Glutaraldehyde treatment of 25I-labeled low density lipoprotein (125I-native-LDL) produced a modified LDL ('lI-gut-LDL) with a molecular weight of 10 X 106 or more. Malondialdehyde treatment of 125I-hative-LDL produced a product (125I-MDA-LDL) with a molecular weight not appreciably different from that of the original lipoprotein. However, the electrophoretic mobility of MDA-LDL indicated a more negative charge than native-LDL. 125I-MDALDL was degraded by two processes: a high-affinity saturable process with maximal velocity at 10-15 pg of proteinper ml and a slower, nonsaturable process. The degradation of 12I-MDA-LDL was readily inhibited by increasing concentrations of nonradioactive MDA-LDL but was not inhibited by acetylated LDL or native-LDL even at concentrations as high as 1600 ptg of protein per ml. After exposure of native-LDL to blood platelet aggregation and release in vitro, 1.73 + 0.19 nmol of malondialdehyde per mg of LDL protein was bound to the platelet-modified-LDL. No detectable malondialdehyde was recovered from native-LDL that had-been treated identically except that the platelets were omitted from the reaction mixture.After incubation with glut-LDL, MDA-LDL, or plateletmodified-LDL for 3 days, human monocyte-macrophages showed a dramatic increase in cholesteryl ester content whereas the cholesteryl ester content of cells incubated with the same concentration of native-LDL did not. Based on these experiments we propose that modification of native-LDL may be a prerequisite to the accumulation of cholesteryl esters within the cells of the atherosclerotic reaction. We further hypothesize that one modification of LDL in vivo may result from malondialdehyde which is released from blood platelets or is produced by lipid peroxidation at the site of arterial injury. There is increasing evidence that the foam cells found in the atherosclerotic reaction are macrophages that are derived from blood-borne monocytes or from smooth muscle cells that have taken on many of the properties of macrophages (1-3). The hallmark of these cells is their high cholesteryl ester content (>50% of total cellular cholesterol) (4, 5). Our objective has been to define the conditions and mechanisms leading to cholesteryl ester accumulation within these cells. We learned from experiments to be reported elsewhere that human monocytes contain very little cholesteryl ester (approximately 2% of total cellular cholesterol), and the conversion of the monocytes into macrophages in vitro did not appreciably increase their cholesteryl ester content. Moreover, these cells did not accumulate cholesteryl esters when incubated in high concentrations of low density lipoprotein (LDL).The experiments reported here demonstrate that LDL must be modified before it will produce cholesteryl ester accumulation in human monocyte-macrophages. Based on these experiments we propose that one modification of LDL in vivo may result from an interaction with malondialdehyde which is released from blood platelets or is produced by lipid peroxidation...
These results indicate that 1) Section C, in particular Phe 288 , may be involved in the second step of catalysis, 2) Tyr 171 of Section A is essential for catalysis, most likely for the first reaction, 3) the two Asp residues in Section B are essential for the activity and most likely bind the substrate via magnesium salt bridges. Based on these results, a mechanism for the first reaction is proposed.
