A practical Java tool for small-molecule compound appraisal

The increased use of small-molecule compound screening by new users from a variety of different academic backgrounds calls for adequate software to administer, appraise, analyse and exchange information obtained from screening experiments. While software and spreadsheet solutions exist, there is a need for software that can be easily deployed and is convenient to use.The Java application cApp addresses this need and aids in the handling and storage of information on small-molecule compounds. The software is intended for the appraisal of compounds with respect to their physico-chemical properties, analysis in relation to adherence to likeness rules as well as recognition of pan-assay interference components and cross-linking with identical entries in the PubChem Compound Database. Results are displayed in a tabular form in a graphical interface, but can also be written in an HTML or PDF format. The output of data in ASCII format allows for further processing of data using other suitable programs. Other features include similarity searches against user-provided compound libraries and the PubChem Compound Database, as well as compound clustering based on a MaxMin algorithm.cApp is a personal database solution for small-molecule compounds which can handle all major chemical formats. Being a standalone software, it has no other dependency than the Java virtual machine and is thus conveniently deployed. It streamlines the analysis of molecules with respect to physico-chemical properties and drug discovery criteria; cApp is distributed under the GNU Affero General Public License version 3 and available from http://www.structuralchemistry.org/pcsb/. To download cApp, users will be asked for their name, institution and email address. A detailed manual can also be downloaded from this site, and online tutorials are available at http://www.structuralchemistry.org/pcsb/capp.php

Expression variation in lysosomal storage disorder genes

Metachromatic leukodystrophy (MLD) and Gaucher disease (GD) are caused by a deficiency of arylsulphatase A (ASA) and b-glucocerebrosidase (GBA), respectively. They are lysosomal storage disorders with a heterogeneous clinical spectrum encompassing visceral, skeletal and neurologic involvement resulting in high morbidity and mortality. The overall aim of this study is to elucidate the genetic component/s of high ASA and GBA enzyme activity in normal healthy individuals with the ultimate goal of using this information to produce greater protein activity from a recombinant protein. A wide variation in ASA and GBA enzyme activity levels has been observed in the normal population. The first objective of this project was to identify and characterise single nucleotide polymorphisms (SNPs) in the arylsulphatase A (ARSA) and glucocerebrosidase (GBA) genes that are responsible for determining the levels of expressed enzyme activity in the normal population. The second objective was to assess the contribution of transcriptional regulation and TCP80 mediated translational control to normal enzyme variation. TCP80, a translational control protein that interacts with the GBA coding region, is a splice variant of the interleukin binding factor 3 (ILF3) gene. Ten samples from individuals with high ASA activity and twenty samples from individuals with high GBA activity were screened for polymorphisms via denaturing high pressure liquid chromatography (dHPLC) and sequencing. The frequency of these polymorphisms in the normal population was determined using dot-blot hybridisation. Fifteen ARSA polymorphisms (4 promoter, 5 coding, 5 intronic and 1 poly(A) signal) and two GBA polymorphisms (1 intronic and 1 in 3¢-UTR) were identified. Two low frequency ASA polymorphisms (2723A > G, W193C) were found to be correlated with low activity, while another low frequency ASA polymorphism (1101+123C > T) was found to be correlated with high activity in a population of 113 individuals. Real time PCR was used to measure mRNA levels of GBA, ASA and LF3 along with enzyme activity levels of GBA and ASA in two cell types (leucocytes and skin fibroblasts) from four healthy individuals and seven cell lines (HL60, THP1, Huh7, U118, SW1353, Hep G2, and B-cells). Transcriptional control was evident for all three genes with GBA mRNA levels varying over 30 fold, ASA mRNA levels varying over seven fold and ILF3 levels varying more than 24 fold. The 5¢-flanking region of GBA was investigated for the cis-elements responsible for tissue-specific expression. However, it was not possible to demonstrate that the cis-element region was influencing GBA expression. Translational efficiency was measured using the magnitude of the mRNA:enzyme activity ratio as an indicator. GBA translational inefficiency was most pronounced in B cells which require four times more mRNA molecules than hepatocytes (Hep G2) and over 25 times more mRNA molecules than chondrocytes (SW1353) to produce one unit of GBA enzyme activity. Except in B-cells, GBA translational efficiency appears to increase as ILF3 mRNA levels decrease. The tissue-specific variation observed in the protein levels of the ILF3 splice variants, TCP80 and DRBP76, may play a role. The correlation of several low frequency SNPs with low ASA enzyme activity or high ASA activity indicates a role in determining the distribution of enzyme activity levels in the normal population. However, there do not appear to be any common high activity polymorphisms. Knowledge of the exact mechanisms responsible for the observed transcriptional and translational control of these lysosomal genes will greatly enhance the understanding of genotype-phenotype correlation and the contribution of genetic variants to natural variation

