Acylation stabilizes a protease-resistant conformation of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides.
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Protein acylation is an important way in which a number of proteins with a variety of functions are modified. The physiological role of the acylation of cellular proteins is still poorly understood. Covalent binding of fatty acids to nonintegral membrane proteins is thought to produce transient or permanent enhancement of the association of the polypeptide chains with biological membranes. In this paper, we investigate the functional role for the palmitoylation of an atypical membrane-bound protein, yeast protoporphyrinogen oxidase, which is the molecular target of diphenyl ether-type herbicides. Palmitoylation stabilizes an active heat- and protease-resistant conformation of the protein. Palmitoylation of protoporphyrinogen oxidase has been demonstrated to occur in vivo both in yeast cells and in a heterologous bacterial expression system, where it may be inhibited by cerulenin leading to the accumulation of degradation products of the protein. The thiol ester linking palmitoleic acid to the polypeptide chain was shown to be sensitive to hydrolysis by hydroxylamine and also by the widely used serine-protease inhibitor phenylmethylsulfonyl fluoride. (+info)
Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids.
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The ability of the Src family kinases Fyn and Lck to participate in signaling through the T cell receptor is critically dependent on their dual fatty acylation with myristate and palmitate. Here we identify a palmitate analog, 2-bromopalmitate, that effectively blocks Fyn fatty acylation in general and palmitoylation in particular. Treatment of COS-1 cells with 2-bromopalmitate blocked myristoylation and palmitoylation of Fyn and inhibited membrane binding and localization of Fyn to detergent-resistant membranes (DRMs). In Jurkat T cells, 2-bromopalmitate blocked localization of the endogenous palmitoylated proteins Fyn, Lck, and LAT to DRMs. This resulted in impaired signaling through the T cell receptor as evidenced by reductions in tyrosine phosphorylation, calcium release, and activation of mitogen-activated protein kinase. We also examined the ability of long chain polyunsaturated fatty acids (PUFAs) to inhibit protein fatty acylation. PUFAs have been reported to inhibit T cell signaling by excluding Src family kinases from DRMs. Here we show that the PUFAs arachidonic acid and eicosapentaenoic acid inhibit Fyn palmitoylation and consequently block Fyn localization to DRMs. We propose that inhibition of protein palmitoylation represents a novel mechanism by which PUFAs exert their immunosuppressive effects. (+info)
Mycobacterium tuberculosis H37Rv parietal and cellular lipoarabinomannans. Characterization of the acyl- and glyco-forms.
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Mannosylated lipoarabinomannans are multifaceted molecules. They have been shown to exert an immunosuppressive role in the immunopathogenesis of tuberculosis. They are also described as antigens of host double negative alphabeta T-cells. Delimitation of ManLAMs epitopes require knowledge of the precise structure of these molecules. The two major functional domains (the cap motifs and the phosphatidylinositol anchor) of the parietal and cellular ManLAMs of Mycobacterium tuberculosis H37Rv were investigated here. Using capillary electrophoresis, we established that parietal and cellular ManLAMs share the same capping motifs, mono-, di-, and trimannosyl units with the same relative abundance. By (31)P NMR analysis of the native LAMs in Me(2)SO-d(6), the major acyl-form of both parietal and cellular H37Rv ManLAM anchors, typified by the P3 phosphorus resonance, comprised a diacylglycerol unit. Three other acyl-forms were characterized in the cellular ManLAMs. Comparative analysis of the cellular Mycobacterium bovis BCG and M. tuberculosis ManLAM acyl-forms revealed the presence of the same populations, but with different relative abundance. The biological importance of the H37Rv ManLAM acyl-form characterization is discussed, particularly concerning the molecular mechanisms of binding of ManLAMs to the CD1 proteins involved in the presentation of ManLAMs to T-cell receptors. (+info)
A differential scanning calorimetry study of phosphocholines mixed with paclitaxel and its bromoacylated taxanes.
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High sensitivity differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of disaturated phosphocholines (PCs) and alpha-bromoacyl taxane derivatives. The alpha-bromoacyl taxanes were synthesized as hydrolyzable hydrophobic prodrugs of paclitaxel. The PCs used were 1, 2-dimyristoyl-sn-glycero-3-phosphatidyl-choline (DMPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The bromoacyl chain lengths of the taxane prodrugs were varied from 6 to 12 or 16 carbons. For comparison, paclitaxel and PC mixtures were also examined. DSC data from DPPC and bromoacyl taxane mixtures showed a complete abolition of the pretransition and significant broadening of the main phase transition with increasing amounts of bromoacyl taxane prodrugs. The effects were more pronounced with the long-chain compared to the short-chain prodrugs. Under equivalent DSC conditions, the short-chain DMPC showed greater changes in thermotropic phase behavior than with DPPC on taxane addition, suggesting an enhanced degree of association with the fluid-type bilayers. Under similar conditions, the long-chain DSPC bilayers showed a far less significant change in phase behavior on taxane addition than DPPC. These changes were also chain length-dependent for both the PCs and the taxane prodrugs. In contrast, PC and paclitaxel (lacking the acyl chain) mixtures under similar conditions showed insignificant changes in the endotherms, suggesting only slight insertion of the molecule into the PC bilayers. From the DSC data it is apparent that taxane prodrugs solvated in DMPC bilayers more than in DPPC and DSPC bilayers, and taxane prodrugs with longer acyl chains were able to associate with PCs better than those with shorter chain prodrugs. DSC data also suggest that paclitaxel was poorly associated with any of the PCs. In general, the amount of taxane association with bilayers decreased in order: DMPC > DPPC >> DSPC. In contrast, the transition enthalpy (DeltaH) of DMPC, DPPC, and DSPC mixtures with paclitaxel showed significantly lower enthalpies than with taxane prodrugs. Taken together, the DSC data suggest that the acyl chains of paclitaxel prodrugs have some access into the bilayers via alignment with the acyl chain of the PC component. (+info)
Lipid-dependent targeting of G proteins into rafts.
