Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide.

Ordered lipid domains enriched in sphingolipids and cholesterol (lipid rafts) have been implicated in numerous functions in biological membranes. We recently found that lipid domain/raft formation is dependent on the sterol component having a structure that allows tight packing with lipids having saturated acyl chains (Xu, X., and London, E. (2000) Biochemistry 39, 844-849). In this study, the domain-promoting activities of various natural sterols were compared with that of cholesterol using both fluorescence quenching and detergent insolubility methods. Using model membranes, it was shown that, like cholesterol, both plant and fungal sterols promote the formation of tightly packed, ordered lipid domains by lipids with saturated acyl chains. Surprisingly ergosterol, a fungal sterol, and 7-dehydrocholesterol, a sterol present in elevated levels in Smith-Lemli-Opitz syndrome, were both significantly more strongly domain-promoting than cholesterol. Domain formation was also affected by the structure of the sphingolipid (or that of an equivalent "saturated" phospholipid) component. Sterols had pronounced effects on domain formation by sphingomyelin and dipalmitoylphosphatidylcholine but only a weak influence on the ability of cerebrosides to form domains. Strikingly it was found that a small amount of ceramide (3 mol %) significantly stabilized domain/raft formation. The molecular basis for, and the implications of, the effects of different sterols and sphingolipids (especially ceramide) on the behavior and biological function of rafts are discussed.

Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis.

Tuberculosis treatment is shortened to six months by the indispensable addition of pyrazinamide (PZA) to the drug regimen that includes isoniazid and rifampin. PZA is a pro-drug of pyrazinoic acid (POA) (ref. 3), whose target of action has never been identified. Although PZA is active only against Mycobacterium tuberculosis, the PZA analog 5-chloro-pyrazinamide (5-Cl-PZA) displays a broader range of anti-mycobacterial activity. We have found that the eukaryotic-like fas1 gene (encoding fatty acid synthetase I, FASI) from M. avium, M. bovis BCG or M. tuberculosis confers resistance to 5-Cl-PZA when present on multi-copy vectors in M. smegmatis. 5-Cl-PZA and PZA markedly inhibited the activity of M. tuberculosis FASI, the biosynthesis of C16 to C24/C26 fatty acids from acetyl-CoA (ref. 6). Importantly, PZA inhibited FASI in M. tuberculosis in correlation with PZA susceptibility. These results indicate that FASI is a primary target of action for PZA in M. tuberculosis. Further characterization of FASI as a drug target for PZA may allow the development of new drugs to shorten the therapy against M. tuberculosis and may provide more options for treatment against M. bovis, M. avium and drug resistant M. tuberculosis.

Inactivation of the inhA-encoded fatty acid synthase II (FASII) enoyl-acyl carrier protein reductase induces accumulation of the FASI end products and cell lysis of Mycobacterium smegmatis.

The mechanism of action of isoniazid (INH), a first-line antituberculosis drug, is complex, as mutations in at least five different genes (katG, inhA, ahpC, kasA, and ndh) have been found to correlate with isoniazid resistance. Despite this complexity, a preponderance of evidence implicates inhA, which codes for an enoyl-acyl carrier protein reductase of the fatty acid synthase II (FASII), as the primary target of INH. However, INH treatment of Mycobacterium tuberculosis causes the accumulation of hexacosanoic acid (C(26:0)), a result unexpected for the blocking of an enoyl-reductase. To test whether inactivation of InhA is identical to INH treatment of mycobacteria, we isolated a temperature-sensitive mutation in the inhA gene of Mycobacterium smegmatis that rendered InhA inactive at 42 degrees C. Thermal inactivation of InhA in M. smegmatis resulted in the inhibition of mycolic acid biosynthesis, a decrease in hexadecanoic acid (C(16:0)) and a concomitant increase of tetracosanoic acid (C(24:0)) in a manner equivalent to that seen in INH-treated cells. Similarly, INH treatment of Mycobacterium bovis BCG caused an inhibition of mycolic acid biosynthesis, a decrease in C(16:0), and a concomitant accumulation of C(26:0). Moreover, the InhA-inactivated cells, like INH-treated cells, underwent a drastic morphological change, leading to cell lysis. These data show that InhA inactivation, alone, is sufficient to induce the accumulation of saturated fatty acids, cell wall alterations, and cell lysis and are consistent with InhA being a primary target of INH.

Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase, InhA, in complex with NAD+ and a C16 fatty acyl substrate.

