The adipokine leptin mediates muscle- and liver-derived IGF-1 in aged mice.

Muscle- and liver-derived IGF-1 play important roles in muscle anabolism throughout growth and aging. Yet, prolonged food restriction is thought to increase longevity in part by lowering levels of IGF-1, which in turn reduces the risk for developing various cancers. The dietary factors that modulate IGF-1 levels are, however, poorly understood. We tested the hypothesis that the adipokine leptin, which is elevated with food intake and suppressed during fasting, is a key mediator of IGF-1 levels with aging and food restriction. First, leptin levels in peripheral tissues were measured in young mice fed ad libitum, aged mice fed ad libitum, and aged calorie-restricted (CR) mice. A group of aged CR mice were also treated with recombinant leptin for 10 days. Later, aged mice fed ad libitum were treated with saline (VEH) or with a novel leptin receptor antagonist peptide (Allo-aca) and tissue-specific levels of IGF-1 were determined. On one hand, recombinant leptin induced a three-fold increase in liver-derived IGF-1 and a two-fold increase in muscle-derived IGF-1 in aged, CR mice. Leptin also significantly increased serum growth hormone levels in the aged, CR mice. On the other, the leptin receptor antagonist Allo-aca did not alter body weight or muscle mass in treated mice compared to VEH mice. Allo-aca did, however, produce a significant (20%) decline in liver-derived IGF-1 as well as an even more pronounced (>50%) decrease in muscle-derived IGF-1 compared to VEH-treated mice. The reduced IGF-1 levels in Allo-aca treated mice were not accompanied by any significant change in growth hormone levels compared to VEH mice. These findings suggest that leptin receptor antagonists may represent novel therapeutic agents for attenuating IGF-1 signaling associated with aging, and could potentially mimic some of the positive effects of calorie restriction on longevity.

Leptin and ghrelin prevent hippocampal dysfunction induced by Aß oligomers.

It was recently established that the stomach-derived ghrelin and the adipokine leptin promote learning and memory through actions within the hippocampus. Changes in the peripheral or brain levels of these peptides were described in Alzheimer's disease (AD) patients and were shown to correlate with the severity of cognitive decline. Furthermore, in vivo and in vitro studies demonstrated that leptin or ghrelin can ameliorate amyloid and tau pathologies as well as cognitive deficits. However, the exact role of these peptides in AD is far from being elucidated. To fill this gap, our working hypothesis was that leptin and ghrelin can exert a neuroprotective role in AD suppressing hippocampal dysfunction triggered by synapto- and neurotoxic amyloid-ß oligomers (AßO). Using primary cultured hippocampal neurons, we demonstrated that both peptides reduce AßO-induced production of superoxide and mitochondrial membrane depolarization, improving cell survival, and inhibit cell death through a receptor-dependent mechanism. Furthermore, it was shown that in AßO-treated neurons both leptin and ghrelin prevent glycogen synthase kinase 3ß activation. Therefore, the evidence gathered in this study revealed that leptin and ghrelin can act as neuroprotective agents able to rescue hippocampal neurons from AßO toxicity, thus highlighting their potential therapeutic role in AD.

Leptin-based glycopeptide induces weight loss and simultaneously restores fertility in animal models.

