Role Of Acylation Research Articles

Hemagglutinin Palmitoylation Contributes to Membrane Curvature in Influenza a Virus Assembly and Membrane Fusion

Three cysteine residues in the cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) are covalently modified by three fatty acids and highly conserved among HA subtypes. The importance of these S-acylation post-translational modifications is highlighted by a strain-dependence to their role in virus replication either in assembly or in fusion, but the mechanisms by which the modifications exerts any effect are unknown. We studied the effects of HA acylation on influenza virus-like particle (VLP) morphology, glycoprotein spacing, protein incorporation, HA induced curvature, and membrane fusion using cryo-electron tomography (cET), VLP-cell and cell-cell fusion assays, and molecular dynamics. Acylation has a significant effect on VLP envelope curvature but is not a determinant of either VLP morphology or HA lateral spacing. De-acylated mutant HA is correlated with a flatter envelope curvature of the released particles in the absence of the M1 layer compared to wild type HA. The de-acylated mutant HA failed to incorporate an M1 layer within spherical VLP consistent with altered HA-M1 interactions. In cell-cell fusion assays, fusion pore enlargement was not observed, regardless of which strain of influenza was de-acylated (H2 (A/Japan/305/57), H3 (A/Aichi/2/68), H3 (A/Udorn/72)), suggesting that the role of acylation in membrane fusion is viral strain independent. Fusion without pore enlargement could be partially rescued by the expression of M1 and M2 proteins. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations, and was found to be comparable to curvature values derived from VLP size distributions. Our studies indicate that HA acylation is important for both influenza virus assembly and membrane fusion by controlling membrane curvature and modifying HA's interactions with M1.

Palmitoylation of Pulmonary Surfactant Protein SP-C Is Critical for Its Functional Cooperation with SP-B to Sustain Compression/Expansion Dynamics in Cholesterol-Containing Surfactant Films

Recent data suggest that a functional cooperation between surfactant proteins SP-B and SP-C may be required to sustain a proper compression-expansion dynamics in the presence of physiological proportions of cholesterol. SP-C is a dually palmitoylated polypeptide of 4.2 kDa, but the role of acylation in SP-C activity is not completely understood. In this work we have compared the behavior of native palmitoylated SP-C and recombinant nonpalmitoylated versions of SP-C produced in bacteria to get a detailed insight into the importance of the palmitic chains to optimize interfacial performance of cholesterol-containing surfactant films. We found that palmitoylation of SP-C is not essential for the protein to promote rapid interfacial adsorption of phospholipids to equilibrium surface tensions (∼22 mN/m), in the presence or absence of cholesterol. However, palmitoylation of SP-C is critical for cholesterol-containing films to reach surface tensions ≤1 mN/m at the highest compression rates assessed in a captive bubble surfactometer, in the presence of SP-B. Interestingly, the ability of SP-C to facilitate reinsertion of phospholipids during expansion was not impaired to the same extent in the absence of palmitoylation, suggesting the existence of palmitoylation-dependent and -independent functions of the protein. We conclude that palmitoylation is key for the functional cooperation of SP-C with SP-B that enables cholesterol-containing surfactant films to reach very low tensions under compression, which could be particularly important in the design of clinical surfactants destined to replacement therapies in pathologies such as acute respiratory distress syndrome.

Mutation of the Active Site Carboxy-Lysine (K70) of OXA-1 β-Lactamase Results in a Deacylation-Deficient Enzyme

Class D beta-lactamases hydrolyze beta-lactam antibiotics by using an active site serine nucleophile to form a covalent acyl-enzyme intermediate and subsequently employ water to deacylate the beta-lactam and release product. Class D beta-lactamases are carboxylated on the epsilon-amino group of an active site lysine, with the resulting carbamate functional group serving as a general base. We discovered that substitutions of the active site serine and lysine in OXA-1 beta-lactamase, a monomeric class D enzyme, significantly disrupt catalytic turnover. Substitution of glycine for the nucleophilic serine (S67G) results in an enzyme that can still bind substrate but is unable to form a covalent acyl-enzyme intermediate. Substitution of the carboxylated lysine (K70), on the other hand, results in enzyme that can be acylated by substrate but is impaired with respect to deacylation. We employed the fluorescent penicillin BOCILLIN FL to show that three different substitutions for K70 (alanine, aspartate, and glutamate) lead to the accumulation of significant acyl-enzyme intermediate. Interestingly, BOCILLIN FL deacylation rates (t(1/2)) vary depending on the identity of the substituting residue, from approximately 60 min for K70A to undetectable deacylation for K70D. Tryptophan fluorescence spectroscopy was used to confirm that these results are applicable to natural (i.e., nonfluorescent) substrates. Deacylation by K70A, but not K70D or K70E, can be partially restored by the addition of short-chain carboxylic acid mimetics of the lysine carbamate. In conclusion, we establish the functional role of the carboxylated lysine in OXA-1 and highlight its specific role in acylation and deacylation.

