Antimicrobial peptide mimics for improved therapeutic properties

The relatively recent recognition of the major role played by antimicrobial peptides (AMPs) in sustaining an effective host response to immune challenges was greatly influenced by studies of amphibian peptides. AMPs are also widely regarded as a potential source of future antibiotics owing to a remarkable set of advantageous properties ranging from molecular simplicity to low-resistance swift-kill of a broad range of microbial cells. However, the peptide formula per se, represents less than ideal drug candidates, namely because of poor bioavailability issues, potential immunogenicity, optional toxicity and high production costs. To address these issues, synthetic peptides have been designed, reproducing the critical peptide biophysical characteristic in unnatural sequence-specific oligomers. Thus, the use of peptidomimetics to overcome the limitations inherent to peptides physical characteristics is becoming an important and promising approach for improving the therapeutic potential of AMPs. Here, we review most recent advances in the design strategies and the biophysical properties of the main classes of mimics to natural AMPs, emphasizing the importance of structure-activity relationship studies in fine-tuning of their physicochemical attributes for improved antimicrobial properties.     

Nuclear transport factors in neuronal function

Active nucleocytoplasmic transport of macromolecules requires soluble transport carriers of the importin/karyopherin superfamily. Although the nuclear transport machinery is essential in all eukaryotic cells, neurons must also mobilise importins and associated proteins to overcome unique spatiotemporal challenges. These include switches in importin α subtype expression during neuronal differentiation, localized axonal synthesis of importin β1 to coordinate a retrograde injury signaling complex on axonal dynein, and trafficking of regulatory and signaling molecules from synaptic terminals to cell bodies. Targeting of RNAs encoding critical components of the importins complex and the Ran system to axons allows sophisticated local regulation of the system for mobilization upon need. Finally, a number of importin family members have been associated with mental or neurodegenerative diseases. The extended roles recently discovered for importins in the nervous system might also be relevant in non-neuronal cells, and the localized modes of importin regulation in neurons offer new avenues to interrogate their cytoplasmic functions.     

Identification of antimicrobial peptide regions derived from genomic sequences of phage lysins

This study was designed to test the possibility that antimicrobial peptides could be derived from the genomic sequences of phage lysins. Using two lysins (D3 and PhiKZ) we selected and produced two putative peptides (X and Z, respectively) believed to possess antimicrobial properties based on their physicochemical characteristics. The data presented support this hypothesis in that the peptides and various analogs displayed antibacterial activity, bacteriostatic or bactericidal, either individually or upon combination. These putative peptides are believed to act by a mechanism of action resembling that of conventional antimicrobial peptides when judged by both structural and functional criteria. Thus, the peptides are shown to have the ability to form a helical structure, to bind to model bacterial membranes and permeabilize model liposomes. They also display rapid bactericidal kinetics and their antibacterial potency is increased upon amidation. The possible relevance of these results in contributing to potency of phage lysins is discussed. Such peptides may be used to design new potent antimicrobial compounds much needed in face of the ever threatening drug resistance problems.     

Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA

Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines from damaged DNA. The Schiff base intermediate formed during this reaction between Escherichia coli Fpg and DNA was trapped by reduction with sodium borohydride, and the structure of the resulting covalently cross-linked complex was determined at a 2.1-A resolution. Fpg is a bilobal protein with a wide, positively charged DNA-binding groove. It possesses a conserved zinc finger and a helix-two turn-helix motif that participate in DNA binding. The absolutely conserved residues Lys-56, His-70, Asn-168, and Arg-258 form hydrogen bonds to the phosphodiester backbone of DNA, which is sharply kinked at the lesion site. Residues Met-73, Arg-109, and Phe-110 are inserted into the DNA helix, filling the void created by nucleotide eversion. A deep hydrophobic pocket in the active site is positioned to accommodate an everted base. Structural analysis of the Fpg-DNA complex reveals essential features of damage recognition and the catalytic mechanism of Fpg.     

Parallel topology of genetically fused EmrE homodimers

EmrE is a small H+-coupled multidrug transporter in Escherichia coli. Claims have been made for an antiparallel topology of this homodimeric protein. However, our own biochemical studies performed with detergent-solubilized purified protein support a parallel topology of the protomers. We developed an alternative approach to constrain the relative topology of the protomers within the dimer so that their activity can be assayed also in vivo before biochemical handling. Tandem EmrE was built with two identical monomers genetically fused tail to head (C-terminus of the first to N-terminus of the second monomer) with hydrophilic linkers of varying length. All the constructs conferred resistance to ethidium by actively removing it from the cytoplasm. The purified proteins bound substrate and transported methyl viologen into proteoliposomes by a proton-dependent mechanism. A tandem where one of the essential glutamates was replaced with glutamine transported only monovalent substrates and displayed a modified stoichiometry. The results support a parallel topology of the protomers in the functional dimer. The implications regarding insertion and evolution of membrane proteins are discussed.     