Enoyl-ACP reductases participate in fatty acid biosynthesis by utilizing NADH to reduce the trans double bond between positions C2 and C3 of a fatty acyl chain linked to the acyl carrier protein. The enoyl-ACP reductase from Mycobacterium tuberculosis, known as InhA, is a member of an unusual FAS-II system that prefers longer chain fatty acyl substrates for the purpose of synthesizing mycolic acids, a major component of mycobacterial cell walls. The crystal structure of InhA in complex with NAD+ and a C16 fatty acyl substrate, trans-2-hexadecenoyl-(N-acetylcysteamine)-thioester, reveals that the substrate binds in a general "U-shaped" conformation, with the trans double bond positioned directly adjacent to the nicotinamide ring of NAD+. The side chain of Tyr158 directly interacts with the thioester carbonyl oxygen of the C16 fatty acyl substrate and therefore could help stabilize the enolate intermediate, proposed to form during substrate catalysis. Hydrophobic residues, primarily from the substrate binding loop (residues 196-219), engulf the fatty acyl chain portion of the substrate. The substrate binding loop of InhA is longer than that of other enoyl-ACP reductases and creates a deeper substrate binding crevice, consistent with the ability of InhA to recognize longer chain fatty acyl substrates.

Sterol side chain length and structure affect the clearance of chylomicron-like lipid emulsions in rats and mice.

In previous work we found that sterols such as cholesterol were essential for physiological plasma clearance of lipid emulsions mimicking the structure of mammalian triglyceride-rich lipoproteins. In the present study we compared the clearances of emulsions prepared with sterols of varying alkyl chain length (straight chains, n-C3 to n-C7, or branched chains, i-C5 to i-C10) at the C-17 position. Our studies show that the length of the alkyl chain at the C-17 position of sterols markedly affects the removal of remnant particles from the plasma of rats traced by emulsion cholesteryl oleate label. An alkyl chain of 7 carbons or more was needed for normal remnant clearance. Straight and branched chains of similar length were cleared similarly, showing that the presence of a branch at the end of the alkyl chain had no effect on remnant clearance. For side chains of 7 carbons or less, substitution of sterols with an unsaturation in the alkyl chain close to the terminal carbon markedly decreased the clearance of remnants. Triolein label was used to estimate lipolysis of the injected emulsions. Lipolysis was little affected by the structure of the sterol side chain, except that lipolysis was markedly higher with emulsions containing sterols with an alkyl chain having 4 carbon atoms (n-C4) or with an unsaturation in the 4 carbon alkyl chain. We conclude that the length of the alkyl side chain is an important element in the essentiality of cholesterol as a regulator of metabolism of lipid emulsion models of triglyceride-rich lipoproteins.

The effect of side-chain analogues of cholesterol on the thermotropic phase behavior of 1-stearoyl-2-oleoylphosphatidylcholine bilayers: a differential scanning calorimetric study.

In this study we have examined the effects of analogues of cholesterol differing with respect to alkyl side-chain length and structure on the thermotropic phase behavior of bilayers formed from 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), an important subclass of naturally occurring phosphatidylcholines (PCs). The synthetic sterols we studied contained either a terminally unbranched (n-series) or a single methyl-branched (iso-series) side chain of 3 to 10 carbon atoms. The phase transition behavior was examined by high-sensitivity differential scanning calorimetry (DSC). The main phase transition endotherm of SOPC/sterol bilayers consists of superimposed sharp and broad components, which represent the hydrocarbon chain melting of sterol-poor and sterol-rich phospholipid domains, respectively. The transition temperature and the cooperativity of the sharp component are moderately reduced upon sterol incorporation and the enthalpy decreases to zero when sterol levels of 20-30 mol% are reached. The enthalpy of the broad component transition initially increases to a maximum around 25 or 25-30 mol% sterol and thereafter decreases with further increases in sterol concentration. However, the broad transition of SOPC bilayers containing both short (C-22, i-C5 and n-C3) and long (i-C9 and i-C10) side-chain sterols still persists at levels of 50 mol% sterol. Thus the effective stoichiometry of SOPC-sterol interactions varies with changes in sterol alkyl side-chain length. The incorporation of short linear or branched side-chain sterols (C-22, n-C3, n-C4, i-C5) causes the broad component transition temperature and cooperativity to decrease dramatically, whereas the incorporation of medium- and long-chain sterols in both the n- and iso-series has less effect on the transition temperature and cooperativity of the broad component. Overall, no significant differences were found between the n- and iso-series sterols for a given side-chain length. A comparison of the phase behavior of dipalmitoylphosphatidylcholine (DPPC)/sterol (McMullen et al. (1995) Biophys. J. 69, 169-176) and SOPC/sterol mixtures indicates that the primary factor responsible for changes in the thermotropic phase behavior of these systems is the extent of the hydrophobic mismatch between the sterol and the host lipid bilayer. However, sterol miscibility in PC bilayers, and thus the stoichiometry of lipid-sterol interactions, also appears to depend on the degree of unsaturation of the host lipid bilayer.