Comparison of VILIP-1 and VILIP-3 binding to phospholipid monolayers

International audienceThe neuronal calcium sensor proteins Visinin-like Proteins 1 (VILIP-1) and 3 (VILIP-3) are effectors of guanylyl cyclase and acetyl choline receptors, and transduce calcium signals in the brain. The “calcium-myristoyl” switch, which involves a post-translationally added myristoyl moiety and calcium binding, is thought to regulate their membrane binding capacity and therefore, play a critical role in their mechanism of action. In the present study, we investigated the effect of membrane composition and solvent conditions on the membrane binding mechanisms of both VILIPs using lipid monolayers at the air/buffer interface. Results based on comparison of the adsorption kinetics of the myristoylated and non-myristoylated proteins confirm the pivotal role of calcium and the exposed myristol moiety for sustaining the membrane-bound state of both VILIPs. However, we also observed binding of both VILIP proteins in the absence of calcium and/or myristoyl conjugation. We propose a two-stage membrane binding mechanism for VILIP-1 and VILIP-3 whereby the proteins are initially attracted to the membrane surface by electrostatic interactions and possibly by specific interactions with highly negatively charged lipids head groups. The extrusion of the conjugated myristoyl group, and the subsequent anchoring in the membrane constitutes the second stage of the binding mechanism, and ensures the sustained membrane-bound form of these proteins

Specific interaction to PIP2 increase the kinetic rate of membrane binding of VILIPs, a subfamily of Neuronal Calcium sensors (NCS) proteins.

International audienceVIsinin-LIke Proteins (VILIPs) are a subfamily of the Neuronal Calcium Sensor (NCS) proteins, which possess both N-myristoylation and EF-hand motifs allowing for a putative 'calcium-myristoyl switch' regulation mechanism. It has previously been established that myristoyl conjugation increases the affinity of proteins for membranes, but, in many cases, a second feature such as a cluster of positively-charged residues is needed for stable membrane binding. The interaction of two members of this family, VILIP-1 and VILIP-3, with Langmuir monolayers as membrane models has been investigated in order to study the effects of both myristoylation and the highly basic region containing conserved poly-lysine residues on membrane association kinetics and binding properties. Results show that in the presence of calcium, N-myristoylation significantly increases the kinetic rate of VILIP adsorption to the membrane. Additionally, the proteins bind to negatively charged phospholipids independently of the conjugated myristate moiety. Besides the regulatory effect of calcium on the rate of binding presumably due to exposure of the myristoyl moiety ascribed to their putative 'calcium-myristoyl switch', VILIP-1 and -3 also engage specific interactions with biomimetic membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2). The presence of PIP2 increases the membrane association rates of both VILIPs. Taken together, these results show the major kinetic role of N-myristoylation for membrane binding, and highlight the critical role of specific phosphoinositide interactions for membrane association of members of the VILIP family

Specific interaction to PIP2 increase the kinetic rate of membrane binding of VILIPs, a subfamily of Neuronal Calcium sensors (NCS) proteins.

International audienceVIsinin-LIke Proteins (VILIPs) are a subfamily of the Neuronal Calcium Sensor (NCS) proteins, which possess both N-myristoylation and EF-hand motifs allowing for a putative 'calcium-myristoyl switch' regulation mechanism. It has previously been established that myristoyl conjugation increases the affinity of proteins for membranes, but, in many cases, a second feature such as a cluster of positively-charged residues is needed for stable membrane binding. The interaction of two members of this family, VILIP-1 and VILIP-3, with Langmuir monolayers as membrane models has been investigated in order to study the effects of both myristoylation and the highly basic region containing conserved poly-lysine residues on membrane association kinetics and binding properties. Results show that in the presence of calcium, N-myristoylation significantly increases the kinetic rate of VILIP adsorption to the membrane. Additionally, the proteins bind to negatively charged phospholipids independently of the conjugated myristate moiety. Besides the regulatory effect of calcium on the rate of binding presumably due to exposure of the myristoyl moiety ascribed to their putative 'calcium-myristoyl switch', VILIP-1 and -3 also engage specific interactions with biomimetic membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2). The presence of PIP2 increases the membrane association rates of both VILIPs. Taken together, these results show the major kinetic role of N-myristoylation for membrane binding, and highlight the critical role of specific phosphoinositide interactions for membrane association of members of the VILIP family

