Molecular mechanism of action of the fibrates

Fibrates are old hypolipidemic drugs with pleitropic effects on lipid metabolism. Until, recently their intimate molecular mechanisms of action were mysterious. In the late 5 years, we have shown that the pharmacological effects of fibrates depend on their binding to "Peroxisome Proliferator Activated Receptor alpha" (PPAR alpha). The binding of fibrates to PPAR alpha induces the activation or the inhibition of multiple genes involved in lipid metabolism through the binding of the activated PPAR alpha to "Peroxisome Proliferator Response Element" (PPRE) located in the gene promoters. Fibrates reduce plasma triglyceride levels by altering the expression of numerous genes coding for proteins involved in fatty acid metabolism (fatty acid transport protein, acyl-CoA synthetase, etc.) and also by increasing the lipoprotein lipase synthesis and decreasing the apolipoprotein C-III synthesis. Fibrates increase HDL cholesterol levels by increasing apolipoprotein A-I and apolipoprotein A-II synthesis. Furthermore, we recently demonstrated that fibrates are potent anti-inflammatory molecules through an indirect modulation of the nuclear-factor-kappa B activity. Therefore, we suggest that fibrates inhibit atherosclerosis development not only by improving the plasma lipid profile but also by reducing inflammation in the vascular wall.     

香兰素抗菌机制研究进展

食品安全问题在中国范围内频发,严重威胁民众的健康和安全。为防止此类问题发生,对于由微生物污染食品所产生的危害,可通过添加抗菌剂有效降低安全隐患,随着食品安全法的不断完善,抗菌剂的添加量也愈加严格和规范。香兰素是一种具有抗菌性的传统食品添加剂,但是目前缺乏对其抗菌机制的全面理解,因此,限制了香兰素在抗菌特性方面的广泛应用。有鉴于此,现对香兰素的结构、功能与抗菌活性的相关性及其抗菌机制研究进展予以综述,并对具有抗菌特性的香兰素衍生物进行探讨。     

Structure and mechanism of action of the antimicrobial peptide piscidin

Piscidin, an antibacterial peptide isolated from the mast cells of striped bass, has potent antimicrobial activity against a broad spectrum of pathogens in vitro. We investigated the mechanism of action of this 22-residue cationic peptide by carrying out structural studies and electrophysiological experiments in lipid bilayers. Circular dichroism experiments showed that piscidin was unstructured in water but had a high alpha-helix content in dodecylphosphocholine (DPC) micelles. 1H NMR data in water and TFE confirmed these results and demonstrated that the segment of residues 8-17 adopted an alpha-helical structure in a micellar environment. This molecule has a marked amphipathic character, due to well-defined hydrophobic and hydrophilic sectors. This structure is similar to those determined for other cationic peptides involved in permeabilization of the bacterial membrane. Multichannel experiments with piscidin incorporated into azolectin planar bilayers gave reproducible I-V curves at various peptide concentrations and unambiguously showed that this peptide permeabilized the membrane. This pore forming activity was confirmed by single-channel experiments, with well-defined ion channels obtained at different voltages. The characteristics of the ion channels (voltage dependence, only one or two states of conductance) clearly suggest that piscidin is more likely to permeabilize the membrane by toroidal pore formation rather than via the "barrel-stave" mechanism.     

Molecular insight into mechanism of antimicrobial action of the beta-hairpin peptide arenicin: specific oligomerization in detergent micelles

Arenicins are 21-residue cationic antimicrobial peptides isolated from marine polychaeta Arenicola marina. The peptides exhibit potent broad-spectrum antimicrobial activity. In water solution arenicin-2 adopts a beta-hairpin conformation, stabilized by one disulfide and nine hydrogen bonds. To determine the propensity for the peptide oligomerization in membrane mimetic systems, the recombinant arenicin-2 was overexpressed as a fused form in Escherichia coli. The arenicin-2 oligomerization and intermolecular packing in membrane mimicking environment were investigated using high-resolution NMR spectroscopy. The present studies show that arenicin-2 preserves a beta-hairpin structure and forms asymmetric dimers upon incorporation into the dodecylphosphocholine micelle. Two monomers of arenicin-2 are aligned parallel to each other by the N-terminal strands of the beta-hairpin (CN upward arrow upward arrowNC type of association). Polyacrylamide gel electrophoresis analysis indicated that in environment of anionic SDS micelles the arenicin-2 might undergo further oligomerization and form tetramers. Our results afford further molecular insight into possible mechanism of antimicrobial action of arenicins.     