Lateral domain formation in cholesterol/phospholipid monolayers as affected by the sterol side chain conformation.

The interaction of side-chain variable cholesterol analogues with dipalmitoylphosphatidylcholine (DPPC) or N-palmitoylsphingomyelin (N-PSPM) has been examined in monolayer membranes at the air/water interface. The sterols had either unbranched (n-series) or single methyl-branched (iso-series) side chains, with the length varying between 3 and 10 carbons (C3-C10). The efficacy of interaction between the sterols and the phospholipids was evaluated based on the ability of the sterols to form condensed sterol/phospholipid domains in the phospholipid monolayers. Domain formation was detected with monolayer fluorescence microscopy using NBD-cholesterol as the fluorescent probe. In general, a side chain length of at least 5 carbons was necessary for the unbranched sterols to form visible sterol/phospholipid domains in DPPC or N-PSPM mixed monolayers. With the iso-analogues, a side chain of at least 6 carbons was needed for sterol/phospholipid domains to form. The macroscopic domains were stable up to a certain surface pressure (ranging from 1 to 12 mN/m). At this onset phase transformation pressure, the domain line boundary dissipated, and the monolayer entered into an apparent one phase state (no clearly visible lateral domains). However, with some DPPC monolayers containing short chain sterols (n-C3, n-C4,n-C5, and i-C5), a new condensed phase appeared to form (at 20 mol%) when the monolayer was compressed beyond the phase transformation pressure. These precipitates formed at surface pressures between 6-8.3 mN/m, were clearly observable up to at least 30 mN/m. When the monolayers containing these four sterols were allowed to expand, the condensed precipitates dissolved at the same pressure at which they were formed during monolayer compression. No condensed precipitates were observed with these sterols in corresponding N-PSPM monolayers. Taken together, the results of this study emphasize the importance of the length and conformation of the cholesterol side chain in determining the efficacy of sterol/phospholipid interaction in model membranes. The major difference between DPPC and N-PSPM monolayers at different sterol compositions was mainly the lateral distribution and the size of the domains as well as the onset phase transformation pressure intervals.

Enzymatic characterization of the target for isoniazid in Mycobacterium tuberculosis.

The inhA gene has been recently shown to encode a common protein target for isoniazid and ethionamide action in Mycobacterium tuberculosis. In this paper, we demonstrate that the M. tuberculosis InhA protein catalyzes the NADH-specific reduction of 2-trans-enoyl-ACP, essential for fatty acid elongation. This enzyme preferentially reduces long-chain substrates (12-24 carbons), consistent with its involvement in mycolic acid biosynthesis. Steady-state kinetic studies showed that the two substrates bind to InhA via a sequential kinetic mechanism, with the preferred ordered addition of NADH and the enoyl substrate. The chemical mechanism involves stereospecific hydride transfer of the 4S hydrogen of NADH to the C3 position of the 2-trans-enoyl substrate, followed by protonation at C2 of an enzyme-stabilized enolate intermediate. Kinetic and microcalorimetric analysis demonstrates that the binding of NADH to the S94A mutant InhA, known to confer resistance to both isoniazid and ethionamide, is altered. This difference can account for the isoniazid-resistance phenotype, with the formation of a binary InhA-NADH complex required for drug binding. Isoniazid binding to either the wild-type or S94A mutant InhA could not be detected by titration microcalorimetry, suggesting that this compound is a prodrug, which must be converted to its active form.

Differential scanning calorimetric study of the effect of sterol side chain length and structure on dipalmitoylphosphatidylcholine thermotropic phase behavior.

