Insights into molecular interactions between CaM and its inhibitors from molecular dynamics simulations and experimental data

In order to contribute to the structural basis for rational design of calmodulin (CaM) inhibitors, we analyzed the interaction of CaM with 14 classic antagonists and two compounds that do not affect CaM, using docking and molecular dynamics (MD) simulations, and the data were compared to available experimental data. The Ca²⁺-CaM-Ligands complexes were simulated 20 ns, with CaM starting in the “open” and “closed” conformations. The analysis of the MD simulations provided insight into the conformational changes undergone by CaM during its interaction with these ligands. These simulations were used to predict the binding free energies (ΔG) from contributions ΔH and ΔS, giving useful information about CaM ligand binding thermodynamics. The ΔG predicted for the CaM’s inhibitors correlated well with available experimental data as the r² obtained was 0.76 and 0.82 for the group of xanthones. Additionally, valuable information is presented here: I) CaM has two preferred ligand binding sites in the open conformation known as site 1 and 4, II) CaM can bind ligands of diverse structural nature, III) the flexibility of CaM is reduced by the union of its ligands, leading to a reduction in the Ca²⁺-CaM entropy, IV) enthalpy dominates the molecular recognition process in the system Ca²⁺-CaM-Ligand, and V) the ligands making more extensive contact with the protein have higher affinity for Ca²⁺-CaM. Despite their limitations, docking and MD simulations in combination with experimental data continue to be excellent tools for research in pharmacology, toward a rational design of new drugs.     

Minimizing the biodiversity impact of Neotropical oil palm development

Oil palm agriculture is rapidly expanding in the Neotropics, at the expense of a range of natural and seminatural habitats. A key question is how this expansion should be managed to reduce negative impacts on biodiversity. Focusing on the Llanos of Colombia, a mixed grassland–forest system identified as a priority zone for future oil palm development, we survey communities of ants, dung beetles, birds and herpetofauna occurring in oil palm plantations and the other principal form of agriculture in the region – improved cattle pasture – together with those of surrounding natural forests. We show that oil palm plantations have similar or higher species richness across all four taxonomic groups than improved pasture. For dung beetles, species richness in oil palm was equal to that of forest, whereas the other three taxa had highest species richness in forests. Hierarchical modelling of species occupancy probabilities indicated that oil palm plantations supported a higher proportion of species characteristic of forests than did cattle pastures. Across the bird community, occupancy probabilities within oil palm were positively influenced by increasing forest cover in a surrounding 250 m radius, whereas surrounding forest cover did not strongly influence the occurrence of other taxonomic groups in oil palm. Overall, our results suggest that the conversion of existing improved pastures to oil palm has limited negative impacts on biodiversity. As such, existing cattle pastures of the Colombian Llanos could offer a key opportunity to meet governmental targets for oil palm development without incurring significant biodiversity costs. Our results also highlight the value of preserving remnant forests within these agricultural landscapes, protecting high biodiversity and exporting avian ‘spill‐over’ effects into oil palm plantations.     

African swine fever virus IAP homologue inhibits caspase activation and promotes cell survival in mammalian cells

African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis.     

Loss of neuronal Miro1 disrupts mitophagy and induces hyperactivation of the integrated stress response

Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1/Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Genetic deletion of Miro or block of Miro1 ubiquitination and subsequent degradation lead to delayed translocation of the E3 ubiquitin ligase Parkin onto damaged mitochondria and reduced mitochondrial clearance in both fibroblasts and cultured neurons. Disrupted mitophagy in vivo, upon post‐natal knockout of Miro1 in hippocampus and cortex, leads to a dramatic increase in mitofusin levels, the appearance of enlarged and hyperfused mitochondria and hyperactivation of the integrated stress response (ISR). Altogether, our results provide new insights into the central role of Miro1 in the regulation of mitochondrial homeostasis and further implicate Miro1 dysfunction in the pathogenesis of human neurodegenerative disease.     

