| For medical practitioners and the general public - Cholesterol Journal Article Catalog. |
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| Crosslinked plasmalemmal cholesterol is sequestered to caveolae: analysis with a new cytochemical probe Fujimoto, T., M. Hayashi, et al. (1997), J Histochem Cytochem 45(9): 1197-205. Abstract: symbol: see text-Toxin (perfringolysin O), a cholesterol-binding toxin, was partially proteolyzed and biotinylated (BC theta) to eliminate hemolyzing activity and was used as a cytochemical probe. In fixed cells, binding of BC theta was intense in the plasma membrane, especially at the base of apical microvilli and in lateral processes. The labeling was abolished by pretreatment with filipin, digitonin, or tomatin. When living cultured cells were treated with BC theta and then with either fluorescein-avidin D or colloidal gold-streptavidin, the labeling in fine dots was distributed on the cell surface without local concentration as long as cells were kept on ice. When the temperature was raised to 37 C after treatment, the probe formed discrete large patches and became sequestered to caveolae. Binding of BC theta alone without the secondary reagents did not cause redistribution even at 37 C. Because the plasma membrane maintains integrity even after binding of BC theta, the probe can be used not only for cytochemical labelling of fixed cells but for pursuing the behavior of crosslinked cholesterol molecules in living cells. By use of this new probe, the present study revealed that crosslinked cholesterol in the plasma membrane is sequestered to caveolae. |
| Crosslinking of beta-cyclodextrin on cholesterol removal from milk Kim, S. H., J. Ahn, et al. (2004), Arch Pharm Res 27(11): 1183-7. Abstract: This study was designed to develop crosslinking of beta-cyclodextrin (beta-CD), and determine the optimum conditions of different factors (mixing time, mixing temperature, and mixing speed) on cholesterol reduction from milk. Crosslinked beta-CD was prepared with epichlorohydrin. When milk was treated with different conditions, the cholesterol removal rate was in the range of 79.4 to 83.3% with 1% crosslinked beta-CD addition, which were not significantly different among treatments. After cholesterol removal from milk, the used crosslinked beta-CD was washed for cholesterol dissociation and reused. For recycling study, the cholesterol removal rate in first trial was 81.8%, which was mostly same as that using new crosslinked beta-CD. With five trials repeatedly using the same sample, the mean cholesterol removal rate was 81.2%. The present study indicated that the optimum conditions on cholesterol removal using crosslinked beta-CD were 10 min mixing with 400 rpm speed at 5 degrees C with about 80% cholesterol removal. In addition, crosslinked beta-CD resulted in the effective recycling efficiency almost 100%. |
| Cross-linking of plasmalemmal cholesterol in lymphocytes induces capping, membrane shedding, and endocytosis through coated pits Hagiwara, H., S. Y. Kogure, et al. (1999), Biochem Biophys Res Commun 260(2): 516-21. Abstract: By use of a nicked and biotinylated perfringolysin O (BCtheta), which binds to cholesterol specifically, we studied consequences of cross-linking cholesterol in lymphocytes. When bound with BCtheta and then with labeled avidin or streptavidin, capping occurred in most cells within 30 min at 37 degrees C. It was inhibited by cytochalasin D or NaN3, but not by nocodazole. When BCtheta-cholesterol was capped, Thy-1 and transferrin receptor, a GPI-anchored protein and a transmembrane protein, respectively, remained evenly distributed. By fluorescence and electron microscopy, a cluster of small vesicles bound with BCtheta were observed in the cap. They were then shed in the medium or internalized through coated pits. The result indicates that cross-linking of cholesterol in lymphocytes induces capping, but does not affect distribution of membrane proteins, and that the capped cholesterol molecules are either shed as vesicles or endocytosed. |
