Insect peptides with improved protease-resistance protect mice against bacterial infection

At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against Gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia call infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.

A serine proteinase homolog venom protein from an endoparasitoid wasp inhibits melanization of the host hemolymph

Activation of prophenoloxidase (proPO) in insects is a defense mechanism against intruding microorganisms and parasites. Pattern recognition molecules induce activation of an enzymatic cascade involving serine proteinases, which leads to the conversion of proPO to active phenoloxidase (PO). Phenolic compounds produced by pPO-activation are toxic to invaders. Here, we describe the isolation of a venom protein from the parasitoid, Cotesia rubecula, injected into the host, Pieris rapae, which is homologous to serine proteinase homologs (SPH). The data presented here indicate that the protein interferes with the proteolytic cascade, which under normal circumstances leads to the activation of proPO and melanin formation. (C) 2003 Elsevier Ltd. All rights reserved.

The protease inhibitor cystatin C is differentially expressed among dendritic cell populations, but does not control antigen presentation

Dendritic cells (DC) undergo complex developmental changes during maturation. The MHC class H (MHC H) molecules of immature DC accumulate in intracellular compartments, but are expressed at high levels on the plasma membrane upon DC maturation. It has been proposed that the cysteine protease inhibitor cystatin C (CyC) plays a pivotal role in the control of this process by regulating the activity of cathepsin S, a protease involved in removal of the MHC H chaperone E, and hence in the formation of MHC H-peptide complexes. We show that CyC is differentially expressed by mouse DC populations. CD8(+) DC, but not CD4(+) or CD4(-)CD8(-) DC, synthesize CyC, which accumulates in MHC II(+)Lamp(+) compartments. However, II processing and MHC H peptide loading proceeded similarly in all three DC populations. We then analyzed MHC H localization and Ag presentation in CD8(+) DC, bone marrow-derived DC, and spleen-derived DC lines, from CyC-deficient mice. The absence of CyC did not affect the expression, the subcellular distribution, or the formation of peptide-loaded MHC II complexes in any of these DC types, nor the efficiency of presentation of exogenous Ags. Therefore, CyC is neither necessary nor sufficient to control MHC II expression and Ag presentation in DC. Our results also show that CyC expression can differ markedly between closely related cell types, suggesting the existence of hitherto unrecognized mechanisms of control of CyC expression.

Synthetic studies of the HIV-1 protease inhibitive didemnaketals: Stereocontrolled synthetic approach to the key mother spiroketals

[GRAPHICS] The stereocontrolled synthesis of (2S,4R,6R,8S,10S,1'R,1"R)-2(acetylhydroxymethyl)-4, 10-dimethyl-8(isopropenylhydroxymethyl)-1, 7-dioxaspiro[5,5]-undecane (4a) and its C1"-epimer (4b), the key mother spiroketals of the HIV-1 protease inhibitive didemnaketals from the ascidian Didemnum sp., has been carried out through multisteps from the natural (R)-(+)-pulegone, which involved the diastereoselective construction of four chiral carbon centers(C-2, C-6, C-8, and C-1') by intramolecular chiral induce.

Schistosoma japonicum cathepsin D aspartic protease cleaves human IgG and other serum components

Recombinant cathepsin D aspartic protease of Schistosoma japonicum cleaved human IgG in vitro in a time and dose-dependent manner. Optimal cleavage was seen at pH 3.6-4.5; modest cleavage remained at pH 5.0, and no cleavage was detected above pH 5.0. Amino terminal sequencing of the major cleavage fragments of human IgG identified a Fab fragment from the VH1 domain, and 2 cleavage sites in the CH2 domain below the hinge region. The P1 and P1' residues at the 2 CH2 cleavage sites were Phe254-Leu255 and Leu325-Thr326, indicating a preference by the schistosome protease for bulky hydrophobic residues flanking the scissile bond. No cleavage of the immunoglobulin light chain was detected. In addition, the recombinant schistosome protease indiscriminately degraded the human serum proteins complement C3 and serum albumin into numerous small fragments. These results demonstrate specific cleavage of human IgG by the recombinant schistosome aspartic protease, and highlight the broad range digestive specificity of the enzyme which may play a role in the degradation of host serum proteins ingested as part of the schistosome bloodmeal.

