Active-site specificity of digestive aspartic peptidases from the four species of Plasmodium that infect humans using chromogenic combinatorial peptide libraries

Two targeted chromogenic octapeptide combinatorial libraries, comprised of 38 pools each containing 361 different peptides, were used to analyze the enzyme/substrate interactions of five plasmepsins. The first library (P1 library) was based on a good synthetic aspartic peptidase substrate [Westling, J., Cipullo, P., Hung, S. H., Saft, H., Dame, J. B., and Dunn, B. M. (1999) Protein Sci. 8, 2001-2009; Scarborough, P. E., and Dunn, B. M. (1994) Protein Eng. 7, 495-502] and had the sequence Lys-Pro-(Xaa)-Glu-P1*Nph-(Xaa)-Leu. The second library (P1' library) incorporated results with the plasmepsins from the first library and had the sequence Lys-Pro-Ile-(Xaa)-Nph*P1'-Gln-(Xaa). In both cases, P1 and P1' were fixed residues for a given peptide pool, where Nph was a para-nitrophenylalanine chromogenic reporter and Xaa was a mixture of 19 different amino acids. Kinetic assays monitoring the rates of cleavage of these libraries revealed the optimal P1 and P1' residues for the five plasmepsins as hydrophobic substitutions. Extended specificity preferences were obtained utilizing liquid chromatography-mass spectrometry (LC-MS) to analyze the cleavage products produced by enzyme-catalyzed digestion of the best pools of each peptide library. LC-MS analysis of the P1-Phe and P1'-Phe pools revealed the favored amino acids at the P3, P2, P2', and P3' positions. These analyses have provided new insights on the binding preferences of malarial digestive enzymes that were used to design specific methyleneamino peptidomimetic inhibitors of the plasmepsins. Some of these compounds were potent inhibitors of the five plasmepsins, and their possible binding modes were analyzed by computational methods.     

Cooperative breeders adjust offspring sex ratios to produce helpful helpers

Whether birds and mammals adaptively adjust their offspring sex ratios in response to their environment is much debated. A source of confusion is that different studies show different patterns, with sex ratio adjustment appearing to occur in some cases but not others. The extent to which this reflects interesting biological variation due to differences in the underlying selective forces, as opposed to statistical noise, is not clear. Cooperatively breeding species offer an opportunity to address this problem because the strength of selection on sex ratio adjustment can be estimated. When helping behavior is sex dependent, parents are predicted to overproduce the helping sex when this sex is rare or absent. We show here that the extent of this behavior depends on the benefit that helpers bring to parents: there is greater sex ratio adjustment when helpers bring larger benefits. Variable selection on sex ratio adjustment may thus explain variable empirical findings.     

Spontaneous curvature of phosphatidic acid and lysophosphatidic acid

The formation of phosphatidic acid (PA) from lysophosphatidic acid (LPA), diacylglycerol, or phosphatidylcholine plays a key role in the regulation of intracellular membrane fission events, but the underlying molecular mechanism has not been resolved. A likely possibility is that PA affects local membrane curvature facilitating membrane bending and fission. To examine this possibility, we determined the spontaneous radius of curvature (R(0p)) of PA and LPA, carrying oleoyl fatty acids, using well-established X-ray diffraction methods. We found that, under physiological conditions of pH and salt concentration (pH 7.0, 150 mM NaCl), the R(0p) values of PA and LPA were -46 A and +20 A, respectively. Thus PA has considerable negative spontaneous curvature while LPA has the most positive spontaneous curvature of any membrane lipid measured to date. The further addition of Ca(2+) did not significantly affect lipid spontaneous curvature; however, omitting NaCl from the hydration buffer greatly reduced the spontaneous curvature of PA, turning it into a cylindrically shaped lipid molecule (R(0p) of -1.3 x 10(2) A). Our quantitative data on the spontaneous radius of curvature of PA and LPA at a physiological pH and salt concentration will be instrumental in developing future models of biomembrane fission.     

Identification and characterization of proteins from Bacillus thuringiensis with high toxic activity against the sheep blowfly, Lucilia cuprina

Current control of the sheep blowfly (Lucilia cuprina) relies on chemical insecticides, however, with the development of resistance and increasing concerns about human health and environmental residues, alternative strategies to control this economically important pest are required. In this study, we have identified several isolates of Bacillus thuringiensis (Bt), collected from various Australian soil samples, that produce crystals containing 130 and 28 kDa proteins. These isolates were highly toxic to feeding larvae in both in vitro bioassays and in vivo on sheep. By N-terminal amino acid sequencing, we identified the smaller crystal band (28 kDa) as a cytological (Cyt) protein. Upon solubilization and proteolytic processing by trypsin, the 130 kDa crystal protein yielded among others, a truncated 55-60 kDa toxin moiety which exhibited larvicidal activity against sheep blowfly. The amino-terminal sequence of the trypsin-resistant protein band revealed that this Bt endotoxin was encoded by a new cry gene. The novel cry protein was present in all the strains that were highly toxic in the larval assay. We have also identified from one of the isolates, a novel secretory toxin with larvicidal activity.     