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Domains rich in sphingolipids and cholesterol, or rafts, may organize signal transduction complexes at the plasma membrane. Raft lipids are believed to exist in a state similar to the liquid-ordered phase. It has been proposed that proteins with a high affinity for an ordered lipid environment will preferentially partition into rafts (Melkonian, K. A., Ostermeyer, A. G., Chen, J. Z., Roth, M. G., and Brown, D. A. (1999) J. Biol. Chem. 274, 3910-3917). We investigated the possibility that lipid-lipid interactions between lipid-modified proteins and raft lipids mediate targeting of proteins to these domains. G protein monomers or trimers were reconstituted in liposomes, engineered to mimic raft domains. Assay for partitioning of G proteins into rafts was based on Triton X-100 insolubility. Myristoylation and palmitoylation of Galpha(i) were necessary and sufficient for association with liposomes and partitioning into rafts. Strikingly, the amount of fatty-acylated Galpha(i) in rafts was significantly reduced when myristoylated Galpha(i) was thioacylated with cis-unsaturated fatty acids instead of saturated fatty acids such as palmitate. Prenylated betagamma subunits were excluded from rafts, whether reconstituted alone or with fatty-acylated alpha subunits. These results suggest that the structural difference between lipids that modify proteins is one basis for the selectivity of protein targeting to rafts. (+info)
Huprine X is a novel high-affinity inhibitor of acetylcholinesterase that is of interest for treatment of Alzheimer's disease.
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Inhibitors of the enzyme acetylcholinesterase (AChE) slow and sometimes reverse the cognitive decline experienced by individuals with Alzheimer's disease. Huperzine A, a natural product used in traditional Chinese herbal medicine, and tacrine (Cognex) are among the potent AChE inhibitors used in this treatment, but the search for more selective inhibitors continues. We report herein the synthesis and characterization of (-)-12-amino-3-chloro-9-ethyl-6,7, 10,11-tetrahydro-7,11-methanocycloocta[b]quinoline hydrochloride (huprine X), a hybrid that combines the carbobicyclic substructure of huperzine A with the 4-aminoquinoline substructure of tacrine. Huprine X inhibited human AChE with an inhibition constant K(I) of 26 pM, indicating that it binds to this enzyme with one of the highest affinities yet reported. Under equivalent assay conditions, this affinity was 180 times that of huperzine A, 1200 times that of tacrine, and 40 times that of E2020 (donepezil, Aricept), the most selective AChE inhibitor currently approved for therapeutic use. The association and dissociation rate constants for huprine X with AChE were determined, and the location of its binding site on the enzyme was probed in competition studies with the peripheral site inhibitor propidium and the acylation site inhibitor edrophonium. Huprine X showed no detectable affinity for the edrophonium-AChE complex. In contrast, huprine X did form a ternary complex with propidium and AChE, although its affinity for the free enzyme was found to be 17 times its affinity for the propidium-AChE complex. These data indicated that huprine X binds to the enzyme acylation site in the active site gorge but interferes slightly with the binding of peripheral site ligands. (+info)
Modeling the kinetics of acylation of insulin using a recursive method for solving the systems of coupled differential equations.
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This paper describes a theoretical method for solving systems of coupled differential equations that describe the kinetics of complicated reaction networks in which a molecule having multiple reaction sites reacts irreversibly with multiple equivalents of a ligand (reagent). The members of the network differ in the number of equivalents of reagent that have reacted, and in the patterns of sites of reaction. A recursive algorithm generates series, asymptotic, and average solutions describing this kinetic scheme. This method was validated by successfully simulating the experimental data for the kinetics of acylation of insulin. (+info)
Characterization of chimeric lipopolysaccharides from Escherichia coli strain JM109 transformed with lipooligosaccharide synthesis genes (lsg) from Haemophilus influenzae.
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Previously, we reported the expression of chimeric lipopolysaccharides (LPS) in Escherichia coli strain JM109 (a K-12 strain) transformed with plasmids containing Haemophilus influenzae lipooligosaccharide synthesis genes (lsg) (Abu Kwaik, Y., McLaughlin, R. E., Apicella, M. A., and Spinola, S. M. (1991) Mol. Microbiol. 5, 2475-2480). In this current study, we have analyzed the O-deacylated LPS and free oligosaccharides from three transformants (designated pGEMLOS-4, pGEMLOS-5, and pGEMLOS-7) by matrix-assisted laser desorption ionization, electrospray ionization, and tandem mass spectrometry techniques, along with composition and linkage analyses. These data show that the chimeric LPS consist of the complete E. coli LPS core structure glycosylated on the 7-position of the non-reducing terminal branch heptose with oligosaccharides from H. influenzae. In pGEMLOS-7, the disaccharide Gal1--> 3GlcNAc1--> is added, and in pGEMLOS-5, the structure is extended to Gal1-->4GlcNAc1-->3Gal1-->3GlcNAc1-->. PGEMLOS-5 LPS reacts positively with monoclonal antibody 3F11, an antibody that recognizes the terminal disaccharide of lacto-N-neotetraose. In pGEMLOS-4 LPS, the 3F11 epitope is apparently blocked by glycosylation on the 6-position of the terminal Gal with either Gal or GlcNAc. The biosynthesis of these chimeric LPS was found to be dependent on a functional wecA (formerly rfe) gene in E. coli. By using this carbohydrate expression system, we have been able to examine the functions of the lsg genes independent of the effects of other endogenous Haemophilus genes and expressed proteins. (+info)