AIM: To design, manufacture and test a second generation leptin receptor (ObR) agonist glycopeptide derivative. The major drawback to current experimental therapies involving leptin protein is the appearance of treatment resistance. Our novel peptidomimetic was tested for efficacy and lack of resistance induction in rodent models of obesity and appetite reduction. METHODS: The glycopeptide containing two additional non-proteinogenic amino acids was synthesized by standard solid-phase methods. Normal mice were fed with peanuts until their blood laboratory data and liver histology showed typical signs of obesity but not diabetes. The mice were treated with the peptidomimetic at 0.02, 0.1 or 0.5 mg/kg/day intraperitoneally side-by-side with 0.1 mg/kg/day leptin for 11 days. After termination of the assay, the blood cholesterol and glucose amounts were measured, the liver fat content was visualized and quantified and the remaining mice returned to normal diet and were allowed to mate. In parallel experiments normal rats were treated intranasally with the glycopeptide at 0.1 mg/kg/day for 10 days. RESULTS: The 12-residue glycosylated leptin-based peptidomimetic E1/6-amino-hexanoic acid (Aca) was designed to target a principal leptin/ObR-binding interface. E1/Aca induced leptin effects in ObR-positive cell lines at picomolar concentrations and readily crossed the blood-brain barrier (BBB) following intraperitoneal administration. The peptide initiated typical leptin-dependent signal transduction pathways both in the presence and absence of leptin protein. The peptide also reduced weight gain in mice fed with high-fat peanut diet in a dose-dependent manner. Obese mice receiving peptide E1/Aca at a 0.5 mg/kg/day dose lost weight, corresponding to a net 6.5% total body weight loss, while similar mice treated with leptin protein did not. Upon cessation of the weight loss treatment, several obesity-related pathologies (i.e. abnormal metabolic profile and liver histology as well as infertility) normalized in peptide-, but not leptin-treated, mice. Peptide E1/Aca added intranasally to growing normal rats decelerated normal weight gain corresponding to a net 6.8% net total body weight loss with statistical significance. CONCLUSIONS: No resistance induction to peptide E1/Aca or toxicity in either obese or healthy rodents was observed, indicating the potential for widespread utility of the peptidomimetic in the treatment of leptin-deficiency disorders. We provide additional proof for the hypothesis that difficulties in current leptin therapies reside at the BBB penetration stage, and we document that by either glycosylation or intranasal peptide administration we can overcome this limitation.

The antibacterial effect of a proline-rich antibacterial peptide A3-APO.

Antimicrobial resistance is an emerging worldwide concern in light of the widespread antimicrobial drug use in humans, livestock and companion animals. The treatment of life-threatening infections is especially problematic because clinical strains rapidly acquire multiple-drug resistance. Antimicrobial peptides have long been considered to be viable alternatives to small molecule antibiotics. However, the peptides' parenteral use is frequently hampered by inadequate safety margins and rapid renal clearance leaving them suitable only for topical applications. The proline-rich peptide A3-APO represents a family of a new class of synthetic dimers that kill bacteria by a dual mode of action and carry domains for interaction with both the bacterial membrane and an intracellular target. From a series of designer antibacterial peptides, A3-APO emerged as a viable preclinical candidate by virtue of its superior ability to disintegrate the bacterial membrane, inhibit the 70-kDa heat shock protein DnaK alone or in synergy with small molecule antibiotics, lack of eukaryotic toxicity and withstand proteolytic degradation in body fluids. As many other proline-rich peptides, A3-APO binds to the C-terminal helical lid of bacterial DnaK and inhibits chaperone-assisted protein folding in bacteria but not in mammalian Hsp70. In this review, the structure, pharmacokinetic properties, antimicrobial spectrum of peptide A3-APO and its in vivo metabolite are summarized and the in vitro and in vivo antimicrobial effects (antimicrobial susceptibilities, postantibiotic effects, resistance induction) are discussed in detail.

Synthesis, conformation, receptor binding and biological activities of monobiotinylated human insulin-like peptide 3.