We have investigated the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers containing a series of cholesterol analogues varying in the length and structure of their alkyl side chains. We find that upon the incorporation of up to approximately 25 mol % of any of the side chain analogues, the DPPC main transition endotherm consists of superimposed sharp and broad components representing the hydrocarbon chain melting of sterol-poor and sterol-rich phospholipid domains, respectively. Moreover, the behavior of these components is dependent on sterol side chain length. Specifically, for all sterol/DPPC mixtures, the sharp component enthalpy decreases linearly to zero by 25 mol % sterol while the cooperativity is only moderately reduced from that observed in the pure phospholipid. In addition, the sharp component transition temperature decreases for all sterol/DPPC mixtures; however, the magnitude of the decrease is dependent on the sterol side chain length. With respect to the broad component, the enthalpy initially increases to a maximum around 25 mol % sterol, thereafter decreasing toward zero by 50 mol % sterol with the exception of the sterols with very short alkyl side chains. Both the transition temperature and cooperativity of the broad component clearly exhibit alkyl chain length-dependent effects, with both the transition temperature and cooperativity decreasing more dramatically for sterols with progressively shorter side chains. We ascribe the chain length-dependent effects on transition temperature and cooperativity to the hydrophobic mismatch between the sterol and the host DPPC bilayer (see McMullen, T. P. W., Lewis, R. N. A. H., and McElhaney, R. N. (1993) Biochemistry 32:516-522). Moreover, the effective stoichiometry of sterol/DPPC interactions is altered by a significantly large degree of hydrophobic mismatch between the sterol and the DPPC bilayer. Thus the short chain sterols appear to exhibit considerable immiscibility in gel state DPPC bilayers, effectively limiting their interaction with adjacent phospholipid molecules.

An efficient asymmetric synthesis of diacylglycerols.

A convenient preparation of 1,2-diacyl-sn-glycerol or 2,3-diacyl-sn-glycerol is described starting from allyl bromide. The latter was converted to allyl 4-methoxyphenyl ether, which is dihydroxylated using AD-mix as a catalyst to yield 3-O-(4'-methoxyphenyl)-sn-glycerol or 1-O-(4'-methoxyphenyl)-sn-glycerol in high yield and high optical purity. After diacylation, ceric ammonium nitrate was used to remove the 4-methoxyphenyl group under mild conditions that avoid acyl migration to 1,3-dipalmitoylglycerol. Thus chiral 1,2-diacylglycerol can be prepared from allyl bromide in just four steps in 78% overall yield and high enantiomeric excess. This scheme represents an inexpensive method for the large-scale preparation of chiral 1,2-diacyl-sn-glycerol and 2,3-diacyl-sn-glycerol.

Effect of sterol side-chain structure on sterol-phosphatidylcholine interactions in monolayers and small unilamellar vesicles.

In this study we have characterized the monolayer behavior of analogues of cholesterol having different side-chain structures and their interaction with phosphatidylcholines in mixed monolayers and small unilamellar vesicles (SUVs). Two series of side-chain analogues of cholesterol were synthesized, one with an unbranched side chain (the n-series, from 3 to 7 carbons in length), and the other with a single methyl-branched side chain (the iso-series, from 5 to 10 carbons in length). The length and conformation of the sterol side chain markedly influenced both the mean molecular area of the pure sterols and their monolayer stability (i.e., collapse pressure). Shorter side chains gave smaller mean molecular areas and decreased monolayer stability. The sterols from the n-series also had smaller mean molecular areas than the corresponding sterols in the iso-series. In mixed 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/sterol monolayers (equimolar ratio; at 22 degrees C), all of the sterols tested decreased the monolayer stability as judged by the lower collapse pressure with sterol than without sterol. A similar trend was observed in mixed monolayers containing 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), except that sterols from the iso-series with a chain length of 8 or 10 carbon atoms actually stabilized the monolayer compared with the sterol-free SOPC monolayer. The ability of the sterols to condense the molecular packing of DPPC was similar with all sterols (3-5% condensation at 10 mN/m), irrespective of the length or structure of the side chain. 5-Androsten-3 beta-ol, however, which lacks the side chain, did not at all condense the monolayer packing of DPPC. With SOPC mixed monolayers, all side chain containing sterols caused a 18-20% condensation (at 10 mN/m) of monolayer packing. The condensing effect of 5-androsten-3 beta-ol on SOPC packing was again much smaller (about 10%) compared with that of the side-chain sterols. The rate of sterol oxidation by cholesterol oxidase (at 37 degrees C) in DPPC-containing SUVs increased as a function of increasing the side-chain length (iso-series). With sterols from the n-series, the same trend was seen, except that the n-C7 analogue was oxidized much slower than the n-C4, n-C5, and n-C6 analogues. With SOPC SUVs, a similar side-chain dependent oxidation pattern was observed. Our results support and extend previous knowledge about the importance of the sterol side chain in determining sterol-sterol and sterol-phospholipid interactions, both in mono- and bilayers.