SummaryOur previous morphological studies illustrated the association of sterols with Plasmodium infecting hepatocytes. Because malaria parasites cannot synthesize sterols, they must scavenge these lipids from the host. In this paper, we have examined the source/s of sterols for intrahepatic Plasmodium and evaluated the importance of sterols for liver stage development. We show that Plasmodium continuously diverts cholesterol from hepatocytes until release of merozoites. Removal of plasma lipoproteins from the medium results in a 70% reduction of cholesterol content in hepatic merozoites but these parasites remain infectious in animals. Plasmodium salvages cholesterol that has been internalized by low-density lipoprotein but reduced expression of host low-density lipoprotein receptors by 70% does not influence liver stage burden. Plasmodium is also able to intercept cholesterol synthesized by hepatocytes. Pharmacological blockade of host squalene synthase or downregulation of the expression of this enzyme by 80% decreases by twofold the cholesterol content of merozoites without further impacting parasite development. These data enlighten that, on one hand, malaria parasites have moderate need of sterols for optimal development in hepatocytes and, on the other hand, they can adapt to survive in cholesterol-restrictive conditions by exploitation of accessible sterols derived from alternative sources in hepatocytes to maintain proper infectivity.

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...

No abstract

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...

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.