Anti-inflammatory and anti-bacterial properties of tetramethylhexadecenyl succinyl cysteine (TSC): a skin-protecting cosmetic functional ingredient.

BACKGROUND: The skin is the first line of defence against exposure to microbial, physical, environmental and chemical insults. In mobilizing a protective response, several different cell types located in our skin release and respond to pro-inflammatory cytokines ensuring skin homeostasis and health. However, chronic activation of this response eventually causes damage resulting in premature ageing. Diosodium tetramethylhexadecenyl succinyl cysteine (TSC or SIG1273), an isoprenylcysteine small molecule, down modulates these inflammatory signalling pathways in various cell types (keratinocytes, peripheral blood mononuclear cells (PBMCs) and endothelial cells) and possesses anti-bacterial properties. Thus, TSC represents a novel cosmetic functional ingredient that provides a broad spectrum of benefits for the skin. OBJECTIVE: To assess the anti-inflammatory properties of TSC in several cutaneous cell types and further investigate its anti-microbial activity. METHODS: Cultured normal human epidermal keratinocytes were exposed to chemical irritant phorbol 12-myrisate 13-acetate (TPA) or ultraviolet-B light (UVB) to induce pro-inflammatory cytokine (IL-6, IL-8 and TNF-α) production. T-cell receptor (TCR) activation of PBMCs and nickel (Ni(2+) ) treatments of human dermal microvascular endothelial cells (HDMECs) were performed resulting in IL-4, IL-6, IL-8 and IL-17 production. Streptococcus pyogenes were cultured to determine minimal inhibitory concentration values. RESULTS: In vitro studies demonstrate TSC blocks TPA and UVB-induced cytokine production in cultured keratinocytes. Similarly, TSC inhibits overproduction of IL-4 and IL-17 in T-cell receptor (TCR)-activated PBMCs as well as nickel induction of IL-6 and IL-8 in HDMECs. Lastly, TSC demonstrated anti-microbial properties, inhibiting cell growth of S. pyogenes. CONCLUSIONS: Tetramethylhexadecenyl succinyl cysteine represents a novel cosmetic functional ingredient that provides a dual modulating benefit of skin protection to individuals by reducing inflammation in keratinocytes, endothelial and mononuclear cell types and S. pyogenes counts.

Carboxyl methylation of Ras-related proteins during signal transduction in neutrophils.

In human neutrophils, as in other cell types, Ras-related guanosine triphosphate-binding proteins are directed toward their regulatory targets in membranes by a series of posttranslational modifications that include methyl esterification of a carboxyl-terminal prenylcysteine residue. In intact cells and in a reconstituted in vitro system, the amount of carboxyl methylation of Ras-related proteins increased in response to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP). Activation of Ras-related proteins by guanosine-5'-O-(3-thiotriphosphate) had a similar effect and induced translocation of p22rac2 from cytosol to plasma membrane. Inhibitors of prenylcysteine carboxyl methylation effectively blocked neutrophil responses to FMLP. These findings suggest a direct link between receptor-mediated signal transduction and the carboxyl methylation of Ras-related proteins.

Prenylcysteine analogs mimicking the C-terminus of GTP-binding proteins stimulate exocytosis from permeabilized HIT-T15 cells: comparison with the effect of Rab3AL peptide.

Most guanine nucleotide binding proteins (G-proteins) possess an S-prenylated C-terminal cysteine whose carboxyl group can be reversibly methylated. The prenylcysteine analog N-acetyl-S-geranylgeranyl-cysteine (AGGC) (50 microM), a competitive inhibitor of prenylcysteine methyl transferases, introduced into streptolysin-O permeabilized HIT-T15 cells doubled the rate of basal (0.1 microM Ca2+) and of stimulated (10 microM Ca2+ or 100 microM GTP gamma S) insulin secretion in a reversible and ATP-dependent manner. N-acetyl-S-farnesylcysteine (AFC) was less potent while N-acetyl-S-geranyl-cysteine was inactive. Prenylcysteine action on exocytosis did not involve inhibition of G-protein methylation, since (1) the methyl ester derivative of AFC, an inefficient inhibitor of methyltransferases in HIT-T15 cell fractions, was as potent as AGGC in stimulating exocytosis; (2) S-adenosyl-homocysteine, a general inhibitor of methylation reactions, did not alter basal or GTP gamma S-triggered secretion while inhibiting Ca(2+)-induced insulin release. The binding of G-proteins to Rab/GDP-dissociation inhibitor, Rab3A/GTPase activating protein or rabphilin-3A was not affected by the prenylcysteine analogs. AGGC or AFC had the same effect on insulin release as a synthetic peptide mimicking the amino acid residues 52-67 of the G-protein Rab3A (Rab3AL). Moreover, the action on secretion of the combination of Rab3AL and prenylcysteines was not additive. We propose that the prenylcysteines and the Rab3AL peptide influence exocytosis by affecting the association of Rab3A with different proteins of the exocytotic machinery of insulin-secreting cells.

