Is cholesterol in all cell membranes?

3.3.2 Cholesterol Transfer

Cholesterol needs are very high in the embryo and the fetus. In early pregnancy, the fetus is unable to synthesize cholesterol. Although most fetal cholesterol is derived endogenously at term, placental mechanisms of maternal transfer satisfy fetal cholesterol needs at early stages of pregnancy [12].

Impaired maternal cholesterol metabolism appears related to fetal diseases. Low maternal cholesterol concentration is associated with impaired neurological development [40] and low birth weight in term infants [41]. Increased maternal cholesterol is associated with increased risk of atherosclerotic disease development [42]. Recent data have shown a U-shaped relationship between maternal cholesterol concentration and preterm birth risk [43].

Membrane Structure

Cholesterol is absent from prokaryotic cells. However, cholesterol plays an essential structural role in maintaining the fluidity of eukaryotic cell membranes. Cholesterol is not equally distributed in all membranes; the membranes of mitochondria, peroxisomes, and endoplasmic reticulum are cholesterol-poor, whereas the plasma membrane is enriched in the sterol. However, the concentration of cholesterol varies significantly even within the plasma membrane; it is highly enriched in two specialized areas termed lipid rafts and caveolae. Since many receptors are localized to these cholesterol- and sphingomyelin-rich domains, it has been suggested that lipid rafts and caveolae function as “signaling gateways” into the cell. The myelin sheath that surrounds nerves has the highest cholesterol concentration.

Membrane Structure

Cholesterol is absent from prokaryotic cells. However, cholesterol plays an essential structural role in maintaining the fluidity of eukaryotic cell membranes. Cholesterol is not equally distributed in all membranes; the membranes of mitochondria, peroxisomes, and endoplasmic reticulum are cholesterol poor, whereas the plasma membrane is enriched in the sterol. However, the concentration of cholesterol varies significantly even within the plasma membrane; it is highly enriched in two specialized areas termed lipid rafts and caveolae. Since many receptors are localized to these cholesterol- and sphingomyelin-rich domains, it has been suggested that lipid rafts and caveolae function as signaling gateways into the cell.

Cholesterol

Cholesterol in the intestinal lumen is derived from both, endogenous sources and dietary sources: The human diet provides about 400 mg of cholesterol daily, and the liver secretes 1 g daily. About 50% of the cholesterol in the intestine is absorbed; the remainder is excreted with feces. Most dietary cholesterol exists in the form of the free sterol, with only 10–15% existing as the cholesteryl ester. The latter must be hydrolyzed by cholesterol esterase to release free cholesterol for absorption. Moreover, cholesterol must pass through a diffusion barrier at the intestinal lumen–enterocyte membrane interface before it can interact with the transporter proteins responsible for its uptake and subsequent transport across the cellular brush border. Bile salt micelles facilitate the transfer of cholesterol across the unstirred water layer. Thus, cholesterol absorption depends on the presence of bile acids in the intestinal lumen.

5.1.1.2 Cholesterol

Cholesterol does not form a bilayer structure itself, but can be incorporated into phospholipid membranes in very high concentrations. Cholesterol content exceeds a threshold, the vacant spaces between the bilayers become occupied with the hydroxyl moiety of cholesterol (Henriksen et al., 2004) toward the aqueous surface and aliphatic chain aligned parallel to the acyl chains in the center of the bilayer, thus resulting in an increase in the rigidity of the vesicles as well as entrapment efficiency. The distribution of cholesterol within lipid is dependent on the concentration of cholesterol. At low concentrations, that is, 20 mole % or less, a phase separation occurs in two phases, one is rich in cholesterol and the other poor in cholesterol. At the higher concentration, that is, more than 50 mole % disruption in the regular linear structure occurs, this may be due to a reduction in the number of specific intermolecular interactions and cholesterol tends to destabilize the vesicle, leading to a possible decrease in drug entrapment.

Connection Between Cholesterol and Alzheimer's Disease

Cholesterol plays a central role in the biology of amyloid precursor protein and the toxic peptide generated by its cleavage, Aβ. The ability of cholesterol to modulate Aβ production, combined with the utility of statins in health care, suggests fertile avenues of research for treating AD. Cholesterol metabolism presents multiple targets for inhibiting Aβ production. Furthermore, cholesterol is a very abundant component of the synaptic membrane, where the acetylcholine receptor (AChR) is located. Cholesterol affects AChR proteins on multiple levels. Thus AD may be partly associated with an abnormal crosstalk between the receptor protein and the sterol in the synaptopathy [32–34].

6.1 Cholesterol and other sterols

Cholesterol is an essential component of mammalian membranes and is a precursor for all steroid hormones. However, cholesterol can accumulate in certain tissues and cause serious pathological consequences in the body, such as artherosclerosis. Recently, the analysis of oxidation products of cholesterol (and other lipids) has gained interest due to their putative pathophysiological role [113]. There is also growing interest toward various plant sterols (phytosterols) as their intake significantly reduces plasma cholesterol levels [114].

Sterols cannot be analyzed by ESI–MS without derivatization as they are not readily ionized [115]. Sandhoff et al. have used chemical sulfatation to achieve high-sensitivity detection of cholesterol [116]. Cholesterol has also been derivatized with dimethylglycine, MDMABS [117], or ferrocenecarbamate [118]. Notably, derivatization can be avoided by using APCI or APPI, which have been applied for the analysis of cholesterol and other sterols [119–122] or oxidized cholesterol [123].