IL-8-induced migratory responses through CXCR1 and CXCR2: association with phosphorylation and cellular redistribution of focal adhesion kinase

CXCR1 and CXCR2 mediate migratory activities in response to IL-8 and other ELR+-CXC chemokines (e.g., GCP-2 and NAP-2). In vitro, activation of migration is induced by low IL-8 concentrations (10-50 ng/mL), whereas migratory shut-off is induced by high IL-8 concentrations (1000 ng/mL). The stimulation of CXCR1 and CXCR2 by IL-8 concentrations that result in migratory activation induced focal adhesion kinase (FAK) phosphorylation in a G(alpha)i-dependent manner. The expression of FRNK, a dominant negative mutant of FAK, perturbed migratory responses to the activating dose of 50 ng/mL IL-8. The migration-activating concentrations of 50 ng/mL GCP-2 and NAP-2 induced less potent migratory responses and FAK phosphorylation in CXCR2-expressing cells as compared with IL-8. These results indicate that FAK is phosphorylated, and required, for the chemotactic response under conditions of migratory activation by ELR+-CXC chemokines. In addition, FAK phosphorylation was determined following exposure to migration-attenuating concentrations of IL-8. In CXCR1-RBL cells this treatment resulted in FAK phosphorylation, in similar levels to those induced by activating concentrations of IL-8. In contrast, in CXCR2-RBL cells the migration-attenuating concentrations of IL-8 induced promoted levels of FAK phosphorylation and different patterns of FAK phosphorylation on its six potential tyrosine phosphorylation sites, as compared to activating concentrations of the chemokine. Exposure to IL-8 resulted not only in FAK phosphorylation but also in its cellular redistribution, indicated by the formation of defined contact regions with the substratum, enriched in phosphorylated FAK and vinculin. Overall, FAK phosphorylation was associated with, and found to be differently regulated upon, ELR+-CXC chemokine-induced migration.     

Effective diffusivity of galactose in calcium alginate gels containing immobilized Zymomonas mobilis.

The effective diffusivity of galactose was measured for calcium alginate gel membranes containing immobilized live Zymomonas mobilis cells at concentrations ranging from 0 to 150 g dry wt/L of gel. Since galactose is not taken up by living Z. mobilis organisms, the diffusion of this representative six-carbon sugar could be studied independently of sugar consumption. Various immobilized biomass loadings were achieved by two different techniques: addition of biomass at known concentrations to the sodium alginate solution before membrane formation and growth of cells in the gel to various biomass concentrations. The highest immobilized cell concentration, attained by in situ growth, corresponds to the maximum of this system, as growth beyond this maximum concentration led to disintegration of the gel membrane. The galactose effective diffusivity measurements for both methods of immobilized cell loading overlap within experimental error and follow the same general monotonic decline with entrapped biomass concentration. Most of the data fall below the upper bound predicted by Hashin and Shtrikman (1962) and show good agreement with the random pore model of Wakao and Smith (1962, 1964). Available effective diffusivity data from the literature provide evidence that the random pore model is an excellent predictor of sugar effective diffusivity in gel immobilized cell systems in general.     

Cytosolic phospholipase A2alpha is targeted to the p47phox-PX domain of the assembled NADPH oxidase via a novel binding site in its C2 domain

We have previously demonstrated a physical interaction between cytosolic phospholipase A2alpha (cPLA2) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, F?rster resonance energy transfer analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67phox or p47phox, that cPLA2 specifically binds to p47phox and that p47phox is sufficient to anchor cPLA2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA2 and p47phox demonstrated that the cPLA2-C2 domain and the p47phox-PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify hydrophobic peptides within these two domains that inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA2-C2 domain mutations were designed to explore its interaction p47phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA2-C2 domain caused a slight inhibition of the affinity binding to p47phox, whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47phox-PX domain caused a significant inhibition of the affinity binding to cPLA2. Thus, Ile67 of the cPLA2-C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47phox-PX domain.