Biotin-avidin immobilization has been routinely used as a tool to study peptide-receptor and peptide-antibody interactions. Biotinylated peptides can also be employed to localize cells that express the peptides' receptor, and to analyse ligand-receptor binding. Insulin-like peptide 3 (INSL3) is a peptide hormone which contains A- and B-chains connected by two disulphide bonds and plays a role in testicular descent during sexual development. In order to study the interaction of INSL3 with its receptor LGR8, a G protein-coupled receptor, we chemically synthesized Nalpha-mono-biotinylated human INSL3 (B-hINSL3) and compared it structurally and biologically with hINSL3. Both peptides exhibited similar, but high, receptor binding affinities on human foetal kidney fibroblast 293T cells transfected human LGR8 based on a competition radioreceptor assay with 33P-labelled relaxin H2 (B33). The modified B-hINSL3 showed full biological activity as determined by the stimulation of gubernacular cell proliferation. The labelled B-hINSL3 contains a higher alpha-helix content, and this increased helical structure is accompanied by an increase in ability to stimulate cAMP accumulation in 293T cells expressing LGR8. Our results suggest that the N-terminal region of the A-chain is not involved in the interaction of INSL3 with its receptor. However, the introduction of biotin onto the N-terminus of the A-chain promoted conformational stability which, in turn, permitted better receptor activation.

The short proline-rich antibacterial peptide family.

From the many peptide families that are induced upon bacterial infection and can be isolated from all classes of animals, the short, proline-rich antibacterial peptides enjoy particular interest. These molecules were shown to inactivate an intracellular biopolymer in bacteria without destroying or remaining attached to the bacterial cell membrane, and as such emerged as viable candidates for the treatment of mammalian infections. These peptides were originally isolated from insects, they kill mostly gram-negative bacteria with high efficiency and they show structural similarities with longer insect- and mammal-derived antimicrobial peptides. However, while the distant relatives appear to carry multiple functional domains, apidaecin, drosocin, formaecin and pyrrhocoricin consist of only minimal determinants needed to penetrate across the cell membrane and bind to the target biopolymer. These peptides appear to inhibit metabolic processes, such as protein synthesis or chaperone-assisted protein folding. Pyrrhocoricin derivatives protect mice from experimental infections in vivo, suggesting the utility of modified analogs in the clinical setting. Sequence variations of the target protein at the peptide-binding site may allow the development of new peptide variants that kill currently unresponsive strains or species.

Intracellular targets of antibacterial peptides.

The recent past witnessed a decrease in the number of new antibacterial compounds approved by the regulatory agencies and an almost complete lack of molecules killing bacteria by novel mechanisms of action. The broad spectrum antimicrobial agents currently on the market carry the potential, and indeed victims, of resistance developed against them. The need for new types of antimicrobial drugs coincides with the desire of developing lead molecules that act selectively on a single strain, or perhaps on a few closely related strains. Such selectivity would exclude the likelihood of the emergence of broad-range resistance. Intracellular bacterial targets most prevalently proteins needed for the life cycle of bacteria, carry the potential to be a resourceful target for a new family of antimicrobial compounds. Inhibition of proteinaceous functions requires stereospecificity, and a drug structurally similar to the target proteins themselves. Indeed, some antibacterial peptides show selective inhibition of intracellular targets. A few native peptides and their designed analogs appear to kill only a limited number of bacterial strains. Identification of the binding sites on the target proteins would allow the design of strain-specific antibacterial and antifungal peptides without the fear of development of common resistance to these agents.

Chemical synthesis and biological activity of rat INSL3.

The recently identified protein, insulin 3 (INSL3), has structural features that make it a bona fide member of the insulin superfamily. Its predicted amino acid sequence contains the classic two-peptide chain (A- and B-) structure with conserved cysteine residues that results in a disulphide bond disposition identical to that of insulin. Recently, the generation of insl3 knockout mice has demonstrated that testicular descent is blocked due to the failure of a specific ligament, the gubernaculum, to develop. The mechanism by which INSL3 exerts its action on the gubernaculum is currently unknown. The purpose of this study was to, for the first time, synthesize rat INSL3 and test its action on organ cultures of foetal rat gubernaculum. INSL3 also contains a cassette of residues Arg-X-X-X-Arg within the B-chain, a motif that is essential for characteristic activity of another related member of the superfamily, relaxin. Hence, the relaxin activity of rat INSL3 was also tested in two different relaxin bioassays. The primary structure of rat INSL3 was determined by deduction from its cDNA sequence and successfully prepared by solid phase peptide synthesis of the two constituent chains followed by their combination in solution. Following confirmation of its chemical integrity by a variety of analytical techniques, circular dichroism spectroscopy confirmed the presence of high beta-turn and alpha-helical content, with a remarkable spectral similarity to the synthetic ovine INSL3 peptide and to synthetic rat relaxin. The synthetic rat INSL3 bound with very low affinity to rat relaxin receptors and had no activity in a relaxin bioassay. Furthermore, it did not augment or antagonize relaxin activity. The rat INSL3 did however induce growth of foetal rat gubernaculum in whole organ cultures demonstrating that INSL3 has a direct action on this structure.

