Direct Molecular Interaction Research Articles

V1b and CRHR1 Receptor Heterodimerization Mediates Synergistic Biological Actions of Vasopressin and CRH

Vasopressin (AVP) and CRH synergistically regulate adrenocorticotropin and insulin release at the level of the pituitary and pancreas, respectively. Here, we first extended these AVP and CRH coregulation processes to the adrenal medulla. We demonstrate that costimulation of chromaffin cells by AVP and CRH simultaneously induces a catecholamine secretion exceeding the one induced by each hormone alone, thus demonstrating a net potentiation. To further elucidate the molecular mechanisms underlying this synergism, we coexpressed human V1b and CRH receptor (CRHR)1 receptor in HEK293 cells. In this heterologous system, AVP also potentiated CRH-stimulated cAMP accumulation in a dose-dependent and saturable manner. This effect was only partially mimicked by phorbol ester or inhibited by a phospholipase C inhibitor respectively. This finding suggests the existence of an new molecular mechanism, independent from second messenger cross talk. Similarly, CRH potentiated the AVP-induced inositol phosphates production. Using bioluminescence resonance energy transfer, coimmunoprecipitation, and receptor rescue experiments, we demonstrate that V1b and CRHR1 receptors assemble as heterodimers. Moreover, new pharmacological properties emerged upon receptors cotransfection. Taken together, these data strongly suggest that direct molecular interactions between V1b and CRHR1 receptors play an important role in mediating the synergistic interactions between these two receptors.

Anti-inflammatory effects of pomegranate (Punica granatum L.) husk ellagitannins in Caco-2 cells, an in vitro model of human intestine

This study aimed at evaluating the anti-inflammatory properties of a pomegranate fruit husk (PomH) polyphenolic extract, rich in punicalagin, using Caco-2 cells, an in vitro model of human intestinal epithelium. Differentiated cells in bicameral inserts were pretreated or not with a PomH extract or punicalagin, as reference, at the apical side, representing the intestinal lumen. Inflammation was then induced with a cocktail of cytokines (Il-1β, TNFα and IFNγ) and LPS. After 24 h incubation, 3 pro-inflammatory markers, i.e., interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1, were assayed both at their gene transcription (qRT-PCR) and secretion (ELISA) levels. As previously described, the pro-inflammatory cocktail significantly stimulated these 3 markers, at the gene transcript and secretion levels. In inflamed cells, a significant down-regulation of the transcription of the genes encoding IL-6 and MCP-1 was observed in the presence of the PomH extract or punicalagin, while IL-8 transcription was unaffected. Both treatments also decreased the amounts of the 3 proteins with dose-response effects, but only in the apical compartment. A lowered ELISA response was also observed when either IL-6, IL-8 or MCP-1 were mixed with punicalagin in a cell-free culture medium, indicating a direct molecular interaction. In conclusion, the punicalagin-rich PomH extract tested showed anti-inflammatory properties in the Caco-2 in vitro intestinal model. It acted both on the pro-inflammatory gene transcription and protein levels, the later phenomenon being possibly due to a direct molecular trapping. These data suggest that pomegranate husk could be an interesting natural source contributing to prevent intestinal chronic inflammation.

The major central endocannabinoid directly acts at GABA A receptors

GABA(A) receptors are the major ionotropic inhibitory neurotransmitter receptors. The endocannabinoid system is a lipid signaling network that modulates different brain functions. Here we show a direct molecular interaction between the two systems. The endocannabinoid 2-arachidonoyl glycerol (2-AG) potentiates GABA(A) receptors at low concentrations of GABA. Two residues of the receptor located in the transmembrane segment M4 of β(2) confer 2-AG binding. 2-AG acts in a superadditive fashion with the neurosteroid 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC) and modulates δ-subunit-containing receptors, known to be located extrasynaptically and to respond to neurosteroids. 2-AG inhibits motility in CB(1)/CB(2) cannabinoid receptor double-KO, whereas β(2)-KO mice show hypermotility. The identification of a functional binding site for 2-AG in the GABA(A) receptor may have far-reaching consequences for the study of locomotion and sedation.