The histidine protein kinase superfamily.

Signal transduction in microorganisms and plants is often mediated by His-Asp phosphorelay systems. Two conserved families of proteins are centrally involved: histidine protein kinases and phospho-aspartyl response regulators. The kinases generally function in association with sensory elements that regulate their activities in response to environmental signals. A sequence analysis with 348 histidine kinase domains reveals that this family consists of distinct subgroups. A comparative sequence analysis with 298 available receiver domain sequences of cognate response regulators demonstrates a significant correlation between kinase and regulator subfamilies. These findings suggest that different subclasses of His-Asp phosphorelay systems have evolved independently of one another.

Do G protein subunits associate via a three-stranded coiled coil?

We used a computer-based prediction algorithm to identify probable coiled-coil segments at the N-termini of G protein alpha, beta and gamma subunits. This result indicates that G protein trimers may form via a three-stranded coiled coil. Previous biochemical results had shown that the N-termini of alpha and beta are involved in subunit interactions. Here we present a structural model for the N-terminal domain of beta gamma and a hypothesis for the reversible association of alpha to beta gamma.

Kinetics of CheY phosphorylation by small molecule phosphodonors.

The chemotaxis response regulator CheY can acquire phosphoryl groups either from its associated autophosphorylating protein kinase, CheA, or from small phosphodonor molecules such as acetyl phosphate. We report a stopped-flow kinetic analysis of CheY phosphorylation by acetyl phosphate. The results show that CheY has a very low affinity for this phosphodonor (K(s)&z.Gt;0.1 M), consistent with the conclusion that, whereas CheY provides catalytic functions for the phosphotransfer reaction, the CheA kinase may act simply to increase the effective phosphodonor concentration at the CheY active site.

Prenylcysteine analogs to study function of carboxylmethylation in signal transduction.

Carboxylmethylation of ras-related proteins is stimulated immediately on exposure of myeloid cells to inflammatory agonists. When the methylation reaction is inhibited with prenylcysteine analogs, G-protein-mediated signal transduction responses are disrupted, but responses to phorbol ester, calcium ionophore, and phospholipase C (PLC) remain intact. Furthermore, prenylcysteine analogs block GTP gamma S-induced aggregation of permeabilized platelets. Together, these results suggest that protein prenylcysteine methylation can play a role in signal transduction. A number of studies with AdoMet antagonists have suggested a role for methylation in cell-cycle regulation and stimulus-response coupling. Because the compounds generally inhibit all cellular methylation events, however, their effects have been difficult to interpret. On the other hand, prenylcysteine analogs have proved to be specific inhibitors of protein prenylcysteine methylation, as opposed to other types of methylation reactions. This enables the segregation of the role of methylation at C-terminal prenylcysteine residues from methylation at other sites, such as the carboxyl terminus of the catalytic subunit of PP2A. It should be emphasized, however, that prenylcysteine tails of proteins may interact with other target sites in addition to the methyltransferase enzyme(s), and prenylcysteine analogs may compete for these sites as well. One cannot assume that the inhibition of a response by the drugs necessarily implicates the involvement of a prenylcysteine methylation reaction. Studies with the analogs must be interpreted in conjunction with other results to ascertain the locus of their effects.

pH sensing in bacterial chemotaxis.

Bacteria are able to sense a broad range of chemical and energetic stimuli and modulate their swimming behaviour to migrate to more favourable environments. Signal transduction in bacterial chemotaxis is mediated by a two-component system composed of a protein histidine kinase, CheA, and a response regulator, CheY. The phosphorylated response regulator, P approximately CheY, binds to a protein at the flagellar motor, FliM, to cause reversals in flagellar motor rotation. The level of P approximately CheY is controlled by the activity of the kinase CheA, which is in turn regulated by membrane receptors at the cell surface. Membrane receptors such as the aspartate receptor, Tar, are composed of two distinct regions: an extracellular sensing domain that binds stimulatory ligands, aspartate in the case of Tar; and an intracellular signalling domain that forms a complex with the protein kinase CheA. What is the mechanism of transmembrane signalling? How does aspartate binding to the sensing domain at the outside surface of the membrane translate into a change in kinase activity at the membrane cytosol interface? Recent results suggest that the mechanism depends on perturbations in lateral packing within an extensive array of receptors localized to patches at the cell poles. Receptor patching appears to depend on higher-order associations with the kinase CheA as well as an adaptor protein, CheW. It is difficult to assess the locus of pH effects within the context of even a simple signal transduction system like that involved in bacterial chemotaxis. Previous results with mutant strains have indicated that the serine receptor, Tsr, is critical for pH sensing, but in vitro results do not support such a straightforward interpretation of the genetic data.

Resistance determinants for beta-lactam antibiotics in laboratory mutants of Streptococcus pneumoniae that are involved in genetic competence.

