Malaria parasite liver stages render host hepatocytes susceptible to mitochondria-initiated apoptosis

Intracellular eukaryotic parasites and their host cells constitute complex, coevolved cellular interaction systems that frequently cause disease. Among them, Plasmodium parasites cause a significant health burden in humans, killing up to one million people annually. To succeed in the mammalian host after transmission by mosquitoes, Plasmodium parasites must complete intracellular replication within hepatocytes and then release new infectious forms into the blood. Using Plasmodium yoelii rodent malaria parasites, we show that some liver stage (LS)-infected hepatocytes undergo apoptosis without external triggers, but the majority of infected cells do not, and can also resist Fas-mediated apoptosis. In contrast, apoptosis is dramatically increased in hepatocytes infected with attenuated parasites. Furthermore, we find that blocking total or mitochondria-initiated host cell apoptosis increases LS parasite burden in mice, suggesting that an anti-apoptotic host environment fosters parasite survival. Strikingly, although LS infection confers strong resistance to extrinsic host hepatocyte apoptosis, infected hepatocytes lose their ability to resist apoptosis when anti-apoptotic mitochondrial proteins are inhibited. This is demonstrated by our finding that B-cell lymphoma 2 family inhibitors preferentially induce apoptosis in LS-infected hepatocytes and significantly reduce LS parasite burden in mice. Thus, targeting critical points of susceptibility in the LS-infected host cell might provide new avenues for malaria prophylaxis.     

Interactions of cationic-hydrophobic peptides with lipid bilayers: a Monte Carlo simulation method

We present a computational model of the interaction between hydrophobic cations, such as the antimicrobial peptide, Magainin2, and membranes that include anionic lipids. The peptide's amino acids were represented as two interaction sites: one corresponds to the backbone alpha-carbon and the other to the side chain. The membrane was represented as a hydrophobic profile, and its anionic nature was represented by a surface of smeared charges. Thus, the Coulombic interactions between the peptide and the membrane were calculated using the Gouy-Chapman theory that describes the electrostatic potential in the aqueous phase near the membrane. Peptide conformations and locations near the membrane, and changes in the membrane width, were sampled at random, using the Metropolis criterion, taking into account the underlying energetics. Simulations of the interactions of heptalysine and the hydrophobic-cationic peptide, Magainin2, with acidic membranes were used to calibrate the model. The calibrated model reproduced structural data and the membrane-association free energies that were measured also for other basic and hydrophobic-cationic peptides. Interestingly, amphipathic peptides, such as Magainin2, were found to adopt two main membrane-associated states. In the first, the peptide resided mostly outside the polar headgroups region. In the second, which was energetically more favorable, the peptide assumed an amphipathic-helix conformation, where its hydrophobic face was immersed in the hydrocarbon region of the membrane and the charged residues were in contact with the surface of smeared charges. This dual behavior provides a molecular interpretation of the available experimental data.     

Characterization of internal motions of Escherichia coli ribonuclease H by Monte Carlo simulation

The backbone dynamics of Escherichia coli ribonuclease H (RNase H) is studied by a recently developed off-lattice Monte Carlo/Metropolis simulation technique. A low-resolution model (virtual-bond model) is used together with knowledge-based potentials. The calculated mean-square fluctuations in alpha carbons are in good agreement with crystallographic temperature factors. The conformations generated around the native state are analyzed by time-dependent orientational and conformational correlation functions to study the internal motions of RNase at different time windows. A correlation between the free-energy changes for native-state hydrogen exchange (HX) and the extent of the autocorrelation in the rotations of the virtual bonds at long times has been observed. Cross-correlations between the rotations of the bonds, which are near-neighbor in the sequence, are effective in all time windows and help the secondary structures to preserve their kinetic stability. On the other hand, the existence of cross-correlations at long times help the tertiary contacts be maintained. The order parameter of NH bond vector for each residue has been calculated and compared with 15N-NMR relaxation measurements.     

Interactions of hydrophobic peptides with lipid bilayers: Monte Carlo simulations with M2delta

We introduce here a novel Monte Carlo simulation method for studying the interactions of hydrophobic peptides with lipid membranes. Each of the peptide's amino acids is represented as two interaction sites: one corresponding to the backbone alpha-carbon and the other to the side chain, with the membrane represented as a hydrophobic profile. Peptide conformations and locations in the membrane and changes in the membrane width are sampled using the Metropolis criterion, taking into account the underlying energetics. Using this method we investigate the interactions between the hydrophobic peptide M2delta and a model membrane. The simulations show that starting from an extended conformation in the aqueous phase, the peptide first adsorbs onto the membrane surface, while acquiring an ordered helical structure. This is followed by formation of a helical-hairpin and insertion into the membrane. The observed path is in agreement with contemporary understanding of peptide insertion into biological membranes. Two stable orientations of membrane-associated M2delta were obtained: transmembrane (TM) and surface, and the value of the water-to-membrane transfer free energy of each of them is in agreement with calculations and measurements on similar cases. M2delta is most stable in the TM orientation, where it assumes a helical conformation with a tilt of 14 degrees between the helix principal axis and the membrane normal. The peptide conformation agrees well with the experimental data; average root-mean-square deviations of 2.1 A compared to nuclear magnetic resonance structures obtained in detergent micelles and supported lipid bilayers. The average orientation of the peptide in the membrane in the most stable configurations reported here, and in particular the value of the tilt angle, are in excellent agreement with the ones calculated using the continuum-solvent model and the ones observed in the nuclear magnetic resonance studies. This suggests that the method may be used to predict the three-dimensional structure of TM peptides.     