Molecular mechanism of the juvenile hormone action.

Structures, physiological role and level regulation of the juvenile hormones are described. A scheme of juvenile hormone mode of action at the molecular level, which includes transport of hormone via its binding protein, is presented.     

Molecular mechanism of antimicrobial peptides: the origin of cooperativity

Based on very extensive studies on four peptides (alamethicin, melittin, magainin and protegrin), we propose a mechanism to explain the cooperativity exhibited by the activities of antimicrobial peptides, namely, a non-linear concentration dependence characterized by a threshold and a rapid rise to saturation as the concentration exceeds the threshold. We first review the structural basis of the mechanism. Experiments showed that peptide binding to lipid bilayers creates two distinct states depending on the bound-peptide to lipid ratio P/L. For P/L below a threshold P/L*, all of the peptide molecules are in the S state that has the following characteristics: (1) there are no pores in the membrane, (2) the axes of helical peptides are oriented parallel to the plane of membrane, and (3) the peptide causes membrane thinning in proportion to P/L. As P/L increases above P/L*, essentially all of the excessive peptide molecules occupy the I state that has the following characteristics: (1) transmembrane pores are detected in the membrane, (2) the axes of helical peptides are perpendicular to the plane of membrane, (3) the membrane thickness remains constant for P/L> or =P/L*. The free energy based on these two states agrees with the data quantitatively. The free energy also explains why lipids of positive curvature (lysoPC) facilitate and lipids of negative curvature (PE) inhibit pore formation.     

Molecular mechanism of action of the vasoconstrictor peptide endothelin

Endothelin, one of the most potent vasoconstrictor known, has been suggested to act as an endogenous agonist of L-type Ca2+ channels. In this paper we show that endothelin stimulates the metabolism of inositol phosphates and induces the mobilization of intracellular Ca2+ stores. The transient activation of Ca2+-sensitive K+ channel provokes an hyperpolarization of the membrane. It is followed by a sustained depolarization which is due to the opening of a non-specific cation channel which is permeable to Ca2+ and Mg2+. The depolarization then activates L-type Ca2+ channels. This mechanism of action explains why part of the endothelin-induced vasocontriction is eliminated by L-type Ca2+ channel blockers.     

Molecular mechanism of Endosulfan action in mammals

Endosulfan is a broad-spectrum organochlorine pesticide, speculated to be detrimental to human health in areas of active exposure. However, the molecular insights to its mechanism of action remain poorly understood. In two recent studies, our group investigated the physiological and molecular aspects of endosulfan action using in vitro, ex vivo and in vivo analyses. The results showed that apart from reducing fertility levels in male animals, Endosulfan induced DNA damage that triggers compromised DNA damage response leading to undesirable processing of broken DNA ends. In this review, pesticide use especially of Endosulfan in the Indian scenario is summarized and the importance of our findings, especially the rationalized use of pesticides in the future, is emphasized.     

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.     

Elucidating the bactericidal mechanism of action of the linear antimicrobial tetrapeptide BRBR-NH2

Linear antimicrobial peptides, with their rapid bactericidal mode of action, are well-suited for development as topical antibacterial drugs. We recently designed a synthetic linear 4-residue peptide, BRBR-NH₂, with potent bactericidal activity against Staphylococcus aureus (MIC 6.25 μM), the main causative pathogen of human skin infections with an unknown mechanism of action. Herein, we describe a series of experiments conducted to gain further insights into its mechanism of action involving electron microscopy, artificial membrane dye leakage, solution- and solid-state NMR spectroscopy followed by molecular dynamics simulations. Experimental results point towards a SMART (Soft Membranes Adapt and Respond, also Transiently) mechanism of action, suggesting that the peptide can be developed as a topical antibacterial agent for treating drug-resistant Staphylococcus aureus infections.     

Molecular mechanism of action of the radioprotective substance WR 2721

The radioprotective effect of WR 2721 on catalase and the type and loci of its interaction with the enzyme have been investigated by means of spectrophotometric and electron spin resonance, (ESR) methods. The radiation damage, indicated by a change in enzymatic activity and in the Soret absorption band, has been the less the larger the WR 2721 concentration. In the case of ESR investigations, addition of WR 2721 has resulted in a reduction of the spin concentration of Cu-2+. Since cysteamine has exhibited similar results, however, to a lesser extent, it can be assumed that the RS-ions are responsible for the protective effect. From the results obtained it can be concluded that (the dephosphorilized) WR 2721 forms a complex with the enzyme and acts as an electron donor.     

