addition, the following amino acid deletions (delta) were generated: delta 113-124, delta 148-186, delta 212-233, and delta 213-243. The apoA-I form isolated from the culture medium of C127 cells was analyzed for its ability to activate lecithin-cholesterol acyltransferase (LCAT) and to bind to phospholipid vesicles and high density lipoprotein (HDL). Compared with the wild type (WT) apoA-I, the relative activation of LCAT achieved by the point mutations Gln-1, Gln-2----Arg,Arg, Pro99----His, and Pro121----His were 106 +/- 7, 92 +/- 6, and 77 +/- 9%, respectively. Kinetic analysis of one mutant apoA-I form showed similar Vmax but a 15-fold increase in the Km of the mutant apoA-I form. Furthermore, the activation achieved by the internal deletion mutants delta 113-124, delta 148-186, delta 212-233, and delta 213-243 was 47 +/- 3, 0.5 +/- 0.4, 28 +/- 4 and 13 +/- 5%, respectively. Mutants deficient in their ability to activate LCAT displayed alterations in liposome and HDL binding, compared with WT as determined by density gradient ultracentrifugation analysis of the culture medium. Thus, the peak recovery (approximately 50%) of apoA-I bound to HDL was at density 1.14 g/ml for the WT apoA-I, at 1.18 g/ml for the mutants delta 113-124 and delta 148-186, and at d greater than 1.21 g/ml for the delta 212-233 and delta 213-243. Electron microscopy of the proteoliposome LCAT substrate generated by WT and mutant apoA-I forms showed that the carboxyl-terminal deletion mutants which displayed aberrant binding to HDL also displayed reduced ability to convert the spherical lecithin-cholesterol vesicles into discs compared with WT. The findings suggest that (a) the importance of the carboxyl terminus of apoA-I for LCAT activation is related to its ability to bind to lipid and/or to form discoidal substrate for LCAT, and (b) the interaction of several domains of apoA-I are required for the activation of LCAT.

Algorithms↗
PubMed1992

Organization of the regulatory elements and nuclear activities participating in the transcription of the human apolipoprotein B gene.

The human apolipoprotein B (apoB) promoter region (-150 to +124) is transcriptionally active in HepG2 and CaCo-2 but not in HeLa cells. In the present study, we demonstrate that transcription of the human apoB gene in hepatic cells is achieved through the interaction of proteins that bind to five regulatory elements, I (-118 to -98), II (-112 to -94), III (-86 to -62), IV (-72 to -53), and V (-53 to -33). Elements I, IV, and V can bind to bacterially expressed enhancer-binding protein (C/EBP) although with different affinities. In addition, elements I, IV, and V interact with at least two other heat-stable nuclear factors, designated NF-BA2 and NF-BA3, that are distinct from C/EBP. These activities have been separated by ion exchange chromatography and shown to produce footprints in elements I, IV, and V that are similar to those obtained with C/EBP. Element II is recognized by three activities designated NF-BCB1, NF-BCB2, and NF-BCB3, which have been separated by heparin-Sepharose chromatography. Saturation mutagenesis of the region established that they belong to a family of proteins recognizing the same motif, 5'-AAAAGCAAACAG-3'. Substitution mutations in element II that abolish binding of all three activities in vitro also reduce hepatic transcription in vivo to 10% of control, indicating that the binding of NF-BCB1, NF-BCB2, and NF-BCB3 activities is required for efficient transcription of the apoB gene. Furthermore, element III is recognized by the previously characterized nuclear factor NF-BA1, the binding of which is essential for the transcriptional activation of the apoB gene in hepatic and intestinal cells.

Animals↗
PubMed1992

Expression of human apolipoprotein E but not that of apolipoprotein A-I by mouse C127 cells is associated with increased secretion of lipids in the form of vesicles and discs.

Transfected mouse mammary-derived cells (C127) expressing human apolipoprotein (apo) E (C127E) were used a) to determine whether the lipid-binding character of apoE is sufficient to promote its assembly with lipid to form lipoprotein-like particles when expressed by cells not normally expressing apolipoproteins; b) to characterize the secreted complexes in terms of morphology, size, and composition; and finally c) to determine whether apoE or apoA-I gene expression by these transfected cells has any effect on the levels and the profiles of the synthesized and secreted lipids. The findings of the present study demonstrate that: a) as determined by density gradient ultracentrifugation and gel filtration chromatography, about 20% of the secreted [35S]methionine-labeled apoE expressed by C127E cells is lipid-associated. b) Negative-stain electron microscopic analysis of the lipid-protein complexes recovered in the lipoprotein fractions (d less than 1.21 g/ml) revealed that approximately 13% of the total population of particles were discs (16 +/- 5 nm mean diameter and 4-6 nm thick), resembling nascent high density lipoproteins (HDL). The majority of the particles however (greater than 82%) appeared vesicular with varying diameters (48 +/- 40 nm mean diameter). The discoidal and the vesicular appearance of the particles secreted by C127E cells is consistent with the composition of lipids. These consisted mostly of surface lipids, phospholipids (45 +/- 18%), diacylglycerols (36 +/- 17%), and free cholesterol (17 +/- 7%) (by weight). c) Expression of apoE by C127E cells was associated with an increased release of [35S]methionine-labeled protein and [3H]glycerol-labeled lipid (3- to 5- and 4- to 8-fold, respectively) compared to nontransfected C127 cells. Expression of mutant apoE or normal apoA-I, however, was not associated with increased release of the major lipid classes compared to the parent C127 cells, strongly suggesting that this character of C127E cells is specific to apoE expression. The release of lipids by C127E cells could be reduced considerably by the addition of the metabolic inhibitors, colchicine or cycloheximide (10 and 1 microM, respectively), suggesting that lipid release by C127E cells is an active process requiring both protein synthesis and functional secretory mechanisms. Taken together the findings suggest that apoE expression by C127 cells promotes the formation of nascent discoidal lipoprotein-like particles and enhances the release of vesicular lipids, possibly by promoting shedding of cell plasma membrane fragments.

Animals↗