Allosteric Dynamic Control of Binding

Proteins have a highly dynamic nature and there is a complex interrelation between their structural dynamics and binding behavior. By assuming various conformational ensembles, they perform both local and global fluctuations to interact with other proteins in a dynamic infrastructure adapted to functional motion. Here, we show that there is a significant association between allosteric mutations, which lead to high-binding-affinity changes, and the hinge positions of global modes, as revealed by a large-scale statistical analysis of data in the Structural Kinetic and Energetic Database of Mutant Protein Interactions (SKEMPI). We further examined the mechanism of allosteric dynamics by conducting studies on human growth hormone (hGH) and pyrin domain (PYD), and the results show how mutations at the hinge regions could allosterically affect the binding-site dynamics or induce alternative binding modes by modifying the ensemble of accessible conformations. The long-range dissemination of perturbations in local chemistry or physical interactions through an impact on global dynamics can restore the allosteric dynamics. Our findings suggest a mechanism for the coupling of structural dynamics to the modulation of protein interactions, which remains a critical phenomenon in understanding the effect of mutations that lead to functional changes in proteins.     

Efficient regeneration system from wheat leaf base segments

Efficient plant regeneration system from leaf base segments of wheat (Triticum aestivum L.) was developed. The factors affecting the callus formation and regeneration capacity of leaf segments of two genotypes; Bobwhite and Pavon 76, were investigated. The highest number of somatic embryos (SE) was obtained on Murashige and Skoog medium supplemented with 2 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 1 mg dm⁻³ naphthalenacetic acid (14.7 SE per segment). Highest frequency of embryogenic callus (96 %) and somatic embryo formation (24.3 SE per segment) were achieved in the first segments. The highest plantlet regeneration was obtained after transfer of embryogenic calli to regeneration medium supplemented with 1 mg dm⁻³ kinetin (6.3 plantlets per segment).     

A model-structure of a periplasm-facing state of the NhaA antiporter suggests the molecular underpinnings of pH-induced conformational changes

The Escherichia coli NhaA antiporter couples the transport of H(+) and Na(+) (or Li(+)) ions to maintain the proper pH range and Na(+) concentration in cells. A crystal structure of NhaA, solved at pH 4, comprises 12 transmembrane helices (TMs), arranged in two domains, with a large cytoplasm-facing funnel and a smaller periplasm-facing funnel. NhaA undergoes conformational changes, e.g. after pH elevation to alkaline ranges, and we used two computational approaches to explore them. On the basis of pseudo-symmetric features of the crystal structure, we predicted the structural architecture of an alternate, periplasm-facing state. In contrast to the crystal structure, the model presents a closed cytoplasmic funnel, and a periplasmic funnel of greater volume. To examine the transporter functional direction of motion, we conducted elastic network analysis of the crystal structure and detected two main normal modes of motion. Notably, both analyses predicted similar trends of conformational changes, consisting of an overall rotational motion of the two domains around a putative symmetry axis at the funnel centers, perpendicular to the membrane plane. This motion, along with conformational changes within specific helices, resulted in closure at the cytoplasmic end and opening at the periplasmic end. Cross-linking experiments, performed between segments on opposite sides of the cytoplasmic funnel, revealed pH-dependent interactions consistent with the proposed conformational changes. We suggest that the model-structure and predicted motion represent alkaline pH-induced conformational changes, mediated by a cluster of evolutionarily conserved, titratable residues, at the cytoplasmic ends of TMs II, V, and IX.     

Conformational transition pathways explored by Monte Carlo simulation integrated with collective modes

Conformational transitions between open/closed or free/bound states in proteins possess functional importance. We propose a technique in which the collective modes obtained from an anisotropic network model (ANM) are used in conjunction with a Monte Carlo (MC) simulation approach, to investigate conformational transition pathways and pathway intermediates. The ANM-MC technique is applied to adenylate kinase (AK) and hemoglobin. The iterative method, in which normal modes are continuously updated during the simulation, proves successful in accomplishing the transition between open-closed conformations of AK and tense-relaxed forms of hemoglobin (C^α− root mean square deviations between two end structures of 7.13 Å and 3.55 Å, respectively). Target conformations are reached by root mean-square deviations of 2.27 Å and 1.90 Å for AK and hemoglobin, respectively. The intermediate conformations overlap with crystal structures from the AK family within a 3.0-Å root mean-square deviation. In the case of hemoglobin, the transition of tense-to-relaxed passes through the relaxed state. In both cases, the lowest-frequency modes are effective during transitions. The targeted Monte Carlo approach is used without the application of collective modes. Both the ANM-MC and targeted Monte Carlo techniques can explore sequences of events in transition pathways with an efficient yet realistic conformational search.