Abstract-HDL mimetics have been constructed from a number of peptides and proteins with varying structures, all of which bind lipids found in HDL. HDL mimetics containing a peptide or protein have been constructed with as few as 4 and as many as 243 amino acid residues. Some HDL mimetics have been constructed with lipid but without a peptide or protein component. Some HDL mimetics promote cholesterol efflux, some have been shown to have a remarkable ability to bind oxidized lipids compared to human apolipoprotein A-I (apoA-I). Many of these peptides have been shown to have antiinflammatory properties. Based on studies in a number of animal models and in early human clinical trials, HDL mimetics appear to have promise as diagnostic and therapeutic agents. Separating HDL-Cholesterol Levels From HDL FunctionSimply increasing the amount of circulating HDL-cholesterol does not reduce the risk of coronary heart disease (CHD) events, CHD deaths, or total deaths. 1 Heinecke 2 has noted that HDL-cholesterol does not define the proteins associated with HDL and suggests that the HDL proteome is a marker, and perhaps a mediator, of CHD. Zheng et al 3 reported that apoA-I, the major protein in HDL, is a selective target for myeloperoxidase-catalyzed oxidation, which results in impairment of the ability of HDL to promote cholesterol efflux. Singh et al 4 suggested that HDL could be a therapeutic target by modifying its lipid and protein cargo to improve its antiinflammatory properties. One method that has been reported to modify the lipid and protein cargo of HDL involves treatment with apolipoprotein mimetic peptides. 5 The Development of Apolipoprotein Mimetic Peptides as Therapeutic AgentsThe efficacy of apoA-I in improving atherosclerosis in animal models 6,7 and in preliminary human studies 8 made it an attractive therapeutic candidate. However, human apoA-I has 243 amino acid residues, making it not only difficult and expensive to synthesize but necessitating that it be given intravenously. The initial promise 8 of therapeutic benefit from weekly intravenous doses for 5 to 6 weeks does not seem to have been borne out by subsequent larger clinical trials. 9 It is likely that longer periods of intravenous administration will be required, making this an unlikely therapy for the millions of patients with atherosclerosis.The laboratories of Segrest and Anantharamaiah designed an 18-aa peptide that did not have sequence homology with apoA-I but mimicked the class A amphipathic helixes contained in apoA-I. 10 -12 This peptide was called 18A because it contained 18 amino acids and formed a class A amphipathic helix. When the amino and carboxyl termini were blocked by addition of an acetyl group and amide group, respectively, stability and lipid-binding properties were improved and the peptide was called 2F because of the 2 phenylalanine residues on the hydrophobic face. The 2F peptide mimicked many of the lipid binding properties of apoA-I but failed to alter lesions in a mouse model of atherosclerosis. 13 Using a cell-based ...
In our present study, we demonstrate that mutation of this HSS-SRE-1 element significantly reduced, but did not abolish, the response of HSS promoter to change in sterol concentration. Mutation scanning indicates that two additional DNA promoter sequences are involved in sterol-mediated regulation. The first sequence contains an inverted SRE-3 element (Inv-SRE-3) and the second contains an inverted Y-box (Inv-Y-box) sequence. A single mutation in any of these sequences reduced, but did not completely remove, the response to sterols. Combination mutation studies showed that the HSS promoter activity was abolished only when all three elements were mutated simultaneously. Co-expression of SRE-1-or SRE-2-binding proteins (SREBP-1 or SREBP-2) with HSS promoter-luciferase reporter resulted in a dramatic increase of HSS promoter activity. Gel mobility shift studies indicate differential binding of the SREBPs to regulatory sequences in the HSS promoter. These results indicate that the transcription of the HSS gene is regulated by multiple regulatory elements in the promoter.Squalene synthase (farnesyl diphosphate-farnesyl diphosphate farnesyltransferase, EC 2.5.1.21) catalyzes the reductive head-to-head condensation of two molecules of farnesyl diphosphate (FPP) 1 to form squalene, the first specific intermediate in the cholesterol biosynthesis pathway. The expression of squalene synthase, as that of several other key enzymes in the pathway, such as 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and FPP synthase, is highly regulated by the cholesterol homeostasis in cells (1-4). It has previously been demonstrated that the expression of squalene synthase is regulated transcriptionally (5), but the mechanism for this regulation is unknown. Low density lipoprotein receptor (LDLR) and HMG-CoA synthase are regulated by interaction between recently described transcriptional factors, sterol regulatory element-binding-proteins (SREBPs). These interact with sequences called sterol regulatory elements (SRE-1 and SRE-2) that exist in the promoters of the two genes (6 -8). Two functionally related SREBPs, SREBP-1 and SREBP-2, have been purified from human cells and hamster cells, and the mechanism by which they regulate the expression of LDLR and HMG-CoA synthase has been studied extensively (9 -11). Human SREBP-1 and SREBP-2 are 47% identical. At the NH 2 -terminal region of each protein, there is a basic-helix-loop-helix leucine zipper (bHLH-Zip) structure that serves as a transcriptionally active domain. Next to the bHLH-Zip domain there are two membrane attachment domains. Nascent SREBP-1 and SREBP-2 are localized in the ER by these domains, and they are inactive in stimulating transcription. At lower concentrations of sterol in cells, an ER-associated, sterol-sensitive protease is activated and proteolytically activates the SREBPs by a cleavage at a site between the leucine zipper and the membrane attachment domains to release the bHLH-Zip domain (12). The active bHLH-Zip segment of SREBP-1 was...