Leishmania major metacaspase can replace yeast metacaspase in programmed cell death and has arginine-specific cysteine peptidase activity

The human protozoan parasite Leishmania major has been shown to exhibit several morphological and biochemical features characteristic of a cell death program when differentiating into infectious stages and under a variety of stress conditions. Although some caspase-like peptidase activity has been reported in dying parasites, no caspase gene is present in the genome. However, a single metacaspase gene is present in L. major whose encoded protein harbors the predicted secondary structure and the catalytic dyad histidine/cysteine described for caspases and other metacaspases identified in plants and yeast. The Saccharomyces cerevisiae metacaspase YCA1 has been implicated in the death of aging cells, cells defective in some biological functions, and cells exposed to different environmental stresses. In this study, we describe the functional heterologous complementation of a S. cerevisiae yca1 null mutant with the L. major metacaspase (LmjMCA) in cell death induced by oxidative stress. We show that LmjMCA is involved in yeast cell death, similar to YCA1, and that this function depends on its catalytic activity. LmjMCA was found to be auto-processed as occurs for caspases, however LmjMCA did not exhibit any activity with caspase substrates. In contrast and similarly to Arabidopsis thaliana metacaspases, LmjMCA was active towards substrates with arginine in the P1 position, with the activity being abolished following H147A and C202A catalytic site mutations. These results suggest that metacaspases are members of a family of peptidases with a role in cell death conserved in evolution notwithstanding possible differences in their catalytic activity.     

Direct demonstration of an acid-labile phosphoenzyme in the cycle of the sarcoplasmic reticulum Ca2(+)-dependent adenosinetriphosphatase

The Ca2(+)-dependent adenosinetriphosphatase (Ca2(+)-ATPase) from the sarcoplasmic reticulum (SR) of rat skeletal muscles is phosphorylated by inorganic phosphate (Pi) in the absence of Ca2+. The reaction can be described by the following simplified scheme: [formula: see text] where E-P is a covalent, acid-stable and ADP-insensitive phosphoenzyme, and E.Pi is a noncovalent and acid-labile complex. The reaction is Mg2(+)-dependent. Membrane fragments deposited on Millipore filters were successively perfused with two solutions, at constant flow. The effluent samples were analyzed. The perfused solutions were Ca2+ free and always contained 40% dimethylsulfoxide (DMSO), plus other reactants. Following the successive perfusion of solutions without and with [32P]Pi, 32P binding is only detected in the presence of Mg2+, indicating the formation of the phosphoenzymes (E.Pi and E-P). Following perfusions of the phosphoenzymes with 5% trichloroacetic acid, 32P release indicates the amount of the acid-labile moiety (E.Pi). After phosphorylations, the filters were washed with acid and unlabeled Pi, and the remaining radioactivity was measured to evaluate the acid-stable phosphoenzyme (E-P). The acid-labile and acid-stable phosphoenzymes amounted, respectively, 0.72 +/- 0.12, and 1.48 +/- 0.10 nmol of Pi/mg of protein ( +/- S.E., n = 5), after phosphorylations with 20 microM Pi. The results indicate: (1) The method allowed the evaluation of the acid-labile intermediate of the SR Ca2(+)-ATPase cycle. Keq = k2/k-2), in the above scheme, approaches 2.0. (2) The substrate of the phosphorylation reaction, in the presence of DMSO, is likely to be the Mg.Pi complex, since Mg2+ is necessary for step 1 in the above scheme.     

Expression of a Bacillus thuringiensisdelta-endotoxin cry1Ab gene in Bacillus subtilis and Bacillus licheniformis strains that naturally colonize the phylloplane of tomato plants (Lycopersicon esculentum,Mills)

AIMS: To introduce a cry gene into microorganisms that naturally colonize the phylloplane of tomato plants to improve the persistence of the Cry proteins for controlling a South American tomato moth (Tuta absoluta, Meyrick, 1917). METHODS AND RESULTS: A cry1Ab gene isolated from a native Bacillus thuringiensis strain (LM-466), showing a relevant activity against T. absoluta larvae, was cloned into the shuttle vector pHT315 (Arantes and Lereclus 1991). The construct was introduced by electroporation into native Bacillus subtilis and Bacillus licheniformis strains, both natural inhabitants of the tomato phylloplane. Western analysis and toxicity assays against the target larvae proved that the successful expression of the gene was accomplished in host bacteria. Recombinant toxin displayed a similar LC50 value in comparison to native donor strain LM-466. Both transformed Bacillus survived for at least 45 days on the tomato leaf surface. CONCLUSIONS: Plant-associated microorganisms that naturally colonize the phylloplane could be useful as recombinant microbial delivery systems of toxin genes of B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Modified microorganisms capable of surviving on leaf surfaces for several weeks with insecticidal activity should allow for a reduction in pesticide application.