Molecular mechanism for the antigonococcal action of lysosomal cathepsin G

Human lysosomal cathepsin G (cat G) appears to be an important mediator of non-oxidative killing of Neisseria gonorrhoeae ingested by human polymorphonuclear leucocytes (PMNLs). Nearly isogenic strains of gonococci having variations in the structure of penicillin-binding protein 2 (PBP2) also exhibit different levels of susceptibility to the lethal action of cat G in vitro. Accordingly, we examined the relationship between gonococcal susceptibility to cat G and PBP2 structure. The results of this study suggest that cat G has the capacity to interact with PBP2, as evidenced by its ability to inhibit binding of [3H]-benzylpenicillin to PBP2. We also found that changes in the amino acid sequence within the transpeptidase domain of PBP2, because of certain penA mutations, modulated such interactions. We propose that PBP2 is an intracellular target for cat G and that levels of gonococcal susceptibility to cat G may be related to PBP2 structure and/or intracellular availability.     

Molecular mechanism of antimicrobial peptides: The origin of cooperativity

Based on very extensive studies on four peptides (alamethicin, melittin, magainin and protegrin), we propose a mechanism to explain the cooperativity exhibited by the activities of antimicrobial peptides, namely, a non-linear concentration dependence characterized by a threshold and a rapid rise to saturation as the concentration exceeds the threshold. We first review the structural basis of the mechanism. Experiments showed that peptide binding to lipid bilayers creates two distinct states depending on the bound-peptide to lipid ratio P/L. For P/L below a threshold P/L*, all of the peptide molecules are in the S state that has the following characteristics: (1) there are no pores in the membrane, (2) the axes of helical peptides are oriented parallel to the plane of membrane, and (3) the peptide causes membrane thinning in proportion to P/L. As P/L increases above P/L*, essentially all of the excessive peptide molecules occupy the I state that has the following characteristics: (1) transmembrane pores are detected in the membrane, (2) the axes of helical peptides are perpendicular to the plane of membrane, (3) the membrane thickness remains constant for P/L ≥ P/L*. The free energy based on these two states agrees with the data quantitatively. The free energy also explains why lipids of positive curvature (lysoPC) facilitate and lipids of negative curvature (PE) inhibit pore formation.     

Free-radical mechanism of antimicrobial action of xanthine oxidase and lactoperoxidase]

The interaction between milk xanthine oxidase (XO) and lactoperoxidase (LP) in model system and antimicrobial action of these enzymes on Escherichia coli 0-111 were studied. It was shown, that bacterial superoxide dismutase (SOD), which transforms O2-. (XO-reaction product) into H2O2 (substrate of LP), is necessary for binding of the reaction sequence: XO-->LP-->antimicrobial products. It is suggested, that these enzymes unite in the protective system in intestinal infections of newborns. Bacterial SOD in this case acts as the key factor, creating the system.     

Fluorescence anisotropy analysis of the mechanism of action of mesenterocin 52A: speculations on antimicrobial mechanism

Mesenterocin 52A (Mes 52A) is a class IIa bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52, active against Listeria sp. The interaction of Mes 52A with bacterial membranes of two sensitive Listeria strains has been investigated. The Microbial Adhesion to Solvents test used to study the physico-chemical properties of the surface of the two strains indicated that both surfaces were rather hydrophilic and bipolar. The degree of insertion of Mes 52A in phospholipid bilayer was studied by fluorescence anisotropy measurements using two probes, 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and DPH, located at different positions in the membrane. TMA-DPH reflects the fluidity at the membrane surface and DPH of the heart. With Listeria ivanovii CIP 12510, Mes 52A induced an increase only in the TMA-DPH fluorescence anisotropy, indicating that this bacteriocin affects the membrane surface without penetration into the hydrophobic core of the membrane. No significant K⁺ efflux was measured, whereas the ΔΨ component of the membrane potential was greatly affected. With Listeria innocua CIP 12511, Mes 52A caused an increase in the fluorescence of TMA-DPH and DPH, indicating that this peptide inserts deeply in the cytoplasmic membrane of this sensitive strain. This insertion led to K⁺ efflux, without perturbation of ΔpH and a weak modification of ΔΨ, and is consistent with pore formation. These data indicate that Mes 52A interacts at different positions of the membrane, with or without pore formation, suggesting two different mechanisms of action for Mes 52A depending on the target strain.