Squalene synthase (farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21) is the first enzyme specific to the cholesterol biosynthetic pathway. The activity of rat hepatic squalene synthase is regulated by dietary cholesterol and by the dietary 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) 1 reductase inhibitors, lovastatin, or fluvastatin (1, 2). The activity of human squalene synthase (HSS) and the level of its mRNA are regulated by sterols in the human hepatoma cell line HepG2. Sterol-mediated regulation has been localized to a 10-base pair (bp) element in the 5Ј-flanking region of other sterolregulated genes. This 10-bp sterol regulatory element 1 (SRE-1) mediates increased transcription of the genes encoding HMG-CoA synthase and the low density lipoprotein (LDL) receptor in sterol-depleted cells, and its activity is inhibited by sterols (3, 4). Proteins that bind to the SRE-1 of the LDL receptor (SREBPs) were purified by DNA affinity chromatography from nuclear extracts of HeLa cells. A cDNA for SREBP-1 was isolated from adipocyte cDNA library (5). This cDNA, designated ADD1, activated transcription of a reporter gene containing an "E-box" sequence present in the promoter of fatty acid synthase in transfected NIH 3T3 cells. Cloned SREBP cDNA contain two major classes of proteins, SREBP-1 (5) and SREBP-2 (8). Three different cDNAs for SREBP-1 were isolated, suggesting multiple forms of the mRNA and perhaps different proteins as well. The physiological significance of these subclasses is unclear (6). Different SREBP-1 proteins may have specific physiological roles because mRNAs for the various isoforms are differentially regulated by sterol depletion in HepG2 cells (8).Proteolytic cleavage of the C-terminal membrane-associated domain of the nascent SREBP-1 (125 kDa) forms its nuclear form (68 kDa). This proteolytic maturation was proposed to be accomplished by a sterol-inhibited protease. The calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) induced the mRNA for HMG-CoA synthase and was proposed to inhibit the degradation of the mature SREBP-1 (9).In other sterol-regulated genes, the SRE-1 is not involved in sterol-mediated transcriptional regulation. Although the promoter region of farnesyl diphosphate synthase contains multiple forms of the SRE-1 element, these elements are not involved in the sterol-mediated transcriptional regulation (10). Similarly, the promoter of the hamster HMG-CoA reductase contains unique sites for sterol regulation. Red 25, a nuclear hamster liver protein, binds to this regulatory region but did not bind to the sterol regulatory regions of the LDL receptor and HMG-CoA synthase promoters (11).In this report we characterize the 5Ј region of the HSS gene. The promoter activity and the sterol-mediated regulation of this DNA were assessed by fusing 5Ј-flanking DNA to a luciferase reporter gene and transfecting it into HepG2 cells and Chinese hamster ovary (CHO-K1) cells. A 69 bp DNA sequence confers transcriptional competence and sterol regulation. ADD1...
No abstract
Several cues for cell proliferation, migration, and survival are transmitted through lipid rafts, membrane microdomains enriched in sphingolipids and cholesterol. Cells obtain cholesterol from the circulation but can also synthesize cholesterol de novo through the mevalonate/isoprenoid pathway. This pathway, however, has several branches and also produces non-sterol isoprenoids. Squalene synthase (SQS) is the enzyme that determines the switch toward sterol biosynthesis. Here we demonstrate that in prostate cancer cells SQS expression is enhanced by androgens, channeling intermediates of the mevalonate/isoprenoid pathway toward cholesterol synthesis. Interestingly, the resulting increase in de novo synthesis of cholesterol mainly affects the cholesterol content of lipid rafts, while leaving non-raft cholesterol levels unaffected. Conversely, RNA interference-mediated SQS inhibition results in a decrease of raft-associated cholesterol. These data show that SQS activity and de novo cholesterol synthesis are determinants of membrane microdomain-associated cholesterol in cancer cells. Remarkably, SQS knock down also attenuates proliferation and induces death of prostate cancer cells. Similar effects are observed when cancer cells are treated with the chemical SQS inhibitor zaragozic acid A. Importantly, although the anti-tumor effect of statins has previously been attributed to inhibition of protein isoprenylation, the present study shows that specific inhibition of the cholesterol biosynthesis branch of the mevalonate/isoprenoid pathway also induces cancer cell death. These findings significantly underscore the importance of de novo cholesterol synthesis for cancer cell biology and suggest that SQS is a potential novel target for antineoplastic intervention.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.