Boswellic acids from frankincense inhibit lipopolysaccharide functionality through direct molecular interference

Lipophilic extracts of gum resins of Boswellia species (BSE) are used in folk medicine to treat various inflammatory disorders and infections. The molecular background of the beneficial pharmacological effects of such extracts is still unclear. Various boswellic acids (BAs) have been identified as abundant bioactive ingredients of BSE. Here we report the identification of defined BAs as direct inhibitors of lipopolysaccharide (LPS) functionality and LPS-induced cellular responses. In pull-down experiments, LPS could be precipitated using an immobilized BA, implying direct molecular interactions. Binding of BAs to LPS leads to an inhibition of LPS activity which was observed in vitro using a modified limulus amoebocyte lysate assay. Analysis of different BAs revealed clear structure-activity relationships with the classical β-BA as most potent derivative (IC 50 = 1.8 μM). In RAW264.7 cells, LPS-induced expression of inducible nitric oxide synthase (iNOS, EC 1.14.13.39) was selectively inhibited by those BAs that interfered with LPS activity. In contrast, interferon-γ-induced iNOS induction was not affected by BAs. We conclude that structurally defined BAs are LPS inhibiting agents and we suggest that β-BA may contribute to the observed anti-inflammatory effects of BSE during infections by suppressing LPS activity.

P53 inhibits autophagy by interacting with the human ortholog of yeast Atg17, RB1CC1/FIP200

The tumor suppressor protein p53 tonically suppresses autophagy when it is present in the cytoplasm. This effect is phylogenetically conserved from mammals to nematodes, and human p53 can inhibit autophagy in yeast, as we show here. Bioinformatic investigations of the p53 interactome in relationship to the autophagy-relevant protein network underscored the possible relevance of a direct molecular interaction between p53 and the mammalian ortholog of the essential yeast autophagy protein Atg17, namely RB1-inducible coiled-coil protein 1 (RB1CC1), also called FAK family kinase-interacting protein of 200 KDa (FIP200). Mutational analyses revealed that a single point mutation in p53 (K382R) abolished its capacity to inhibit autophagy upon transfection into p53-deficient human colon cancer or yeast cells. In conditions in which wild-type p53 co-immunoprecipitated with RB1CC1/FIP200, p53K382R failed to do so, underscoring the importance of the physical interaction between these proteins for the control of autophagy. In conclusion, p53 regulates autophagy through a direct molecular interaction with RB1CC1/FIP200, a protein that is essential for the very apical step of autophagy initiation.

Progressive Myoclonic Epilepsy-Associated Gene KCTD7 is a Regulator of Potassium Conductance in Neurons

The potassium channel tetramerization domain-containing protein 7 (KCTD7) was named after the structural homology of its predicted N-terminal broad complex, tramtrack and bric à brac/poxvirus and zinc finger domain with the T1 domain of the Kv potassium channel, but its expression profile and cellular function are still largely unknown. We have recently reported a homozygous nonsense mutation of KCTD7 in patients with a novel form of autosomal recessive progressive myoclonic epilepsy. Here, we show that KCTD7 expression hyperpolarizes the cell membrane and reduces the excitability of transfected neurons in patch clamp experiments. We found the expression of KCTD7 in the hippocampal and Purkinje cells of the murine brain, an expression profile consistent with our patients' phenotype. The effect on the plasma membrane resting potential is possibly mediated by Cullin-3, as we demonstrated direct molecular interaction of KCTD7 with Cullin-3 in co-immunoprecipitation assays. Our data link progressive myoclonic epilepsy to an inherited defect of the neuron plasma membrane's resting potential in the brain.

Adaptor protein Nck1 interacts with p120 Ras GTPase-activating protein and regulates its activity

Adaptor protein Nck1 binds a number of intracellular proteins and influences various signaling pathways. Here we show that Nck1 directly binds and activates the GTPase-activating protein of Ras (RasGAP), which is responsible for the down-regulation of Ras. The first and the third SH3 domains of Nck1 and the NH 2-terminal proline-rich sequence of RasGAP contribute most to the complex formation causing direct molecular interaction between the two proteins. Cell adhesion to the substrate is obligatory for the Nck1 and RasGAP association, as cell detachment makes RasGAP incapable of associating with Nck1. This leads to the complex dissipation, decrease of RasGAP activity and the increase of H-Ras-GTP level in the detached cells. Our findings reveal unexpected feature of adaptor protein Nck1 as the regulator of RasGAP activity.

