Quantifying RNA–protein interactions in situ using modified-MTRIPs and proximity ligation

The stabilization, translation and degradation of RNA are regulated by interactions between trans-acting factors, such as microRNA and RNA-binding proteins (RBP). In order to investigate the relationships between these events and their significance, a method that detects the localization of these interactions within a single cell, as well as their variability across a cell population, is needed. To visualize and quantify RNA–protein interactions in situ, we developed a proximity ligation assay (PLA) that combined peptide-modified, multiply-labelled tetravalent RNA imaging probes (MTRIPs), targeted to sequences near RBP binding sites, with proximity ligation and rolling circle amplification (RCA). Using this method, we detected and quantified, with single-interaction sensitivity, the localization and frequency of interactions of the human respiratory syncytial virus (hRSV) nucleocapsid protein (N) with viral genomic RNA (gRNA). We also described the effects of actinomycin D (actD) on the interactions of HuR with β-actin mRNA and with poly(A)+ mRNA at both native and increased HuR expression levels.     

An integrated in silico approach to understand protein–protein interactions: human meprin-β with fetuin-A

Abstract

Human meprin-β, a zinc metalloprotease belonging to the astacin family, have been found to be associated with many pathological conditions like inflammatory bowel disease, fibrosis and neurodegenerative disease. The inhibition of meprin-β by various inhibitors, both macromolecular and small molecules, is crucial in the control of several diseases. Human fetuin-A, a negative acute phase protein involved in inflammatory disease, has recently been identified as an endogenous inhibitor for meprin-β. In this computational study, an integrated in silico approach was performed using existing structural information of meprin-β coupled with ab initio modelling of human fetuin-A to predict a rational model of the complex through protein–protein docking. Further, the models were optimized and validated to generate an ensemble of conformations through extensive molecular dynamics simulation. Virtual alanine scanning mutagenesis was explored to identify hotspot residues on both proteins significant for protein–protein interaction (PPI). The results of the study provide structural insight into PPI between meprin-β and fetuin-A which can be useful in designing molecules to modulate meprin-β activity.

Communicated by Ramaswamy H. Sarma     

Application of a novel in silico high-throughput screen to identify selective inhibitors for protein–protein interactions

Increasing numbers of target protein structures available for computational studies makes the structure-based screening paradigm more attractive for initial hit indentification. We have developed a novel in silico screening methodology incorporating Molecular Mechanics (MM)/implicit solvent methods to evaluate binding free energies and applied this technology to the identification of inhibitors of the TLR4/MD-2 interaction.     

Dual metabolomics: A novel approach to understanding plant–pathogen interactions

One of the most well-characterised plant pathogenic interactions involves Arabidopsis thaliana and the bacteria Pseudomonas syringae pathovar tomato (Pst). The standard Pst inoculation procedure involves infiltration of large populations of bacteria into plant leaves which means that metabolite changes cannot be readily assigned to the host or pathogen. A plant cell–pathogen co-culture based approach has been developed where the plant and pathogen cells are separated after 12 h of co-culture via differential filtering and centrifugation. Fourier transform infrared (FT-IR) spectroscopy was employed to assess the intracellular metabolomes (metabolic fingerprints) of both host and pathogen and their extruded (extracellular) metabolites (metabolic footprints) under conditions relevant to disease and resistance. We propose that this system will enable the metabolomic profiling of the separated host and pathogen (i.e. ‘dual metabolomics’) and will facilitate the modelling of reciprocal responses.     

A luciferase complementation assay system using transferable mouse artificial chromosomes to monitor protein–protein interactions mediated by G protein-coupled receptors