S-Myristoylation of a Glycosylphosphatidylinositol-specific Phospholipase C inTrypanosoma brucei

Covalent modification with lipid can target cytosolic proteins to biological membranes. With intrinsic membrane proteins, the role of acylation can be elusive. Herein, we describe covalent lipid modification of an integral membrane glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from the kinetoplastid Trypanosoma brucei. Myristic acid was detected on cysteine residue(s) (i.e. thiomyristoylation). Thiomyristoylation occurred both co- and post-translationally. Acylated GPI-PLC was active against variant surface glycoprotein (VSG). The half-life of fatty acid on GPI-PLC was 45 min, signifying the dynamic nature of the modification. Deacylation in vitro decreased activity of GPI-PLC 18-30-fold. Thioacylation, from kinetic analysis, activated GPI-PLC by accelerating the conversion of a GPI-PLC.VSG complex to product. Reversible thioacylation is a novel mechanism for regulating the activity of a phospholipase C.

Fatty acid composition of myelin proteolipid protein during vertebrate evolution.

The hydrophobic myelin proteolipid protein (PLP) contains covalently bound long-chain fatty acids which are attached to intracellular cysteine residues via thioester linkages. To gain insight into the role of acylation in the structure and function of myelin PLP, the amount and pattern of acyl groups attached to the protein during vertebrate evolution was determined. PLP isolated from brain myelin of amphibians, reptiles, birds and several mammals was subjected to alkaline methanolysis and the released methyl esters were analyzed by gas-liquid chromatography. In all species studied, PLP contained approximately the same amount of covalently bound fatty acids (3% w/w), and palmitic, palmitoleic, oleic and stearic acids were always the major acyl groups. Although the relative proportions of these fatty acids changed during evolution, the changes did not necessarily follow the variations in the acyl chain composition of the myelin free fatty acid pool, suggesting fatty acid specificity. The phylogenetic conservation of acylation suggests that this post-translational modification is critical for PLP function.

The roles of residues Tyr150, Glu272, and His314 in class C beta-lactamases.

Serine beta-lactamases contribute widely to the beta-lactam resistance phenomena. Unfortunately, the intimate details of their catalytic mechanism remain elusive and subject to some controversy even though many "natural" and "artificial" mutants of these different enzymes have been isolated. This paper is essentially focused on class C beta-lactamases, which contain a Tyr (Tyr150) as the first residue of the second conserved element, in contrast to their class A counterparts, in which a Ser is found in the corresponding position. We have modified this Tyr residue by site-directed mutagenesis. On the basis of the three-dimensional structure of the Enterobacter cloacae P99 enzyme, it seemed that residues Glu272 and His314 might also be important. They were similarly substituted. The modified enzymes were isolated and their catalytic properties determined. Our results indicated that His314 was not required for catalysis and that Glu272 did not play an important role in acylation but was involved to a small extent in the deacylation process. Conversely, Tyr150 was confirmed to be central for catalysis, at least with the best substrates. On the basis of a comparison of data obtained for several class C enzyme mutants and in agreement with recent structural data, we propose that the phenolate anion of Tyr150, in conjunction with the alkyl ammonium of Lys315, acts as the general base responsible for the activation of the active-site Ser64 during the acylation step and for the subsequent activation of a water molecule in the deacylation process. The evolution of the important superfamily of penicillin-recognizing enzymes is further discussed in the light of this proposed mechanism.

The roles of residues Tyr150, Glu272, and His314 in class C β‐lactamases

Serine beta-lactamases contribute widely to the beta-lactam resistance phenomena. Unfortunately, the intimate details of their catalytic mechanism remain elusive and subject to some controversy even though many "natural" and "artificial" mutants of these different enzymes have been isolated. This paper is essentially focused on class C beta-lactamases, which contain a Tyr (Tyr150) as the first residue of the second conserved element, in contrast to their class A counterparts, in which a Ser is found in the corresponding position. We have modified this Tyr residue by site-directed mutagenesis. On the basis of the three-dimensional structure of the Enterobacter cloacae P99 enzyme, it seemed that residues Glu272 and His314 might also be important. They were similarly substituted. The modified enzymes were isolated and their catalytic properties determined. Our results indicated that His314 was not required for catalysis and that Glu272 did not play an important role in acylation but was involved to a small extent in the deacylation process. Conversely, Tyr150 was confirmed to be central for catalysis, at least with the best substrates. On the basis of a comparison of data obtained for several class C enzyme mutants and in agreement with recent structural data, we propose that the phenolate anion of Tyr150, in conjunction with the alkyl ammonium of Lys315, acts as the general base responsible for the activation of the active-site Ser64 during the acylation step and for the subsequent activation of a water molecule in the deacylation process. The evolution of the important superfamily of penicillin-recognizing enzymes is further discussed in the light of this proposed mechanism.

The role of acylation in exopolysaccharides including those for food use

Abstract Microbial exopolysaccharides with potential for food usage, frequently carry acyl groups which may profoundly affect their interactive properties. Thus xanthan with or without acylation shows marked differences in synergis‐tic gelling with plant gluco‐ and galacto‐mannans, although the poly‐saccharides with different acylation patterns show similar viscosity. Similarly gellan from Auromonas elodea is of greater potential value after deacetylation, when it provides a valuable gelling agent, than it is as a viscosifier in the natural acylated form. The Enterobacter XM6 polymer only forms gels when it, too, has been chemically deacetylated. Both these polysaccharides form gels due to the enhanced interaction with cations following deacylation. Recent work in our laboratory suggests that deacetylation of certain bacterial alginates also significantly increases ion binding.