The role of food plant and pathogen-induced behaviour in the persistence of a nucleopolyhedrovirus

Insect baculoviruses can survive between epidemics as infectious particles external to the host. Many pathogens persist in reservoirs, i.e., microhabitats where survival is enhanced, for example due to protection from the degrading effects of UV irradiation. However, the probability of infecting new susceptible hosts is usually reduced. Persistence of pathogens and their movement in and out of reservoirs is an important, albeit little understood, aspect of insect pathogen ecology. This study investigated interactions between the behaviour of infected insect hosts, virus distribution and plant species on the persistence of the winter moth (Operophtera brumata) nucleopolyhedrovirus. Habitat influenced the persistence of infectious baculovirus in the field: virus on Sitka spruce (Picea sitchensis) and oak (Quercus robur) in forested areas retained more infectivity than virus on heather (Calluna vulgaris) in an unshaded habitat. Plant species per se did not directly affect the persistence of virus on the foliage of potted seedlings. Virally infected insects had altered behaviour and moved down plants relative to control insects, whereas in other systems larvae show height-seeking behaviour. Consequently, the majority of virus particles were distributed on plant stems. In two experiments (one using winter moth NPV and one Mamestra brassicae NPV) virus persisted better on plant stems relative to foliage. Neonate larvae were shown to be able to acquire infections from tree stems contaminated with a low level of virus. These data suggest that plant stems may be important reservoirs for between-year persistence of this pathogen. The observed virus-induced changes in host behaviour in winter moth could enhance the viral persistence by increasing the deposition of occlusion bodies in these reservoirs.     

Islet amyloid polypeptide-induced membrane leakage involves uptake of lipids by forming amyloid fibers

Fibril formation of islet amyloid polypeptide (IAPP) is associated with cell death of the insulin-producing pancreatic beta-cells in patients with Type 2 Diabetes Mellitus. A likely cause for the cytotoxicity of human IAPP is that it destroys the barrier properties of the cell membrane. Here, we show by fluorescence confocal microscopy on lipid vesicles that the process of hIAPP amyloid formation is accompanied by a loss of barrier function, whereby lipids are extracted from the membrane and taken up in the forming amyloid deposits. No membrane interaction was observed when preformed fibrils were used. It is proposed that lipid uptake from the cell membrane is responsible for amyloid-induced membrane damage and that this represents a general mechanism underlying the cytotoxicity of amyloid forming proteins.     

Oxidants inhibit ERK/MAPK and prevent its ability to delay neutrophil apoptosis downstream of mitochondrial changes and at the level of XIAP

Normal spontaneous apoptosis in neutrophils is enhanced by "stress" stimuli such as tumor necrosis factor-alpha, Fas ligand, and oxidants, and this effect is inhibited by anti-apoptotic stimuli including granulocyte-macrophage colony-stimulating factor, lipopolysaccharide, and formylmethionine-leucine-phenylalanine. In this report we demonstrate that anti-apoptotic stimuli protect neutrophils from stress-induced apoptosis via activation of the ERK/MAPK pathway. The protection occurs downstream of mitochondrial alterations assessed as a decrease in membrane potential concomitant with enhanced cytochrome c release. ERK activation was shown to inhibit apoptosis by maintaining levels of XIAP, which is normally decreased in the presence of the pro-apoptotic/stress stimuli. This report also demonstrates that potent intra- and extracellular oxidants inhibit the protective effect of ERK. Oxidant-dependent inhibition of ERK was because of activation of p38 MAPK and activation of the protein phosphatases PP1 and PP2A. Our data suggest that ERK suppresses stress-induced apoptosis downstream of mitochondrial alterations by maintaining XIAP levels and that oxidants block this effect through activation of p38 and protein phosphatases.     

Amyloid fibril formation by lens crystallin proteins and its implications for cataract formation

The alpha-, beta-, and gamma-crystallins are the major structural proteins within the eye lens and are responsible for its exceptional stability and transparency. Under mildly denaturing conditions, all three types of bovine crystallin assemble into fibrillar structures in vitro. Characterization by transmission electron microscopy, dye binding assays, and x-ray fiber diffraction shows that these species have all of the characteristics of fibrils associated with the family of amyloid diseases. Moreover, the full-length proteins are incorporated into the fibrils, (i.e. no protein cleavage is required for these species to form), although for the gamma-crystallins some fragmentation occurs under the conditions employed in this study. Our findings indicate that the inherent stability of the beta-sheet supramolecular structure adopted by the crystallins in the eye lens and the chaperone ability of alpha-crystallin must be crucial for preventing fibril formation in vivo. The crystallins are very stable proteins but undergo extensive post-translational modification with age that leads to their destabilization. The ability of the crystallins to convert into fibrils under destabilizing conditions suggests that this process could contribute to the development of cataract with aging.