Vaccine efficacy of recombinant cathepsin D aspartic protease from Schistosoma japonicum

Mice were vaccinated with recombinant Schistosoma japonicum cathepsin D aspartic protease, expressed in both insect cells and bacteria, in order to evaluate the vaccine efficacy of the schistosome protease. Mean total worm burdens were significantly reduced in vaccinated mice by 21-38%, and significant reductions in female worm burdens were also recorded (22-40%). Vaccination did not reduce fecundity; rather, we recorded increased egg output per female worm in vaccinated animals, suggesting a crowding effect. Vaccinated mice developed high levels of antibodies (predominantly IgG1, IgG2a and IgG2b isotypes), but there was no correlation between antibody levels and protective efficacy. Immune sera from vaccinated mice did not inhibit the in vitro degradation of human haemoglobin by the recombinant protease, and passive transfer of serum or antibodies from vaccinated animals, before and after parasite challenge, did not significantly reduce worm or egg burdens in recipient animals. These results suggest that antibodies may not play a key role in the protective effect elicited, and that protection may be due to a combination of humoral and cell-mediated responses.

beta-strand mimicking macrocyclic amino acids: Templates for protease inhibitors with antiviral activity

New amino acids are reported in which component macrocycles are constrained to mimic tripeptides locked in a beta-strand conformation. The novel amino acids involve macrocycles functionalized with both an N- and a C-terminus enabling addition of appendages at either end to modify receptor affinity, selectivity, or membrane permeability. We show that the cycles herein are effective templates within inhibitors of HIV-1 protease. Eleven compounds originating from such bifunctionalized cyclic templates are potent inhibitors of HIV-1 protease (Ki 0.3-50 nM; pH 6.5, I = 0.1 M). Unlike normal peptides comprising amino acids, five of these macrocycle-containing compounds are potent antiviral agents with sub-micromolar potencies (IC50 170-900 nM) against HIV-1 replication in human MT2 cells. The most active antiviral agents are the most lipophilic, with calculated values of LogD(6.5) greater than or equal to 4. All molecules have a conformationally constrained 17-membered macrocyclic ring that has been shown to structurally mimic a tripeptide segment (Xaa)-(Val/Ile)-(Phe/Tyr) of a peptide substrate in the extended conformation. The presence of two trans amide bonds and a para-substituted aromatic ring prevents intramolecular hydrogen bonds and fixes the macrocycle in the extended conformation. Similarly constrained macrocycles may be useful templates for the creation of inhibitors for the many other proteins and proteases that recognize peptide beta-strands.

Cellular responses to Schistosoma japonicum cathepsin D aspartic protease

Lymphocyte proliferation and cytokine production were measured in groups of mice vaccinated (but not subsequently challenge infected) with recombinant forms of Schistosoma japonicum cathepsin D aspartic protease, rSjASP1 (expressed in bacteria; enzymatically inactive) and rSjASP2 (expressed in insect cells; enzymatically active). Both forms of the schistosome enzyme induced significant proliferation of splenocytes recovered from vaccinated mice, and expression of interferon (IFN)-gamma, interleukin (IL)-4 and IL-10 mRNA in these cells was detected using reverse transcriptase-polymerase chain reaction. Secretion of IFN-gamma, IL-4 and IL-10 by splenocytes from vaccinated mice was confirmed and quantified using enzyme-linked immunosorbent assay. IFN-gamma was the most abundant cytokine produced, followed by IL-4 and IL-10 in rank order. These findings indicated that vaccination of mice with the schistosome protease induces a mixed Th1/Th2 cytokine response, which may explain the modest level of protection after challenge infection in cathepsin d-vaccinated mice, reported previously.