G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors that interact with the β-arrestin family, particularly β-arrestin 1 (ARRB1). GPCRs interact with 33% of small molecule drugs. Ligand screening is promising for drug discovery concerning GPCR-related diseases. Luciferase complementation assay (LCA) enables detection of protein–protein complementation via bioluminescence following complementation of N- and C-terminal luciferase fragments (NEluc and CEluc) fused to target proteins, but it is necessary to co-express the two genes. Here, we developed LCAs with mouse artificial chromosomes (MACs) that have unique characteristics such as stable maintenance and a substantial insert-carrying capacity. First, an NEluc-ARRB1 was inserted into MAC4 by Cre-loxP recombination in CHO cells, named ARRB1-MAC4. Second, a parathyroid hormone receptor 2 (PTHR2)-CEluc or prostaglandin EP4 receptor (hEP4)-CEluc were inserted into ARRB1-MAC4, named ARRB1-PTHR2-MAC4 and ARRB1-hEP4-MAC4, respectively, via the sequential integration of multiple vectors (SIM) system. Each MAC was transferred into HEK293 cells by microcell-mediated chromosome transfer (MMCT). LCAs using the established HEK293 cell lines resulted in 35,000 photon counts upon somatostatin stimulation for ARRB1-MAC4 with transient transfection of the somatostatin receptor 2 (SSTR2) expression vector, 1800 photon counts upon parathyroid hormone stimulation for ARRB1-PTHR2-MAC4, and 35,000 photon counts upon prostaglandin E2 stimulation for ARRB1-hEP4-MAC4. These MACs were maintained independently from host chromosomes in CHO and HEK293 cells. HEK293 cells containing ARRB1-PTHR2-MAC4 showed a stable reaction for long-term. Thus, the combination of gene loading by the SIM system into a MAC and an LCA targeting GPCRs provides a novel and useful platform to discover drugs for GPCR-related diseases.     

Topology and weights in a protein domain interaction network – a novel way to predict protein interactions

Background

While the analysis of unweighted biological webs as diverse as genetic, protein and metabolic networks allowed spectacular insights in the inner workings of a cell, biological networks are not only determined by their static grid of links. In fact, we expect that the heterogeneity in the utilization of connections has a major impact on the organization of cellular activities as well.

Results

We consider a web of interactions between protein domains of the Protein Family database (PFAM), which are weighted by a probability score. We apply metrics that combine the static layout and the weights of the underlying interactions. We observe that unweighted measures as well as their weighted counterparts largely share the same trends in the underlying domain interaction network. However, we only find weak signals that weights and the static grid of interactions are connected entities. Therefore assuming that a protein interaction is governed by a single domain interaction, we observe strong and significant correlations of the highest scoring domain interaction and the confidence of protein interactions in the underlying interactions of yeast and fly.Modeling an interaction between proteins if we find a high scoring protein domain interaction we obtain 1, 428 protein interactions among 361 proteins in the human malaria parasite Plasmodium falciparum. Assessing their quality by a logistic regression method we observe that increasing confidence of predicted interactions is accompanied by high scoring domain interactions and elevated levels of functional similarity and evolutionary conservation.

Conclusion

Our results indicate that probability scores are randomly distributed, allowing to treat static grid and weights of domain interactions as separate entities. In particular, these finding confirms earlier observations that a protein interaction is a matter of a single interaction event on domain level. As an immediate application, we show a simple way to predict potential protein interactions by utilizing expectation scores of single domain interactions.

     

Intracellular antibody capture: A molecular biology approach to inhibitors of protein–protein interactions

Many proteins of interest in basic biology, translational research studies and for clinical targeting in diseases reside inside the cell and function by interacting with other macromolecules. Protein complexes control basic processes such as development and cell division but also abnormal cell growth when mutations occur such as found in cancer. Interfering with protein–protein interactions is an important aspiration in both basic and disease biology but small molecule inhibitors have been difficult and expensive to isolate. Recently, we have adapted molecular biology techniques to develop a simple set of protocols for isolation of high affinity antibody fragments (in the form of single VH domains) that function within the reducing environment of higher organism cells and can bind to their target molecules. The method called Intracellular Antibody Capture (IAC) has been used to develop inhibitory anti-RAS and anti-LMO2 single domains that have been used for target validation of these antigens in pre-clinical cancer models and illustrate the efficacy of the IAC approach to generation of drug surrogates. Future use of inhibitory VH antibody fragments as drugs in their own right (we term these macrodrugs to distinguish them from small molecule drugs) requires their delivery to target cells in vivo but they can also be templates for small molecule drug development that emulate the binding sites of the antibody fragments. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

CCN2/decorin interactions: a novel approach to combating fibrosis?

