Mechanism of action of Drosophila Reaper in mammalian cells: Reaper globally inhibits protein synthesis and induces apoptosis independent of mitochondrial permeability

Drosophila Reaper can bind inhibitor of apoptosis proteins (IAP) and thereby rescue caspases from proteasomal degradation. In insect cells, this is sufficient to induce apoptosis. Reaper can also induce apoptosis in mammalian cells, in which caspases need to be activated, usually via the mitochondrial pathway. Nevertheless, we find that Reaper efficiently induces apoptosis in mammalian cells in the absence of mitochondrial permeabilisation and cytochrome c release. Moreover, this capacity was only marginally affected by deletion of Reaper's amino-terminal IAP-binding motif. Independent of this motif, Reaper could globally suppress protein synthesis. Deletion of 20 amino acids from the carboxy-terminus of Reaper fully abrogated its potential to inhibit protein synthesis and to induce apoptosis in the absence of IAP-binding. Our findings indicate that the newly identified capacity of Reaper to suppress protein translation can operate in mammalian cells and may be key to its pro-apoptotic activity.     

Central assumptions of predator-prey models fail in a semi-natural experimental system

The relationship between the encounter rate of predators with prey and the density of this prey is fundamental to models of predator-prey interactions. The relationship determines, among other variables, the rate at which prey patches are depleted, and hence the impact of predator populations on their prey, and the optimal spatial distribution of foraging effort. Two central assumptions that are made in many models are that encounter rate is directly proportional to prey density and that it is independent of the proportion of prey already removed, other than via the decreased density. We show here, using captive great tits searching for winter moth caterpillars in their natural hiding positions, that neither of these assumptions hold. Encounter rate increased less than directly in proportion to prey density, and it depended not only on the current density of prey, but also on the proportion of prey already removed by previous foragers. Both of these effects are likely to have major consequences for the outcome of predator-prey interactions.     

Cleavage of DNA without loss of genetic information by incorporation of a disaccharide nucleoside

A ribose residue inserted between the 3′-OH of one nucleotide and the 5′-phosphate group of the next nucleotide, functions as a site-specific cleavage site within DNA. This extra ribose does not interrupt helix formation and it protects duplex DNA against cleavage by restriction enzymes. Cleavage can be obtained with periodate and all ribose fragments can be removed with sodium hydroxide. As a result of this, an intact natural oligodeoxynucleotide is obtained after ligation reaction, which means that site-specific cleavage and recovering of intact DNA occurs without loss of genetic information.     

Canalization of development and ecdysteroid timing during the last instar in lubber grasshoppers

Development in many phytophagous, holometabolous insects is flexible at the beginning but inflexible at the end of the last larval instar. A prominent feature of the inflexible period is a peak in hemolymph levels of ecdysteroids. We tested whether this pattern holds true for the final molt of a phytophagous, hemimetabolous insect, Romalea microptera (the Eastern lubber grasshopper). We fed one group of grasshoppers a high quantity diet (H) throughout the 5th (final) instar and a second group a low quantity diet (L) throughout the instar. Three other diet treatments involved starting the instar on the high diet and then abruptly switching to the low diet at 3, 8, or 13 days (H3L, H8L, and H13L respectively) and continuing the low diet until adult molt. Diet treatment did not affect the maximum hemolymph level of ecdysteroids (E(max)); this peak typically reached ~4000 ng/ml. Ecdysteroid levels were elevated for ~4 days in all groups. In contrast, diet significantly affected age at adult molt and age at E(max) such that H = H13L = H8L < H3L = L. We identified estimates of thresholds for weight gain (20% initial weight) and hemolymph ecdysteroids (100 ng/ml), after which diet did not affect the time to the adult molt. The weight gain threshold was less precise than the ecdysteroid threshold. These results suggest that R. microptera has an extended period of inflexible (canalized) development during the final instar that includes a peak of ecdysteroids. We hypothesize this pattern holds for many phytophagous, hemimetabolous insects.     

Immunogenicity of peptide-vaccine candidates predicted by molecular dynamics simulations

We present an in silico, structure-based approach for design and evaluation of conformationally restricted peptide-vaccines. In particular, we designed four cyclic peptides of ten or 11 residues mimicking the crystallographically observed beta-turn conformation of a predicted immunodominant loop of PorA from Neisseria meningitidis. Conformational correctness and stability of the peptide designs, as evaluated by molecular dynamics simulations, correctly predicted the immunogenicity of the peptides. We observed a peptide-induced functional antibody response that, remarkably, exceeded the response induced by the native protein in outer membrane vesicles, without losing specificity for related strains. The presented approach offers tools for a priori design and selection of peptide-vaccine candidates with full biological activity. This approach could be widely applicable: to outer membrane proteins of Gram-negative bacteria, and to other epitopes in a large range of pathogens.     

Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. New insights into the phosphoryl transfer mechanism

The chemical synthesis of new compounds designed as inhibitors of Mycobacterium tuberculosis TMP kinase (TMPK) is reported. The synthesis concerns TMP analogues modified at the 5-position of the thymine ring as well as a novel compound with a six-membered sugar ring. The binding properties of the analogues are compared with the known inhibitor azido-TMP, which is postulated here to work by excluding the TMP-bound Mg(2+) ion. The crystallographic structure of the complex of one of the compounds, 5-CH(2)OH-dUMP, with TMPK has been determined at 2.0 A. It reveals a major conformation for the hydroxyl group in contact with a water molecule and a minor conformation pointing toward Ser(99). Looking for a role for Ser(99), we have identified an unusual catalytic triad, or a proton wire, made of strictly conserved residues (including Glu(6), Ser(99), Arg(95), and Asp(9)) that probably serves to protonate the transferred PO(3) group. The crystallographic structure of the commercially available bisubstrate analogue P(1)-(adenosine-5')-P(5)-(thymidine-5')-pentaphosphate bound to TMPK is also reported at 2.45 A and reveals an alternative binding pocket for the adenine moiety of the molecule compared with what is observed either in the Escherichia coli or in the yeast enzyme structures. This alternative binding pocket opens a way for the design of a new family of specific inhibitors.     