| Cross-sectional and longitudinal associations between high density lipoprotein cholesterol and women's employment Haertel, U., G. Heiss, et al. (1992), Am J Epidemiol 135(1): 68-78. Abstract: This study examined the association between women's employment and high density lipoprotein (HDL) cholesterol. Subjects were 1.998 women aged 25-64 years who were sampled by the first MONICA Augsburg Survey (Monitoring of Trends and Determinants in Cardiovascular Disease). The women were sampled from the population of Augsburg, Federal Republic of Germany, in 1984-1985, were followed up for 3 years, and were reexamined in 1987-1988. In cross-sectional analysis (1984-1985), the mean HDL cholesterol level of employed women was 3.4 mg/dl higher than that of full-time homemakers (p less than 0.001). After adjustment for age, body mass, cigarette smoking, consumption of coffee and alcohol, use of sex hormones, leisure-time physical activity, and reproductive history, this difference decreased to 2.1 mg/dl and remained statistically significant (p less than 0.01). As was predicted from the cross-sectional findings, the mean HDL cholesterol levels of women who gave up employment and became full-time homemakers during the follow-up period decreased by 3.04 mg/dl (p less than 0.01), whereas homemakers who became employed showed no significant change in HDL cholesterol levels. The change in mean HDL cholesterol of employed women who had become homemakers could be explained in part by changes in alcohol consumption and in number of pregnancies. The authors conclude that giving up employment is related to life-style changes that are associated with a decrease in HDL cholesterol levels. Furthermore, the findings suggest that employment may exert a beneficial influence on coronary risk in women that is consistent with a positive association between employment and HDL cholesterol. |
| Cross-sectional and longitudinal changes in total and high-density-lipoprotein cholesterol levels over a 20-year period in elderly men: the Honolulu Heart Program Abbott, R. D., D. S. Sharp, et al. (1997), Ann Epidemiol 7(6): 417-24. Abstract: PURPOSE: The purpose of this report is to describe levels of total cholesterol and high-density-lipoprotein cholesterol (HDL-C) in a group of elderly men and to compare these levels to those that were observed 20 years earlier. METHODS: From 1965-1968, the Honolulu Heart Program began following 8006 men of Japanese ancestry living on the island of Oahu, Hawaii, in a prospective study of coronary heart disease and stroke. This report presents data for 971 men who participated in a separate fasting study of lipids and lipoproteins that first occurred from 1970-1972 and in those who received repeat examinations 10 and 20 years later. Men were aged 71-93 years at the last examination. RESULTS: Over the 20-year period, total cholesterol declined by 1.6-1.8 mg/dL per year (P < 0.001), from average baseline values of 219-222 mg/dL. Levels of HDL-C rose 0.2-0.3 mg/dL per year (P < 0.001), from average baseline values of 44-46 mg/dL. After adjustment for baseline cholesterol levels, men with prevalent coronary heart disease at the end of the 20-year follow-up experienced significantly greater reductions in total cholesterol levels than men without disease (P < 0.001). Men who developed coronary heart disease within the first 10 years of follow-up had the greatest yearly decline in total cholesterol (1.9 mg/dL), followed by men who developed heart disease later (1.8 mg/dL) and men who remained disease free (1.5 mg/dL). Differences between men with recent and earlier disease were not statistically significant, although men without coronary disease experienced a significantly smaller decrease in total cholesterol than either of these groups (P < 0.05). CONCLUSIONS: Changes in total cholesterol and HDL-C levels with advancing age may be part of a natural aging process. Some changes, however, such as large reductions in total cholesterol, may signal occult disease or declines in overall health. Selective survival may contribute to these findings since improvements in lipid and lipoprotein levels that are beneficial in younger ages were common in this long-lived cohort of men. |