Exploring the molecular mechanisms of parasite-host interactions with a view towards new therapeutics and vaccines

Despite the massive disease burden worldwide caused by parasitic nematodes and other infectious pathogens, the molecular basis of many infectious diseases caused by these pathogens has been unduly neglected for a long time. Therefore, accelerated progress towards novel therapeutics, and ultimately control of such infectious diseases, is of crucial importance. Capitalising on the wealth of data becoming available from proteomic and genomic studies, new protein targets at the pathogen-host interface can be identified and subjected to protein-based explorations of the molecular basis of pathogen-host interactions. By combining the use of systems and structural biology methodologies, insights into the structural and molecular mechanisms of these interactions can assist in the development of therapeutics and/or vaccines. This brief review examines two different proteins from the body wall of blood flukes - annexins and the stress-induced phosphoprotein 1 - both of which are presently interesting targets for the development of therapeutics

Alpha-1 giardin is an annexin with highly unusual calcium-regulated mechanisms

Alpha-giardins constitute the annexin proteome (group E annexins) in the intestinal protozoan parasite Giardia and, as such, represent the evolutionary oldest eukaryotic annexins. The dominance of alpha-giardins in the cytoskeleton of Giardia with its greatly reduced actin content emphasises the importance of the alpha-giardins for the structural integrity of the parasite, which is particularly critical in the transformation stage between cyst and trophozoite. In this study, we report the crystal structures of the apo- and calcium-bound forms of α1-giardin, a protein localised to the plasma membrane of Giardia trophozoites that has recently been identified as a vaccine target. The calcium-bound crystal structure of α1-giardin revealed the presence of a type III site in the first repeat as known from other annexin structures, as well as a novel calcium binding site situated between repeats I and IV. By means of comparison, the crystal structures of three different alpha-giardins known to date indicate that these proteins engage different calcium coordination schemes, among each other, as well as compared to annexins of groups A-D. Evaluation of the calcium-dependent binding to acidic phosphoplipid membranes revealed that this process is not only mediated but also regulated by the environmental calcium concentration. Uniquely within the large family of annexins, α1-giardin disengages from the phospholipid membrane at high calcium concentrations possibly due to formation of a dimeric species. The observed behaviour is in line with changing calcium levels experienced by the parasite during excystation and may thus provide first insights into the molecular mechanisms underpinning the transformation and survival of the parasite in the host

Adsorption of VILIPs to monolayers of different lipid compositions under varying buffer conditions.

<p>Histograms of averaged Δ∏ for three different lipid monolayers in the absence or presence of calcium: myr-VILIP-1 (blue), VILIP-1 (red), myr-VILIP-3 (green), and VILIP-3 (purple). The phospholipid monolayers are composed of A) DMPS/DMPC (at molar ratio 3:1), B) DOPS/DOPC (3:1), and C) DOPS/DOPC (1:3) as indicated. The subphase consisted of 10 mM Hepes at pH 7.4, 150 mM NaCl, 2 mM CaCl₂ or 2 mM EDTA. Conditions in the presence or absence of calcium are shown in full and striped color, respectively. The error bars indicate the standard deviation of between two and five technical repeats.</p

Adsorption of myristoylated VILIPs and BSA to phospholipid monolayers.

<p>Surface pressure (Δ∏) change vs. time after injection of myr-VILIP-1, myr-VILIP-3 or Bovine Serum Albumine (BSA) into subphase beneath phospholipid monolayers of DOPS/DOPC (at molar ratio 3:1) compressed at an initial surface pressure (∏i) of about 16 mN/m. The final concentration of myr-VILIP-1 (red,○?), myr-VILIP-3 (blue, ⋄), and BSA (black, □) was 30 nM. The subphase consisted of 10 mM HEPES at pH 7.4, 150 mM NaCl, 2 mM CaCl2. After proteins injection (indicated by an arrow), the surface pressure increased until saturation at the equilibrium adsorption pressure (∏_e). Note that the dotted line is the tangent to the curve at initial time of protein injection and corresponds to the initial rate of binding to lipid monolayer.</p