3.3 Cholesterol deficiency and sonic hedgehog signaling

Cholesterol is an end product of the mevalonate pathway [58] and is required for normal development. Ethanol has been shown to inhibit the mevalonate pathway and thus cholesterol biosynthesis [59,60]. In mice, the lack of availability of cholesterol results in a drastic decline in sonic hedgehog (shh) signals transduction [58]. Moreover, chicken and mouse embryos exposed to ethanol displayed reduced shh signaling [61]. Therefore, alcohol-dependent inhibition of the cholesterol modification of shh produces morphologic defects which are similar to FASD phenotypes. In zebrafish embryos, ethanol treatment causes a dose-dependent reduction in cholesterol content, decreased cholesterol modification of shh, and a loss of shh signal transduction resulting in cyclopia, craniofacial hypoplasia, and holoprosencephaly [60]. Further, supplementation of cholesterol is able to rescue the zebrafish embryos from these phenotypic defects.

Cell Cholesterol Homeostasis [49]

Exogenous cholesterol and triacylglycerides in our diet are introduced to the entero-hepatic circulation, and delivered to the liver as chylomicron remnants. The liver, which is the central organ involved in cholesterol biosynthesis, is also involved in cholesterol and triacylglycerol transport, and repackaging them into very-low-density lipoprotein (VLDL) particles, which are secreted to the hepato-peripheral circulation. The VLDV in circulation enters a cascade of lipoysis as a substrate for lipoprotein lipase so that triacylglycerol is progressively removed with a concomitant increase in density until LDL particles are formed. LDL particles are the primary carrier of cholesterol in the plasma and deliver cholesterol to peripheral cells via apolipoproteins B and E are also present in the liver so that particles formed by lipolysis of chylomicrons and VLDL are also cleared by the liver. Here it should be noted that Peng et al [50] demonstrated that LDL and VLDL are major carriers of the oxysterols that cause lethal injury to the endothelial and smooth muscle cells. Thus the popular name coined for LDL-cholesterol, the “Bad Cholesterol,” may be appropriate. On the other hand, the popular name the “Good Cholesterol” for HDL may be equally appropriate, since HDL carry considerably fewer oxysterols, and it is this lipoprotein species that is involved in transporting cholesterol molecules from peripheral tissues to the liver as mediated by HDL is called the reverse cholesterol transport pathway. It is our hypothesis that some oxysterols can cause obstruction in the reverse transportation of cholesterol, and thereby instrumental in the development of AP.

It has been shown that free cholesterol molecules can transfer between membranes by diffusion through the intervening aqueous layer [17]. Desorption of free cholesterol molecules from the donor lipid-water interface is rate-limiting for the overall transfer process and the rate of this step is influenced by interactions of free cholesterol molecules with neighboring phospholipid molecules. The influence of phospholipid unsaturation and sphingomyelin content on the rate of free cholesterol exchange are known in pure phospholipid bilayers and similar effects probably occur in cell membranes. The rate of free cholesterol clearance from cells is determined by the structure of the plasma membrane [17] It follows that the physical state of free cholesterol in the plasma membrane is important for the kinetics of cholesterol clearance and cell cholesterol homeostasis, as well as the structure of the plasma membrane.

Bi-directional flux of free cholesterol between cells and lipoproteins occurs, and rate constants characteristic of influx and efflux can be measured [17]. The direction of any net transfer of free cholesterol is determined by the relative free cholesterol/phospholipid molar ratios of the donor and acceptor particles. Cholesterol diffuses down its gradient of chemical potential generally partitioning to the phospholipid-rich particle. Such a surface transfer process can lead to delivery of cholesterol to cells. This mechanism operates independently of any lipoprotein internalization by the receptor-mediated endocytosis. The influence of enzymes such as lecithin-cholesterol acyltransferase and hepatic lipase on the direction of net transfer of free cholesterol between lipoproteins and cells can be understood in terms of their effects of the pool sizes and the rate constants for influx and efflux.

Excess accumulation of free cholesterol in cells stimulates the rate of cholesterol ester formation and induces deposition of cholestryl ester inclusions in the cytoplasm similar to the situation in the ‘foam’ cells of atherosclerotic plaque.

Abstract

Cholesterol is a major component of the plasma membranes (PMs) of animal cells, comprising 35–40 mol% of total PM lipids. Recent studies using cholesterol-binding bacterial toxins such as domain 4 of Anthrolysin O (ALOD4) and fungal toxins such as Ostreolysin A (OlyA) have revealed new insights into the organization of PM cholesterol. These studies have defined three distinct pools of PM cholesterol—a fixed pool that is essential for membrane integrity, a sphingomyelin (SM)-sequestered pool that can be detected by OlyA, and a third pool that is accessible and can be detected by ALOD4. Accessible cholesterol is available to interact with proteins and transport to the endoplasmic reticulum (ER), and controls many cellular signaling processes including cholesterol homeostasis, Hedgehog signaling, and bacterial and viral infection. Here, we provide detailed descriptions for the use of ALOD4 and OlyA, both of which are soluble and non-lytic proteins, to study cholesterol organization in the PMs of animal cells. Furthermore, we describe two new versions of ALOD4 that we have developed to increase the versatility of this probe in cellular studies. One is a dual His6 and FLAG epitope-tagged version and the other is a fluorescent version where ALOD4 is fused to Neon, a monomeric fluorescent protein. These new forms of ALOD4 together with previously described OlyA provide an expanded collection of tools to sense, visualize, and modulate levels of accessible and SM-sequestered cholesterol on PMs and study the role of these cholesterol pools in diverse membrane signaling events.