| Cross-talk between fatty acid and cholesterol metabolism mediated by liver X receptor-alpha Tobin, K. A., H. H. Steineger, et al. (2000), Mol Endocrinol 14(5): 741-52. Abstract: LXR alpha (liver X receptor, also called RLD-1) is a nuclear receptor, highly expressed in tissues that play a role in lipid homeostasis. In this report we show that fatty acids are positive regulators of LXR alpha gene expression and we investigate the molecular mechanisms underlying this regulation. In cultured rat hepatoma and primary hepatocyte cells, fatty acids and the sulfur-substituted fatty acid analog, tetradecylthioacetic acid, robustly induce LXR alpha (up to 3.5- and 7-fold, respectively) but not LXR beta (also called OR-1) mRNA steady state levels, with unsaturated fatty acids being more effective than saturated fatty acids. RNA stability and nuclear run-on studies demonstrate that changes in the transcription rate of the LXR alpha gene account for the major part of the induction of LXR alpha mRNA levels. A similar induction of protein level was also seen after treatment of primary hepatocytes with the same fatty acids. Consistent with such a transcriptional effect, transient transfection studies with a luciferase reporter gene, driven by 1.5 kb of the 5'-flanking region of the mouse (m)LXR alpha gene, show a peroxisome proliferator-activated receptor-alpha-dependent increase in luciferase activity upon treatment with tetradecylthioacetic acid and the synthetic peroxisome proliferator-activated receptor-alpha activator, Wy 14.643, suggesting that the mLXR alpha 5'-flanking region contains the necessary sequence elements for fatty acid responsiveness. In addition, in vivo LXR alpha expression was induced by fatty acids, consistent with the in vitro cell culture data. These observations demonstrate that LXR alpha expression is controlled by fatty acid signaling pathways and suggest an important cross-talk between fatty acid and cholesterol regulation of lipid metabolism. |
| Crosstalk between LXR and toll-like receptor signaling mediates bacterial and viral antagonism of cholesterol metabolism Castrillo, A., S. B. Joseph, et al. (2003), Mol Cell 12(4): 805-16. Abstract: The liver X receptors (LXR) alpha and beta are regulators of cholesterol metabolism and determinants of atherosclerosis susceptibility. Viral and bacterial pathogens have long been suspected to be modulators of atherogenesis; however, mechanisms linking innate immunity to cholesterol metabolism are poorly defined. We demonstrate here that pathogens interfere with macrophage cholesterol metabolism through inhibition of the LXR signaling pathway. Activation of Toll-like receptors (TLR) 3 and 4 by microbial ligands blocks the induction of LXR target genes including ABCA1 in cultured macrophages as well as in aortic tissue in vivo. As a consequence of these transcriptional effects, TLR3/4 ligands strongly inhibit cholesterol efflux from macrophages. Crosstalk between LXR and TLR signaling is mediated by IRF3, a specific effector of TLR3/4 that inhibits the transcriptional activity of LXR on its target promoters. These findings highlight a common mechanism whereby bacterial and viral pathogens may modulate macrophage cholesterol metabolism and cardiovascular disease. |
| Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER Yang, T., P. J. Espenshade, et al. (2002), Cell 110(4): 489-500. Abstract: Using coimmunoprecipitation and tandem mass spectrometry, we identify INSIG-1 as an ER protein that binds the sterol-sensing domain of SREBP cleavage-activating protein (SCAP) and facilitates retention of the SCAP/SREBP complex in the ER. In sterol-depleted cells, SCAP escorts SREBPs from ER to Golgi for proteolytic processing, thereby allowing SREBPs to stimulate cholesterol synthesis. Sterols induce binding of SCAP to INSIG-1, as determined by blue native-PAGE, and this is correlated with the inhibition of SCAP exit from the ER. Overexpression of INSIG-1 increases the sensitivity of cells to sterol-mediated inhibition of SREBP processing. Mutant SCAP(Y298C) fails to bind INSIG-1 and is resistant to sterol-mediated inhibition of ER exit. By facilitating sterol-dependent ER retention of SCAP, INSIG-1 plays a central role in cholesterol homeostasis. |