Point mutations identify the glutamate binding pocket of the N-methyl-D-aspartate receptor as major site of conantokin-G inhibition.

Conantokin-G (Con-G), a gamma-carboxylglutamate (Gla) containing peptide derived from the venom of the marine cone snail Conus geographus, acts as a selective and potent inhibitor of N-methyl-D-aspartate (NMDA) receptors. Here, the effect of Con-G on recombinant NMDA receptors carrying point mutations within the glycine and glutamate binding pockets of the NR1 and NR2B subunits was studied using whole-cell voltage-clamp recording from cRNA injected Xenopus oocytes. At wild-type receptors, glutamate-induced currents were inhibited by Con-G in a dose-dependent manner at concentrations of 0.1-100 microM. Substitution of selected residues within the NR2B subunit reduced the inhibitory potency of Con-G, whereas similar mutations in the NR1 subunit had little effect. These results indicate a selective interaction of Con-G with the glutamate binding pocket of the NMDA receptor. Homology-based molecular modeling of the glutamate binding region based on the known structure of the glutamate binding site of the AMPA receptor protein GluR2 suggests how selected amino acid side chains of NR2B might interact with specific residues of Con-G.

Structural and biosensor analyses of a synthetic biotinylated peptide probe for the isolation of adenomatous polyposis coli tumor suppressor protein complexes.

Large numbers of colon tumors stem from mutations in the gene coding for the production of the adenomatous polyposis coli (APC) tumor suppressor protein. This protein contains a coiled-coil N-terminal domain that is known to be responsible for homodimerization. Previous work by others has led to the design of a specific 54-residue anti-APC peptide (anti-APCp1) that dimerizes preferentially with this domain. We have undertaken the chemical synthesis of a modified form of this peptide (anti-APCp2) that bears a biotin moiety at its N-terminus for use in subsequent ligand-binding analysis studies. The peptide was subjected to comprehensive chemical characterization to confirm its purity. Secondary structural analysis by circular dichroism spectroscopy and Fourier transform infrared spectroscopy indicated that the peptide could assume a wide range of potential conformations, depending upon the precise microenvironment. Significantly, a stable alpha-helical structure was generated when the solvent conditions supported intramolecular salt-bridge formation along the helix barrel. The biotinylated anti-APCp2 was immobilized onto a streptavidin sensor surface, in a specific orientation leaving all amino acids available to form a coiled structure. In one experiment, injection of colonic cell lysate extracts (LIM1215) onto a size-exclusion column resulted in the isolation of a high molecular mass protein peak (> 600 kDa) that reacted specifically with the immobilized anti-APCp2 on the biosensor surface. In another experiment, a high molecular mass protein (M(r) > 250 kDa on SDS-PAGE) could be specifically immunoprecipitated from this peak using either the anti-APCp2 peptide or an anti-APC polyclonal antibody. This demonstrates the specific interaction between the anti-APCp2 peptide and native APC and highlights the potential use of the former peptide in a multidimensional micropreparative chromatographic/biosensor/proteomic protocol for the purification of APC alone and APC complexed with different biopolymers in various cell lines, and stages of tumor development.