Laboratory mutants of Streptococcus pneumoniae resistant to either cefotaxime or piperacillin reveal defects in competence development independent of the selective beta-lactam. A resistance determinant ciaH encoding a putative histidine kinase of a two-component signal-transducing system that is also involved in competence regulation was recently identified in cefotaxime-resistant mutants. We show now that the CiaH protein can be phosphorylated by ATP in vitro, and that it also phosphorylates the cognate response regulator CiaR. The mutant C306 containing the CiaH mutation Thr-230-Pro is completely noncompetent. It does not release competence-inducing activity (competence factor) into the medium nor can such an activity be released from the cells. Competence in C306 cannot be induced upon addition of external competence factor, in contrast to the competence-defective piperacillin-resistant mutants P506 and P408. A novel resistance determinant cpoA specific for piperacillin was identified in piperacillin-resistant mutants. CpoA is responsible for the competence defect in P506 but not in P408. The results document a tight link between the action of beta-lactams and competence development in the pneumococcus and confirm that the two beta-lactams piperacillin and cefotaxime act via different primary targets.

Changing reactivity of receptor carboxyl groups during bacterial sensing.

A microdistillation procedure has been developed to analyze carboxylmethylation of the Mr = 60,000 chemoreceptor proteins involved in chemotaxis of Salmonella typhimurium and Escherichia coli. Methylation levels obtained by this method are substantially higher than those reported in the literature. In highly motile strains under optimal conditions there are approximately 100,000 methylated receptor residues per cell which are entirely composed of gamma-methylglutamyl esters. Whereas with previously used methods only groups which turn over could be detected, the microdistillation assay provides absolute values. Under steady state conditions, approximately one-half the total number of methyl ester residues are continuously hydrolyzed and resynthesized, while the remainder are sequestered. A mechanism has been devised to explain the observed patterns of methyl ester synthesis and hydrolysis. According to this hypothesis, substrate glutamyl residues on the receptor are located in a restricted region near the active sites of transferase and esterase which are bound to the receptor protein. Small, stimuli-induced changes in receptor conformation effect perturbations in receptor methylation by shifting the focus of activity of the modifying enzymes from one pair of closely spaced groups to another.

Purification and characterization of the CheZ protein of bacterial chemotaxis.

The cheZ gene is the most distal of five genes that comprise the Meche operon of the Salmonella typhimurium chemotaxis system. We have determined the sequence of the cheZ gene along with an 800-nucleotide flanking region at its 3' end. The flanking sequence contains an open reading frame that probably corresponds to the 5' end of flaM. The cheZ coding sequence predicts an extremely acidic, hydrophilic protein with a molecular weight of 23,900. We have purified and characterized this protein. N-terminal analysis of pure CheZ yields an amino acid sequence identical to that predicted by the nucleotide sequence except that the amino-terminal methionine residue is modified by N methylation. The purified CheZ protein exhibits a native molecular weight of 115,000, but in cell extracts the majority of CheZ exists as a much larger aggregate (Mr greater than 500,000). Under these conditions, CheZ appears to be a homopolymer composed of at least 20 monomeric subunits.

Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation.

In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphonenolpyruvate: sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose permease. When strains were constructed which lacked the galactose permease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are unable to carry out facilitated diffusion.

A protein methylesterase involved in bacterial sensing.

A protein methylesterase has been identified in soluble extracts of Salmonella typhimurium and Escherichia coli. This enzyme catalyzes the hydrolysis of gamma-glutamyl methyl ester residues from membrane-bound 60,000-molecular weight proteins that are essential for chemotaxis. Analyses of methylesterase activity in a variety of chemotactically defective strains suggest that the methylesterase is a product of the cheX gene in Salmonella and the cheB gene in E. coli. In addition, the cheT gene product in S. typhimurium seems to play a role in expression of methylesterase activity. Mutant strains lacking the protein methylesterase tumble incessantly in the absence of attractant gradients. This behavior is the converse of that shown by mutant strains defective in methyltransferase activity, which swim smoothly in the absence of repellent gradients. This finding indicates that reversible methylation acts as a control mechanism and that both a methyltransferase and a protein methylesterase are instrumental in bacterial sensing.

Response regulation in bacterial chemotaxis.

The signal transduction system that mediates bacterial chemotaxis allows cells to modulate their swimming behavior in response to fluctuations in chemical stimuli. Receptors at the cell surface receive information from the surroundings. Signals are then passed from the receptors to cytoplasmic chemotaxis components: CheA, CheW, CheZ, CheR, and CheB. These proteins function to regulate the level of phosphorylation of a response regulator designated CheY that interacts with the flagellar motor switch complex to control swimming behavior. The structure of CheY has been determined. Magnesium ion is essential for activity. The active site contains highly conserved Asp residues that are required for divalent metal ion binding and CheY phosphorylation. Another residue at the active site, Lys109, is important in the phosphorylation-induced conformational change that facilitates communication with the switch complex and another chemotaxis component, CheZ. CheZ facilitates the dephosphorylation of phospho-CheY. Defects in CheY and CheZ can be suppressed by mutations in the flagellar switch complex. CheZ is thought to modulate the switch bias by varying the level of phospho-CheY.