| Crystal structure of human cholesterol sulfotransferase (SULT2B1b) in the presence of pregnenolone and 3'-phosphoadenosine 5'-phosphate. Rationale for specificity differences between prototypical SULT2A1 and the SULT2BG1 isoforms Lee, K. A., H. Fuda, et al. (2003), J Biol Chem 278(45): 44593-9. Abstract: The gene for human hydroxysteroid sulfotransferase (SULT2B1) encodes two peptides, SULT2B1a and SULT2B1b, that differ only at their amino termini. SULT2B1b has a predilection for cholesterol but is also capable of sulfonating pregnenolone, whereas SULT2B1a preferentially sulfonates pregnenolone and only minimally sulfonates cholesterol. We have determined the crystal structure of SULT2B1a and SULT2B1b bound to the substrate donor product 3'-phosphoadenosine 5'-phosphate at 2.9 and 2.4 A, respectively, as well as SULT2B1b in the presence of the acceptor substrate pregnenolone at 2.3 A. These structures reveal a different catalytic binding orientation for the substrate from a previously determined structure of hydroxysteroid sulfotransferase (SULT2A1) binding dehydroepiandrosterone. In addition, the amino-terminal helix comprising residues Asp19 to Lys26, which determines the specificity difference between the SULT2B1 isoforms, becomes ordered upon pregnenolone binding, covering the substrate binding pocket. |
| Crystal structure of human squalene synthase. A key enzyme in cholesterol biosynthesis Pandit, J., D. E. Danley, et al. (2000), J Biol Chem 275(39): 30610-7. Abstract: Squalene synthase catalyzes the biosynthesis of squalene, a key cholesterol precursor, through a reductive dimerization of two farnesyl diphosphate (FPP) molecules. The reaction is unique when compared with those of other FPP-utilizing enzymes and proceeds in two distinct steps, both of which involve the formation of carbocationic reaction intermediates. Because FPP is located at the final branch point in the isoprenoid biosynthesis pathway, its conversion to squalene through the action of squalene synthase represents the first committed step in the formation of cholesterol, making it an attractive target for therapeutic intervention. We have determined, for the first time, the crystal structures of recombinant human squalene synthase complexed with several different inhibitors. The structure shows that SQS is folded as a single domain, with a large channel in the middle of one face. The active sites of the two half-reactions catalyzed by the enzyme are located in the central channel, which is lined on both sides by conserved aspartate and arginine residues, which are known from mutagenesis experiments to be involved in FPP binding. One end of this channel is exposed to solvent, whereas the other end leads to a completely enclosed pocket surrounded by conserved hydrophobic residues. These observations, along with mutagenesis data identifying residues that affect substrate binding and activity, suggest that two molecules of FPP bind at one end of the channel, where the active center of the first half-reaction is located, and then the stable reaction intermediate moves into the deep pocket, where it is sequestered from solvent and the second half-reaction occurs. Five alpha helices surrounding the active center are structurally homologous to the active core in the three other isoprenoid biosynthetic enzymes whose crystal structures are known, even though there is no detectable sequence homology. |
| Crystal structure of the human RORalpha Ligand binding domain in complex with cholesterol sulfate at 2.2 A Kallen, J., J. M. Schlaeppi, et al. (2004), J Biol Chem 279(14): 14033-8. Abstract: The retinoic acid-related orphan receptor alpha (RORalpha) is an orphan member of the subfamily 1 of nuclear hormone receptors. Our recent structural and functional studies have led to the hypothesis that cholesterol or a cholesterol derivative is the natural ligand of RORalpha. We have now solved the x-ray crystal structure of the ligand binding domain of RORalpha in complex with cholesterol-3-O-sulfate following a ligand exchange experiment. In contrast to the 3-hydroxyl of cholesterol, the 3-O-sulfate group makes additional direct hydrogen bonds with three residues of the RORalpha ligand