Solution structures by 1H NMR of the novel cyclic trypsin inhibitor SFTI-1 from sunflower seeds and an acyclic permutant.

SFTI-1 is a recently discovered cyclic peptide trypsin inhibitor from sunflower seeds comprising 14 amino acid residues. It is the most potent known Bowman-Birk inhibitor and the only naturally occurring cyclic one. The solution structure of SFTI-1 has been determined by 1H-NMR spectroscopy and compared with a synthetic acyclic permutant. The solution structures of both are remarkably similar. The lowest energy structures from each family of 20 structures of cyclic and acyclic SFTI-1 have an rmsd over the backbone and heavy atoms of 0.29 A and 0.66 A, respectively. The structures consist of two short antiparallel beta-strands joined by an extended loop containing the active site at one end. Cyclic SFTI-1 also has a hairpin turn completing the cycle. Both molecules contain particularly stable arrangements of cross-linking hydrogen bonds between the beta-strands and a single disulfide bridge, making them rigid and well defined in solution. These stable arrangements allow both the cyclic and acyclic variants of SFTI-1 to inhibit trypsin with very high potencies (0.5 nM and 12.1 nM, respectively). The cyclic nature of SFTI-1 appears to have evolved to provide higher trypsin inhibition as well as higher stability. The solution structures are similar to the crystal structure of the cyclic inhibitor in complex with trypsin. The lack of a major conformational change upon binding suggests that the structure of SFTI-1 is rigid and already pre-organized for maximal binding due to minimization of entropic losses compared to a more flexible ligand. These properties make SFTI-1 an ideal platform for the design of small peptidic pharmaceuticals or pesticides.

Phosphorylated osteopontin peptides suppress crystallization by inhibiting the growth of calcium oxalate crystals.

BACKGROUND: Osteopontin isolated from human urine [uropontin (uOPN)] is a potent inhibitor of calcium oxalate (CaOx) monohydrate (COM) crystallization. However, specific structural features responsible for its effects on CaOx crystallization were not previously known. The present studies were designed to define molecular features responsible for interactions of uOPN with COM crystals and the inhibition of crystallization. METHODS: Peptides and phosphopeptides with sequences corresponding to potential crystal binding domains within the protein sequence of osteopontin were synthesized. Then the effects of these peptides on COM crystal growth and crystal aggregation were investigated and their secondary structures analyzed. RESULTS: Growth of COM crystals was inhibited by approximately 50% at 1000 nmol/L concentrations by the two unmodified peptides with the closest clustering of aspartic acid residues. Growth was not inhibited by the other two unmodified peptides, with aspartic residues more evenly distributed within their sequences. Phosphorylation markedly increased inhibition of COM crystal growth, so that each of the four phosphorylated peptides inhibited growth by at least 50% at concentrations of < or =200 nmol/L. Phosphorylation of these peptides did not cause changes in secondary structure that would favor interaction with COM crystal surfaces. CONCLUSIONS: These studies of synthetic peptides identify molecular features within the osteopontin molecule that contribute to the inhibition of one aspect of COM crystallization. The inhibition of crystal growth induced by phosphorylation appears to result from altered local patterns of charge density, since conformational changes favoring interaction with crystals were not caused by phosphorylation.

Synthesis, conformational studies and biological activity of N(alpha)-mono-biotinylated rat relaxin.