CCN2 (connective tissue growth factor, CTGF), a member of the CCN family is overexpressed in fibrotic disease and is essential for the development of experimental fibrosis. Drugs targeting CCN2 action may therefore prove to be useful anti-fibrotic approaches. CCN2 acts via integrins and heparan sulfate-containing proteoglycans (HSPGs). In a recent study, Vial and colleagues (2011) show that decorin can bind CCN2. A peptide corresponding to the leucine rich repeats peptide 12 region of decorin can neutralize CCN2-mediated activity on C2C12 cells in vitro. Thus it is conceivable that this peptide could be used in the future as a novel antifibrotic approach.     

Mimicking direct protein–protein and solvent-mediated interactions in the CDP-methylerythritol kinase homodimer: a pharmacophore-directed virtual screening approach

The 2C-methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl pyrophosphate and its isomer dimethylallyl pyrophosphate, which are the precursors of isoprenoids, is present in plants, in the malaria parasite Plasmodium falciparum and in most eubacteria, including pathogenic agents. However, the MEP pathway is absent from fungi and animals, which have exclusively the mevalonic acid pathway. Given the characteristics of the MEP pathway, its enzymes represent potential targets for the generation of selective antibacterial, antimalarial and herbicidal molecules. We have focussed on the enzyme 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase (CMK), which catalyses the fourth reaction step of the MEP pathway. A molecular dynamics simulation was carried out on the CMK dimer complex, and protein–protein interactions analysed, considering also water-mediated interactions between monomers. In order to find small molecules that bind to CMK and disrupt dimer formation, interactions observed in the dynamics trajectory were used to model a pharmacophore used in database searches. Using an intensity-fading matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry approach, one compound was found to interact with CMK. The data presented here indicate that a virtual screening approach can be used to identify candidate molecules that disrupt the CMK–CMK complex. This strategy can contribute to speeding up the discovery of new antimalarial, antibacterial, and herbicidal compounds.     

Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein–protein interactions

We developed “fractionation profiling,” a method for rapid proteomic analysis of membrane vesicles and protein particles. The approach combines quantitative proteomics with subcellular fractionation to generate signature protein abundance distribution profiles. Functionally associated groups of proteins are revealed through cluster analysis. To validate the method, we first profiled >3500 proteins from HeLa cells and identified known clathrin-coated vesicle proteins with >90% accuracy. We then profiled >2400 proteins from Drosophila S2 cells, and we report the first comprehensive insect clathrin-coated vesicle proteome. Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes. We show that it also allows the detailed compositional characterization of complexes, including the delineation of subcomplexes and subunit stoichiometry. Our predictions are presented in an interactive database. Fractionation profiling is a universal method for defining the clathrin-coated vesicle proteome and may be adapted for the analysis of other types of vesicles and particles. In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps.     

Pyruvate kinase M2 in chronic inflammations: a potpourri of crucial protein–protein interactions

Cell Biology and Toxicology - Chronic inflammation (CI) is a primary contributing factor involved in multiple diseases like cancer, stroke, diabetes, Alzheimer’s disease, allergy, asthma,...     

‘RNA walk’ a novel approach to study RNA–RNA interactions between a small RNA and its target

In this study we describe a novel method to investigate the RNA–RNA interactions between a small RNA and its target that we termed ‘RNA walk’. The method is based on UV-induced AMT cross-linking in vivo followed by affinity selection of the hybrid molecules and mapping the intermolecular adducts by RT–PCR or real-time PCR. Domains carrying the cross-linked adducts fail to efficiently amplify by PCR compared with non-cross-linked domains. This method was calibrated and used to study the interaction between a special tRNA-like molecule (sRNA-85) that is part of the trypanosome signal recognition particle (SRP) complex and the ribosome. Four contact sites between sRNA-85 and rRNA were identified by ‘RNA walk’ and were further fine-mapped by primer extension. Two of the contact sites are expected; one contact site mimics the interaction of the mammalian Alu domain of SRP with the ribosome and the other contact sites include a canonical tRNA interaction. The two other cross-linked sites could not be predicted. We propose that ‘RNA walk, is a generic method to map target RNA small RNAs interactions in vivo.     