binding domain, namely NH-Gln(289), NH-Tyr(290), and NH1-Arg(370). When compared with the complex with cholesterol, seven well ordered water molecules have been displaced, and the ligand is slightly shifted toward the hydrophilic part of the ligand binding pocket, which is ideally suited for interactions with a sulfate group. These additional ligand-protein interactions result in an increased affinity of cholesterol sulfate when compared with cholesterol, as shown by mass spectrometry analysis done under native conditions and differential scanning calorimetry. Moreover, mutational studies show that the higher binding affinity of cholesterol sulfate translates into an increased transcriptional activity of RORalpha. Our findings suggest that cholesterol sulfate could play a crucial role in the regulation of RORalpha in vivo. |
| Crystal structure of the Mus musculus cholesterol-regulated START protein 4 (StarD4) containing a StAR-related lipid transfer domain Romanowski, M. J., R. E. Soccio, et al. (2002), Proc Natl Acad Sci U S A 99(10): 6949-54. Abstract: The x-ray structure of the mouse cholesterol-regulated START protein 4 (StarD4) has been determined at 2.2-A resolution, revealing a compact alpha/beta structure related to the START domain present in the cytoplasmic C-terminal portion of human MLN64. The volume of the putative lipid-binding tunnel was estimated at 847 A(3), which is consistent with the binding of one cholesterol-size lipid molecule. Comparison of the tunnel-lining residues in StarD4 and MLN64-START permitted identification of possible lipid specificity determinants in both molecular tunnels. Homology modeling of related proteins, and comparison of the StarD4 and MLN64-START structures, showed that StarD4 is a member of a large START domain superfamily characterized by the helix-grip fold. Additional mechanistic and evolutionary studies should be facilitated by the availability of a second START domain structure from a distant relative of MLN64. |
| Crystalline cholesterol as a factor in thrombus formation Poliakov, A. E. (1995), Fiziol Zh 41(5-6): 97-102. Abstract: The model using crystalline cholesterol as the thrombogenic process inductor was developed. Thrombi that are morphologically equivalent to the human arterial thrombi under atherosclerosis were created in experiment on standard animals. It was shown that the "head" (conglutinational part) of such thrombi includes the thrombocyte mass, organised into the system of branched trabeculae surrounded by leucocytal limbus. The "caudal" (coagulational part) of the experimentally obtained thrombi consists of fibrin and erythrocytes. Data obtained make it possible to consider crystalline cholesterol of atheromatous plaques as the most probable trigger factor of thrombogenesis during atherosclerotic process. |
| Crystallization of free cholesterol in model macrophage foam cells Kellner-Weibel, G., P. G. Yancey, et al. (1999), Arterioscler Thromb Vasc Biol 19(8): 1891-8. Abstract: -The present study examined free cholesterol (FC) crystallization in macrophage foam cells. Model foam cells (J774 or mouse peritoneal macrophages MPMs) were incubated with acetylated low density lipoprotein and FC/phospholipid dispersions for 48 hours, resulting in the deposition of large stores of cytoplasmic cholesteryl esters (CEs). The model foam cells were then incubated for up to 5 days with an acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor (CP-113,818) in the absence of an extracellular FC acceptor to allow intracellular accumulation of FC. FC crystals of various shapes and sizes formed in the MPMs but not in the J774 macrophages. Examination of the MPM monolayers by microscopy indicated that the crystals were externalized rapidly after formation and thereafter continued to increase in size. Incubating J774 macrophages with 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (CPT-cAMP) in addition to CP-113,818 caused FC crystal formation as a consequence of CPT-cAMP stimulation of CE hydrolysis and inhibition of cell growth. In addition, 2 separate cholesterol phases (liquid-crystalline and cholesterol monohydrate) in the plane of the membrane bilayer were