Biotin-avidin immobilization can be a useful tool in structure-function studies of hormone receptors. A crucial step is the preparation of a specifically biotinylated hormone that is able to bind to its receptor while leaving the biotin group free for interaction with avidin. The receptor for relaxin, an ovarian peptidic hormone produced during pregnancy, has not yet been isolated. We therefore undertook to prepare a specifically monobiotinylated rat relaxin for use in ligand-searching strategies. Rat relaxin is a convenient analogue because reliable bioassays exist, thus allowing assessment of the effect of N-biotinylation on bioactivity. To help improve the yield of the two-chain, three-disulfide bond rat relaxin, 2-hydroxy-4-methoxybenzyl (Hmb) backbone protection was used during the solid-phase assembly of the B-chain to help prevent any possible chain aggregation. As a final step, while the protected peptide was still on the resin, the biotin label was introduced at the N-terminus of the B-chain using standard coupling protocols. The chain combination with the A-chain was accomplished in reasonable yield. Secondary structural measurements demonstrated that the biotin caused the starting B-chain to adopt a more ordered conformation. The labelled synthetic relaxin exhibited similar circular dichroism spectra to native and synthetic single B-chain peptides. In addition, the biotinylated relaxin showed no significant difference in its chronotropic activity in the rat isolated heart assay compared with the native peptide. Biosensor studies showed that antibody recognition was retained upon attachment of the synthetic relaxin to the streptavidin-derivatized surface.

Synthesis of a disulfide-linked octameric peptide construct carrying three different antigenic determinants.

In an effort to develop peptide vaccines against the influenza virus, we have successfully synthesized a disulfide-linked octameric homodimer that bears four copies of the influenza virus M2 protein ectodomain as well as two copies each of T-helper cell hemagglutinin epitopes, the I-E(d) restricted S1 and the I-A(d) restricted S2 fragments. Peptide attachment was via intermolecular disulfide formation from free sulfhydryl-bearing cysteine derivatives in solution. This reaction was efficient only when the amino-group of the cysteine was Fmoc-protected.

Mitochondrial targeted cytochrome P450 2E1 (P450 MT5) contains an intact N terminus and requires mitochondrial specific electron transfer proteins for activity.

Hepatic mitochondria contain an inducible cytochrome P450, referred to as P450 MT5, which cross-reacts with antibodies to microsomal cytochrome P450 2E1. In the present study, we purified, partially sequenced, and determined enzymatic properties of the rat liver mitochondrial form. The mitochondrial cytochrome P450 2E1 was purified from pyrazole-induced rat livers using a combination of hydrophobic and ion-exchange chromatography. Mass spectrometry analysis of tryptic fragments of the purified protein further ascertained its identity. N-terminal sequencing of the purified protein showed that its N terminus is identical to that of the microsomal cytochrome P450 2E1. In reconstitution experiments, the mitochondrial cytochrome P450 2E1 displayed the same catalytic activity as the microsomal counterpart, although the activity of the mitochondrial enzyme was supported exclusively by adrenodoxin and adrenodoxin reductase. Mass spectrometry analysis of tryptic fragments and also immunoblot analysis of proteins with anti-serine phosphate antibody demonstrated that the mitochondrial cytochrome P450 2E1 is phosphorylated at a higher level compared with the microsomal counterpart. A different conformational state of the mitochondrial targeted cytochrome P450 2E1 (P450 MT5) is likely to be responsible for its observed preference for adrenodoxin and adrenodoxin reductase electron transfer proteins.

The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding.

Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin, drosocin, and apidaecin interact with the bacterial heat shock protein DnaK, and peptide binding to DnaK can be correlated with antimicrobial activity. In the current report we studied the mechanism of action of these peptides and their binding sites to Escherichia coli DnaK. Biologically active pyrrhocoricin made of L-amino acids diminished the ATPase activity of recombinant DnaK. The inactive D-pyrrhocoricin analogue and the membrane-active antibacterial peptide cecropin A or magainin 2 failed to inhibit the DnaK-mediated phosphate release from adenosine 5'-triphosphate (ATP). The effect of pyrrhocoricin on DnaK's other significant biological function, the refolding of misfolded proteins, was studied by assaying the alkaline phosphatase and beta-galactosidase activity of live bacteria. Remarkably, both enzyme activities were reduced upon incubation with L-pyrrhocoricin or drosocin. D-Pyrrhocoricin, magainin 2, or buforin II, an antimicrobial peptide involved in binding to bacterial nucleic acids, had only negligible effect. According to fluorescence polarization and dot blot analysis of synthetic DnaK fragments and labeled pyrrhocoricin analogues, pyrrhocoricin bound with a K(d) of 50.8 microM to the hinge region around the C-terminal helices D and E, at the vicinity of amino acids 583 and 615. Pyrrhocoricin binding was not observed to the homologous DnaK fragment of Staphylococcus aureus, a pyrrhocoricin nonresponsive strain. In line with the lack of ATPase inhibition, drosocin binding appears to be slightly shifted toward the D helix. Our data suggest that drosocin and pyrrhocoricin binding prevents the frequent opening and closing of the multihelical lid over the peptide-binding pocket of DnaK, permanently closes the cavity, and inhibits chaperone-assisted protein folding. The biochemical results were strongly supported by molecular modeling of DnaK-pyrrhocoricin interactions. Due to the prominent sequence variations of procaryotic and eucaryotic DnaK molecules in the multihelical lid region, our findings pave the road for the design of strain-specific antibacterial peptides and peptidomimetics. Far-fetched applications of the species-specific inhibition of chaperone-assisted protein folding include the control of not only bacteria but also fungi, parasites, insects, and perhaps rodents.

The use of coiled-coil interactions for the analysis and micropreparative isolation of adenomatous polyposis coli protein complexes.

The coiled coil is a common structural motif found both as the dominant structure in fibrous proteins and as an oligomerization domain in a variety of cytoskeletal and extracellular matrix proteins. Coiled-coils typically consist of two to four helices that are supercoiled around one another in either parallel or antiparallel orientations. In the past few years our knowledge of the structure and specificity of coiled coil interactions has increased, allowing the de novo design and preparation of coiled-coils with well-defined structure and specificity. Indeed, the design and synthesis of a peptide that binds specifically to a single coiled-coil-containing protein, adenomatous polyposis coli (APC) has been reported. We have optimized solid-phase synthesis techniques to produce a modified form of the anti-APC peptide that contains a biotin moiety specifically placed so as to allow selective orientation onto the surface of a biosensor or affinity support. These peptide surfaces have been used to both monitor and purify APC and APC complexes from cellular extracts.

Antibacterial peptides isolated from insects.

Insects are amazingly resistant to bacterial infections. To combat pathogens, insects rely on cellular and humoral mechanisms, innate immunity being dominant in the latter category. Upon detection of bacteria, a complex genetic cascade is activated, which ultimately results in the synthesis of a battery of antibacterial peptides and their release into the haemolymph. The peptides are usually basic in character and are composed of 20-40 amino acid residues, although some smaller proteins are also included in the antimicrobial repertoire. While the proline-rich peptides and the glycine-rich peptides are predominantly active against Gram-negative strains, the defensins selectively kill Gram-positive bacteria and the cecropins are active against both types. The insect antibacterial peptides are very potent: their IC50 (50% of the bacterial growth inhibition) hovers in the submicromolar or low micromolar range. The majority of the peptides act through disintegrating the bacterial membrane or interfering with membrane assembly, with the exception of drosocin, apidaecin and pyrrhocoricin which appear to deactivate a bacterial protein in a stereospecific manner. In accordance with their biological function, the membrane-active peptides form ordered structures, e.g. alpha-helices or beta-pleated sheets and often cast permeable ion-pores. Their cytotoxic properties were exploited in in vivo studies targeting tumour progression. Although the native peptides degrade quickly in biological fluids other than insect haemolymph, structural modifications render the peptides resistant against proteases without sacrificing biological activity. Indeed, a pyrrhocoricin analogue shows lack of toxicity in vitro and in vivo and protects mice against experimental Escherichia coli infection. Careful selection of lead molecules based on the insect antibacterial peptides may extend their utility and produce viable alternatives to the conventional antimicrobial compounds for mammalian therapy.