Hemoglobin interactions with αB crystallin: a direct test of sensitivity to protein instability

As a small stress response protein, human αB crystallin, detects protein destabilization that can alter structure and function to cause self assembly of fibrils or aggregates in diseases of aging. The sensitivity of αB crystallin to protein instability was evaluated using wild-type hemoglobin (HbA) and hemoglobin S (HbS), the glutamate-6-valine mutant that forms elongated, filamentous aggregates in sickling red blood cells. The progressive thermal unfolding and aggregation of HbA and HbS in solution at 37°C, 50°C and 55°C was measured as increased light scattering. UV circular dichroism (UVCD) was used to evaluate conformational changes in HbA and HbS with time at the selected temperatures. The changes in interactions between αB crystallin and HbA or HbS with temperature were analyzed using differential centrifugation and SDS PAGE at 37°C, 50°C and 55°C. After only 5 minutes at the selected temperatures, differences in the aggregation or conformation of HbA and HbS were not observed, but αB crystallin bound approximately 6% and 25% more HbS than HbA at 37°C, and 50°C respectively. The results confirmed (a) the remarkable sensitivity of αB crystallin to structural instabilities at the very earliest stages of thermal unfolding and (b) an ability to distinguish the self assembling mutant form of HbS from the wild type HbA in solution.     

The importance of protein–protein interactions for optimising oxygen activity in photosystem II: reconstitution with a recombinant thioredoxin—manganese stabilising protein

In this paper we describe how photosystem II (PSII) from higher plants, which have been depleted, of the extrinsic proteins can be reconstituted with a chimeric fusion protein comprising thioredoxin from Escherichia coli and the manganese stabilising protein from Thermosynechococcus elongatus. Surprisingly, even though E. coli thioredoxin is completely unrelated to PSII, the fusion protein restores higher rates of activity upon rebinding to PSII than either the native spinach MSP, or T. elongatus MSP. PSII reconstituted with the fusion protein also has a lower requirement for calcium than PSII with the small extrinsic proteins removed, or PSII reconstituted with spinach or T. elongatus MSP. The MSP portion of the fusion protein is less thermally stable compared to isolated MSP from T. elongatus, which could be the key to its superior activation capability through greater flexibility. This work reveals the importance of protein–protein interactions in the water splitting activity of PSII and suggests that conformational configurations, which increase flexibility in MSP, are essential to its function, even when these are induced by an unrelated protein.     

NLDB: a database for 3D protein–ligand interactions in enzymatic reactions

NLDB (Natural Ligand DataBase; URL: http://nldb.hgc.jp) is a database of automatically collected and predicted 3D protein–ligand interactions for the enzymatic reactions of metabolic pathways registered in KEGG. Structural information about these reactions is important for studying the molecular functions of enzymes, however a large number of the 3D interactions are still unknown. Therefore, in order to complement such missing information, we predicted protein–ligand complex structures, and constructed a database of the 3D interactions in reactions. NLDB provides three different types of data resources; the natural complexes are experimentally determined protein–ligand complex structures in PDB, the analog complexes are predicted based on known protein structures in a complex with a similar ligand, and the ab initio complexes are predicted by docking simulations. In addition, NLDB shows the known polymorphisms found in human genome on protein structures. The database has a flexible search function based on various types of keywords, and an enrichment analysis function based on a set of KEGG compound IDs. NLDB will be a valuable resource for experimental biologists studying protein–ligand interactions in specific reactions, and for theoretical researchers wishing to undertake more precise simulations of interactions.     

The subcellular distribution of rat liver l-alanine–glyoxylate aminotransferase in relation to a pathway for glucose formation involving glyoxylate

1. The distribution of l-alanine-glyoxylate aminotransferase activity between subcellular fractions prepared from rat liver homogenates was investigated. The greater part of the homogenate activity (about 80%) was recovered in the ;total-particles' fraction sedimented by high-speed centrifugation and the remainder in the cytosol fraction. 2. Subfractionation of the particles by differential sedimentation and on sucrose density gradients revealed a specific association between the aminotransferase and the mitochondrial enzymes glutamate dehydrogenase and rhodanese. 3. The aminotransferase activities in the cytosol and the mitochondria are due to isoenzymes. The solubilized mitochondrial enzyme has a pH optimum of 8.6, an apparent K(m) of 0.24mm with respect to glyoxylate and is inhibited by glyoxylate at concentrations above 5mm. The cytosol aminotransferase shows no distinct pH optimum (over the range 7.0-9.0) and has an apparent K(m) of 1.11mm with respect to glyoxylate; there is no evidence of inhibition by glyoxylate. 4. The mitochondrial location of the bulk of the rat liver l-alanine-glyoxylate aminotransferase activity is discussed in relation to a pathway for gluconeogenesis involving glyoxylate.     