detected after 31 hours of ACAT inhibition by the use of small-angle x-ray diffraction of J774 macrophage foam cells treated with CPT-cAMP. Other compounds reported to inhibit ACAT, namely progesterone (20 microgram/mL) and N-acetyl-D-sphingosine (c(2)-ceramide, 10 microgram/mL), induced cellular toxicity in J774 macrophage foam cells and FC crystallization when coincubated with CPT-cAMP. Addition of the extracellular FC acceptors apolipoproteins (apo) E and A-I (50 microgram/mL) reduced FC crystal formation. In MPMs, lower cell density and frequent changes of medium were conducive to crystal formation. This may be due to "dilution" of apoE secreted by the MPMs and is consistent with our observation that the addition of exogenous apoE or apoA-I inhibits FC crystal formation in J774 macrophage foam cells cotreated with CP-113,818 plus CPT-cAMP. These data demonstrate that FC crystals can form from the hydrolysis of cytoplasmic stores of CEs in model foam cells. FC crystal formation can be modulated by the addition of extracellular FC acceptors or by affecting the cellular rate of CE hydrolysis. This process may contribute to the formation of FC crystals in atherosclerotic plaques. |
| Crystallization of the isobutylphosphocholine-cholesterol-isobutanol (1:3:3) complex and its investigation by X-ray analysis: interaction of phopholipid headgroups with cholesterol Karasev, V. A., V. S. Fundamensky, et al. (2000), Biochim Biophys Acta 1466(1-2): 23-38. Abstract: A crystal complex consisting of the isobutyl analog of phosphatidylcholine (PC) (isobutylphosphocholine), cholesterol, and isobutanol with molecular ratio 1:3:3 was obtained and investigated by means of X-ray analysis. The complex was shown to correspond to the monoclinic system (sp. gr. P2(1)): a=16.994(10), b=11.314(7), c=28.164(15), beta=104.07(3), V=5252.63 A(3), Z=2, D(calc)=1.0273 g/cm(3). The isobutylphosphocholine molecule is the key component of the complex. Pairs of hydrogen bonds are formed between the (-delta)O-P-O(delta-) group of the isobutylphosphocholine molecule and C-OH groups of two cholesterol and two isobutanol molecules. The third molecules of cholesterol and isobutanol are H-bonded with the (-delta)O-P-O(delta-) group of the isobutylphosphocholine molecule via C-OH groups of isobutanol and cholesterol, respectively. The crystal structure is built up by translation of the complex in multiplicate along the two-fold axis in the direction of axis b. It contains bands formed by isobutylphosphocholine molecules alternately changing their direction. They are fixed by virtue of two zones of electrostatic interactions of the type (-delta)O-P-O(delta-)ellipsis(+)N(CH(3))(3) and are more or less parallel to the bc plane. The structure also contains three-layer domains formed by cholesterol molecules perpendicular to isobutylphosphocholine bands. In the direction of the c-axis isobutylphosphocholine bands alternate with the layers of cholesterol molecules herewith reproducing repeated blocks. The obtained structure is compared with that of crystals of phospholipids and cholesterol and its derivatives. |
| CSF collection time at lumbar puncture is influenced by plasma cholesterol and triglycerides Nordin, C., T. Eklundh, et al. (2001), Neuropsychobiology 43(1): 19-22. Abstract: It is a fairly well-known fact that the CSF collection time (tapping time) at lumbar puncture may influence CSF levels of monoamine compounds (e.g. the serotonin metabolite 5-hydroxyindoleacetic acid, 5-HIAA) and some neuropeptides. Since serum levels of cholesterol and triglycerides and low CSF levels of 5-HIAA have been linked to violent behaviour and impulsivity, we investigated retrospectively whether serum cholesterol and triglycerides affect CSF collection time. The series consists of 14 healthy males lumbar punctured at the L(4-5) level. We found that both serum cholesterol and serum triglycerides influenced the CSF collection time for 12 ml of CSF (R = 0.77; p = 0.0067). There was no correlation between cholesterol in serum and CSF, nor between cholesterol in the CSF and collection time. However, we accidentally found a correlation between cholesterol in the CSF and age. The proposed hypothesis tries to explain why cholesterol- and triglyceride-rich lipoprotein particles modify the CSF collection time and influence endothelial function with a subsequent effect on CSF production and/or intraspinal pressure. Thus, it may be of interest to pay attention to serum cholesterol and triglycerides, their effect on CSF collection time and, in the next step, their putative impact on levels of various compounds in the CSF. |