Interaction between heat shock proteins and antimicrobial peptides.

Drosocin, pyrrhocoricin, and apidaecin, representing the short (18-20 amino acid residues) proline-rich antibacterial peptide family, originally isolated from insects, were shown to act on a target bacterial protein in a stereospecific manner. Native pyrrhocoricin and one of its analogues designed for this purpose protect mice from bacterial challenge and, therefore, may represent alternatives to existing antimicrobial drugs. Furthermore, this mode of action can be a basis for the design of a completely novel set of antibacterial compounds, peptidic or peptidomimetic, if the interacting bacterial biopolymers are known. Recently, apidaecin was shown to enter Escherichia coli and subsequently kill bacteria through sequential interactions with diverse target macromolecules. In this paper report, we used biotin- and fluorescein-labeled pyrrhocoricin, drosocin, and apidaecin analogues to identify biopolymers that bind to these peptides and are potentially involved in the above-mentioned multistep killing process. Through use of a biotin-labeled pyrrhocoricin analogue, we isolated two interacting proteins from E. coli. According to mass spectrometry, Western blot, and fluorescence polarization, the short, proline-rich peptides bound to DnaK, the 70-kDa bacterial heat shock protein, both in solution and on the solid-phase. GroEL, the 60-kDa chaperonin, also bound in solution. Control experiments with an unrelated labeled peptide showed that while binding to DnaK was specific for the antibacterial peptides, binding to GroEL was not specific for these insect sequences. The killing of bacteria and DnaK binding are related events, as an inactive pyrrhocoricin analogue made of all-D-amino acids failed to bind. The pharmaceutical potential of the insect antibacterial peptides is underscored by the fact that pyrrhocoricin did not bind to Hsp70, the human equivalent of DnaK. Competition assay with unlabeled pyrrhocoricin indicated differences in GroEL and DnaK binding and a probable two-site interaction with DnaK. In addition, all three antibacterial peptides strongly interacted with two bacterial lipopolysaccharide (LPS) preparations in solution, indicating that the initial step of the bacterial killing cascade proceeds through LPS-mediated cell entry.

Role of phosphorylation in the conformation of tau peptides implicated in Alzheimer's disease.

A series of peptides corresponding to isolated regions of Tau (tau) protein have been synthesized and their conformations determined by (1)H NMR spectroscopy. Immunodominant peptides corresponding to tau(224-240) and a bisphosphorylated derivative in which a single Thr and a single Ser are phosphorylated at positions 231 and 235 respectively, and which are recognized by an Alzheimer's disease-specific monoclonal antibody, were the main focus of the study. The nonphosphorylated peptide adopts essentially a random coil conformation in aqueous solution, but becomes slightly more ordered into beta-type structure as the hydrophobicity of the solvent is increased by adding up to 50% trifluoroethanol (TFE). Similar trends are observed for the bisphosphorylated peptide, with a somewhat stronger tendency to form an extended structure. There is tentative NMR evidence for a small population of species containing a turn at residues 229-231 in the phosphorylated peptide, and this is strongly supported by CD spectroscopy. A proposal that the selection of a bioactive conformation from a disordered solution ensemble may be an important step (in either tubulin binding or in the formation of PHF) is supported by kinetic data on Pro isomerization. A recent study showed that Thr231 phosphorylation affected the rate of prolyl isomerization and abolished tubulin binding. This binding was restored by the action of the prolyl isomerase Pin1. In the current study, we find evidence for the existence of both trans and cis forms of tau peptides in solution but no difference in the equilibrium distribution of cis-trans isomers upon phosphorylation. Increasing hydrophobicity decreases the prevalence of cis forms and increases the major trans conformation of each of the prolines present in these molecules. We also synthesized mutant peptides containing Tyr substitutions preceding the Pro residues and found that phosphorylation of Tyr appears to have an effect on the equilibrium ratio of cis-trans isomerization and decreases the cis content.