ATP-dependent interactions between Escherichia coli Min proteins and the phospholipid membrane in vitro

Proper placement of the division apparatus in Escherichia coli requires pole-to-pole oscillation of the MinC division inhibitor. MinC dynamics involves a membrane association-dissociation cycle that is driven by the activities of the MinD ATPase and the MinE topological specificity factor, which themselves undergo coupled oscillatory localization cycles. To understand the biochemical mechanisms underlying Min protein dynamics, we studied the interactions of purified Min proteins with phospholipid vesicles and the role of ATP in these interactions. We show that (i) the ATP-bound form of MinD (MinD.ATP) readily associates with phospholipid vesicles in the presence of Mg(2+), whereas the ADP-bound form (MinD.ADP) does not; (ii) MinD.ATP binds membrane in a self-enhancing fashion; (iii) both MinC and MinE can be recruited to MinD.ATP-decorated vesicles; (iv) MinE stimulates dissociation of MinD.ATP from the membrane in a process requiring hydrolysis of the nucleotide; and (v) MinE stimulates dissociation of MinC from MinD.ATP-membrane complexes, even when ATP hydrolysis is blocked. The results support and extend recent work by Z. Hu et al. (Z. Hu, E. P. Gogol, and J. Lutkenhaus, Proc. Natl. Acad. Sci. USA 99:6761-6766, 2002) and support models of protein oscillation wherein MinE induces Min protein dynamics by stimulating the conversion of the membrane-bound form of MinD (MinD.ATP) to the cytoplasmic form (MinD.ADP). The results also indicate that MinE-stimulated dissociation of MinC from the MinC-MinD.ATP-membrane complex can, and may, occur prior to hydrolysis of the nucleotide.     

Hemolymph levels of methyl farnesoate increase in response to osmotic stress in the green crab, Carcinus maenas

The salinity of estuarine environments can vary widely, exposing resident organisms to considerable osmotic stress. The green crab Carcinus maenas is well known for its ability to osmoregulate in response to such stress. Therefore, we tested the relationship between osmoregulation and hemolymph levels of methyl farnesoate (MF), a compound previously shown to rise in response to various types of environmental stresses. When crabs were transferred from 100% seawater to dilute (hypo-osmotic) seawater, hemolymph osmolality dropped rapidly, reaching an acclimation level 48 h after transfer. Hemolymph levels of MF also rose in these animals after a delay of 6 h, and reached a maximum level at 48 h. MF levels remained elevated as long as the crabs were maintained in dilute seawater, and quickly returned to basal levels when the animals were returned to full strength seawater. In most (but not all) animals, MF levels were elevated when hemolymph osmolality fell below the isosmotic point (approx. 800 mOsm/kg). These data suggest that MF may have a role in osmoregulation by this species. In addition, the elevation of MF by hypo-osmotic seawater suggests an experimental strategy for manipulating MF levels in crustaceans.     

TRAIL receptor and CD95 signal to mitochondria via FADD,caspase-8/10, Bid,and Bax but differentially regulate events downstream from truncated Bid

The death receptor ligand TRAIL arouses much interest for clinical application. We found that TRAIL receptor could induce cytochrome c (Cyt c) release from mitochondria in cells that failed to respond to CD95. Therefore, we examined whether these two closely related death receptors use different intermediates to convey the apoptotic signal to mitochondria. Dominant negative FADD, FLIP(L), or a Bid mutant lacking cleavage sites for caspase-8/10 completely inhibited Cyt c release in response to either receptor. Depletion of Bid from TRAIL- or CD95-activated cytosols blocked their capacity to mediate Cyt c release from mitochondria in vitro, whereas Bax depletion reduced it. We conclude that FADD, caspase-8/10, and caspase-cleaved Bid are required for TRAIL receptor and CD95 signaling to mitochondria, whereas Bax is a common accessory. In vitro, caspase-8 treatment of cytosol from CD95-resistant cells permitted generation of truncated Bid and its association with mitochondria. However, this cytosol impaired the ability of truncated Bid to liberate Cyt c from exogenous mitochondria. We conclude that the TRAIL receptor can bypass or neutralize the activity of cytosolic factor that blocks truncated Bid function. This may benefit the capacity of TRAIL to break apoptosis resistance in tumor cells.     

Structure of apo-phosphatidylinositol transfer protein alpha provides insight into membrane association

Phosphatidylinositol transfer protein alpha (PITP alpha) is a ubiquitous and highly conserved protein in multicellular eukaryotes that catalyzes the exchange of phospholipids between membranes in vitro and participates in cellular phospholipid metabolism, signal transduction and vesicular trafficking in vivo. Here we report the three-dimensional crystal structure of a phospholipid-free mouse PITP alpha at 2.0 A resolution. The structure reveals an open conformation characterized by a channel running through the protein. The channel is created by opening the phospholipid-binding cavity on one side by displacement of the C-terminal region and a hydrophobic lipid exchange loop, and on the other side by flattening of the central beta-sheet. The relaxed conformation is stabilized at the proposed membrane association site by hydrophobic interactions with a crystallographically related molecule, creating an intimate dimer. The observed open conformer is consistent with a membrane-bound state of PITP and suggests a mechanism for membrane anchoring and the presentation of phosphatidylinositol to kinases and phospholipases after its extraction from the membrane. Coordinates have been deposited in the Protein Data Bank (accession No. 1KCM).