Synthesis and evaluation of a library of 2,5-bisdiamino-benzoquinone derivatives as probes to modulate protein–protein interactions in prions

A small library combining two different benzoquinone cores with seven (l) amino acid methyl esters (alanine, Nω-nitro-arginine, Nε-BOC-lysine, isoleucine, methionine, phenylalanine and tryptophan) was prepared and tested for prion replication inhibition in ScGT1 cells. The most potent hit, 6a, displayed an EC₅₀ value of 0.87 μM, which is very close to that of quinacrine (0.4 μM).     

Fractal Dimension in Butterflies’ Wings: a novel approach to understanding wing patterns ?

The geometrical complexity in the wings of several, taxonomically different butterflies, is analyzed in terms of their fractal dimension. Preliminary results provide some evidence on important questions about the (dis)similarity of the wing patterns in terms of their fractal dimension. The analysis is restricted to two groups which are widely used in the literature as typical examples of mimicry, and a small number of unrelated species, thus implying the consideration of only a fraction of the wing pattern diversity. The members of the first mimicry ring, composed by the species Danaus plexippus (better known as the monarch butterfly), and the two subspecies Basilarchia archippus obsoleta (or northern viceroy) and Basilarchia archippus hoffmanni (or tropical viceroy), are found to have a very similar value for the fractal dimension of their wing patterns, even though they do not look very similar at first sight. It is also found that the female of another species (Neophasia terlootii), which looks similar to the members of the previous group, does not share the same feature, while the Lycorea ilione albescens does share it. For the members of the second group of mimicry related butterflies, the Greta nero nero and the Hypoleria cassotis, it is shown that they also have very close values for the fractal dimension of their wing patterns. Finally, it is shown that other species, which apparently have very similar wing patterns, do not have the same fractal dimension. A possible, not completely tested hypothesis is then conjectured: the formation of groups by individuals whose wing patterns have an almost equal fractal dimension may be due to the fact that they do share the same developmental raw material, and that this common feature is posteriorly modified by natural selection, possibly through predation.We sincerely acknowledge the invaluable help of Adolfo Ibarra Vázquez, senior curator of the Lepidopterous collection at the Instituto de Biología, Universidad Nacional Autónoma de México, and the comments made by two anonymous referees     

Phenotypes of gene disruptants in relation to a putative mitochondrial malate–citrate shuttle protein in citric acid-producing Aspergillus niger

The mitochondrial citrate transport protein (CTP) functions as a malate–citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger.     

Using the concept of Chou’s pseudo amino acid composition to predict protein subcellular localization: an approach by incorporating evolutionary information and von Neumann entropies

The rapidly increasing number of sequence entering into the genome databank has called for the need for developing automated methods to analyze them. Information on the subcellular localization of new found protein sequences is important for helping to reveal their functions in time and conducting the study of system biology at the cellular level. Based on the concept of Chou’s pseudo-amino acid composition, a series of useful information and techniques, such as residue conservation scores, von Neumann entropies, multi-scale energy, and weighted auto-correlation function were utilized to generate the pseudo-amino acid components for representing the protein samples. Based on such an infrastructure, a hybridization predictor was developed for identifying uncharacterized proteins among the following 12 subcellular localizations: chloroplast, cytoplasm, cytoskeleton, endoplasmic reticulum, extracell, Golgi apparatus, lysosome, mitochondria, nucleus, peroxisome, plasma membrane, and vacuole. Compared with the results reported by the previous investigators, higher success rates were obtained, suggesting that the current approach is quite promising, and may become a useful high-throughput tool in the relevant areas.