| CSF taurine level is influenced by plasma cholesterol and the CYP2D6 phenotype Nordin, C., M. L. Dahl, et al. (2003), Eur Neuropsychopharmacol 13(5): 333-5. Abstract: Eight healthy male volunteers, lumbar-punctured before and during simvastatin treatment, were phenotyped for CYP2D6 analysis of the debrisoquine metabolic ratio (the ratio between the urinary recovery of debrisoquine and its 4-hydroxy metabolite) after a single oral dose of debrisoquine. The mean cerebrospinal fluid concentrations of cholesterol and taurine did not differ before and during treatment. During (but not before) treatment taurine in the CSF correlated with the debrisoquine metabolic ratio (r=-0.93; P=0.0007) Our results might indicate an influence of CYP2D6 on the level of taurine in the CSF that was secondary to the change in plasma cholesterol. |
| CT of papillary renal cell carcinomas with cholesterol necrosis mimicking angiomyolipomas Lesavre, A., J. M. Correas, et al. (2003), AJR Am J Roentgenol 181(1): 143-5. |
| C-terminal amino acid residues are required for the folding and cholesterol binding property of perfringolysin O, a pore-forming cytolysin Shimada, Y., M. Nakamura, et al. (1999), J Biol Chem 274(26): 18536-42. Abstract: Perfringolysin O (theta-toxin) is a pore-forming cytolysin whose activity is triggered by binding to cholesterol in the plasma membrane. The cholesterol binding activity is predominantly localized in the beta-sheet-rich C-terminal half. In order to determine the roles of the C-terminal amino acids in theta-toxin conformation and activity, mutants were constructed by truncation of the C terminus. While the mutant with a two-amino acid C-terminal truncation retains full activity and has similar structural features to native theta-toxin, truncation of three amino acids causes a 40% decrease in hemolytic activity due to the reduction in cholesterol binding activity with a slight change in its higher order structure. Furthermore, both mutants were found to be poor at in vitro refolding after denaturation in 6 M guanidine hydrochloride, resulting in a dramatic reduction in cholesterol binding and hemolytic activities. These activity losses were accompanied by a slight decrease in beta-sheet content. A mutant toxin with a five-amino acid truncation expressed in Escherichia coli is recovered as a further truncated form lacking the C-terminal 21 amino residues. The product retains neither cholesterol binding nor hemolytic activities and shows a highly disordered structure as detected by alterations in the circular dichroism and tryptophan fluorescence spectra. These results show that the C-terminal region of theta-toxin has two distinct roles; the last 21 amino acids are involved to maintain an ordered overall structure, and in addition, the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities. |
| Cu(2+)-induced lipid oxidation in plasma: questionable relation between cholesterol oxidation and LDL modification Tallineau, C., R. Pontcharraud, et al. (1992), Biochem Int 27(6): 983-90. Abstract: Oxidatively modified low-density lipoproteins (LDL) may be involved in the process of cholesterol deposition in arteries. Because of their cytotoxicity, oxysterols resulting from cholesterol oxidation could be a contributing factor in this process. Studies in this area have generally been performed on purified LDL, but in our research whole plasma was exposed to the oxidizing action of copper. Oxidation of the ring structure, which is at the origin of oxysterols, apparently occurs once most polyunsaturated fatty acids have disappeared. Hydrated LDL density reaches a value identical to that obtained during oxidation of LDL by endothelial cells, whereas the ring structure remains unmodified. |