Ion transport induced by proteinase-activated receptors (PAR2) in colon and airways

Protease-activated receptors type 2 (PAR2) are activated by serine proteases like trypsin and mast cell tryptase. The function and physiological significance of PAR2 receptors is poorly understood, but recent studies suggest a role during inflammatory processes in both airways and intestine. PAR2 receptors are also likely to participate in the control of ion transport in these tissues. We demonstrate that stimulation of PAR2 in airways and intestine significantly enhanced ion transport. Trypsin induced CI- secretion in both airways and intestine when added to the basolateral but not to the luminal side of these tissues. In both airways and intestine, stimulation of ion transport was largely dependent on the increase in intracellular Ca2+. Effects of trypsin were largely reduced by basolateral bumetanide and barium and by trypsin inhibitor. Thrombin, an activator of proteinase-activated receptors types 1, 3, and 4 had no effects on equivalent short-circuit current in either airways or intestine. Expression of PAR2 in colon and airways was further confirmed by reverse transcription-polymerase chain reaction. We postulate that these receptors play a significant role in the regulation of electrolyte transport, which might be important during inflammatory diseases of airways and intestine.

Synthesis, stability, antiviral activity, and protease-bound structures of substrate-mimicking constrained macrocyclic inhibitors of HIV-1 protease

Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-I, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 mu M. Their activities against HIV-1 protease (K-i 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC50 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC50 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55(gag) to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 Angstrom (1) and 1.85 Angstrom (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.

Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activity-guided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.

Structural basis and prediction of substrate specificity in protein serine/threonine kinases

The large number of protein kinases makes it impractical to determine their specificities and substrates experimentally. Using the available crystal structures, molecular modeling, and sequence analyses of kinases and substrates, we developed a set of rules governing the binding of a heptapeptide substrate motif (surrounding the phosphorylation site) to the kinase and implemented these rules in a web-interfaced program for automated prediction of optimal substrate peptides, taking only the amino acid sequence of a protein kinase as input. We show the utility of the method by analyzing yeast cell cycle control and DNA damage checkpoint pathways. Our method is the only available predictive method generally applicable for identifying possible substrate proteins for protein serine/threonine kinases and helps in silico construction of signaling pathways. The accuracy of prediction is comparable to the accuracy of data from systematic large-scale experimental approaches.

Storage protein mobilization and thiol protease up-regulation by solid matrix priming in loblolly pine (Pinus taeda) seed embryos

Experiments were conducted to investigate physiological mechanisms of solid matrix priming (SMP) on germination enhancement of loblolly pine (Pinus taeda) seeds. During SMP, osmotic potential in the embryo decreased by 0.65 MPa, concentration of crystalloid proteins decreased to 62% and concentrations of buffer soluble proteins and free amino acids increased by 22% and by 166%, respectively. Observations under an electron microscope demonstrated protein bodies in the embryo were mobilized. Inhibitor analysis indicated thiol protease was the dominant enzyme among endopiptidases to degrade the reserved proteins. A fragment of thiol protease was cloned from the primed seed embryos and it has high identities to those thiol proteases responsive to water stress. RNA get blot analysis showed a 1.5 kb thiol protease gene was up-regulated by SMP. Treatment with E64, a thiol protease inhibitor, negated SMP effects on germination performance, water potentials and protein profiles. Based on the experimental results, reserve protein mobilization induced by SMP in the embryo before radicle emergence might be one of the mechanisms to enhance germination in loblolly pine seeds.

Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion

Hookworms are voracious blood-feeders. The cloning and functional expression of an aspartic protease, Na-APR-2, from the human hookworm Necator americanus are described here. Na-APR-2 is more similar to a family of nematode-specific, aspartic proteases than it is to cathepsin D or pepsin, and the term nemepsins for members of this family of nematode-specific hydrolases is proposed. Na-apr-2 mRNA was detected in blood-feeding, developmental stages only of N. americanus, and the protease was expressed in the intestinal lumen, amphids, and excretory glands. Recombinant Na-APR-2 cleaved human hemoglobin (Hb) and serum proteins almost twice as efficiently as the orthologous substrates from the nonpermissive dog host. Moreover, only 25% of the Na-APR-2 cleavage sites within human Hb were shared with those generated by the related N. americanus cathepsin D, Na-APR-1. Antiserum against Na-APR-2 inhibited migration of 50% of third-stage N. americanus larvae through skin, which suggests that aspartic proteases might be effective vaccines against human hookworm disease.