Convergence of Two Independent Mental Disease Genes on the Protein Level: Recruitment of Dysbindin to Cell-Invasive Disrupted-In-Schizophrenia 1 Aggresomes

Both disrupted-in-schizophrenia 1 (DISC1) and dysbindin have been identified as schizophrenia candidate genes in independent genetic linkage studies. The proteins have been assigned distinct subcellular locations and functions. We investigated whether both proteins converge into a common pathway specific for schizophrenia or mental diseases. DISC1 and dysbindin were expressed as recombinant proteins with or without a fluorescent protein-tag in human or mouse neuroblastoma cells and as recombinant proteins in E. coli. Postmortem brains of patients with mental diseases from the Stanley Research Medical Institute's Consortium Collection were used to demonstrate molecular interactions in biochemically purified protein fractions. First, upon overexpression in neuroblastoma cells, DISC1 formed aggresomes that recruited homologous soluble C-terminal DISC1 fragment or heterologous dysbindin. Domains involved in binding could be mapped to DISC1 (316-597) and dysbindin (82-173), indicating a specific interaction. In addition, recruitment was demonstrated when externally added, purified DISC1 aggresomes penetrated recipient cells after coincubation. Second, a direct interaction between soluble DISC1 protein and dysbindin was demonstrated in a cell free system using E. coli-expressed proteins. Third, co-aggregation of DISC1 and dysbindin was demonstrated in postmortem brains for a subgroup of cases with chronic mental disease but not healthy control subjects. A direct interaction of soluble and insoluble DISC1 protein with dysbindin protein demonstrates convergence of so far considered independent mental disease genes by direct molecular interaction. Our findings highlight protein aggregation and recruitment as a biological mechanism in mental disease.

Minocycline Suppresses Activation of Nuclear Factor of Activated T Cells 1 (NFAT1) in Human CD4+ T Cells

Minocycline is a tetracycline family antibiotic that has anti-inflammatory and immunomodulatory properties. These properties have shown promise in the treatment of conditions such as rheumatoid arthritis, Huntington disease, and multiple sclerosis. As lymphocyte activation is involved in the pathogenesis of many of these diseases, T cells are postulated to be a primary target in minocycline therapy. Previous studies have demonstrated attenuation of CD4(+) T cell activation by minocycline, but a specific mechanism has not been elucidated. In this study, we investigated the effect of minocycline on the activity of three key transcription factors regulating CD4(+) T cell activation: NF-κB, AP-1 (activator protein 1), and NFAT (nuclear factor of activated T) cells. Our data demonstrate that minocycline selectively impairs NFAT-mediated transcriptional activation, a result of increased phosphorylation and reduced nuclear translocation of the isoform NFAT1. Minocycline increased the activity of the NFAT kinase GSK3 and decreased intracellular Ca(2+) flux, both of which facilitate NFAT1 phosphorylation. These findings provide a novel mechanism for minocycline induced suppression of CD4(+) T cell activation and may better inform the application of minocycline as an immunomodulatory agent.

Fluorescence Properties of Paracetamol During Interactions with Mitochondria

ABSTRACT Paracetamol interaction with rat liver mitochondria in respiration media in the presence of succinate was the focus of this experiment. Fluorescence of paracetamol in water was studied by three-dimensional synchronous fluorescence fingerprint (SFF) and by excitation emission matrix (EEM). The direct molecular interactions of paracetamol and mitochondria were studied by fluorescence polarization technique. The paracetamol fluorescence maximum of SFF was Δλ = 110/λex = 320 nm, Fmax = 508 nm, and EEM maximum was λex = (320 nm)/λem = 425 nm, Fmax = 508. The fluorescence polarization results showed nonsignificant elevation of fluorescence polarization after addition of paracetamol into mitochondria in comparison to the control mitochondria group without paracetamol at time point t = 0. Paracetamol probably covalently bound to the mitochondrial surface proteins at time point t = 0, but paracetamol also entered mitochondria, which was observed as nonsignificant decline of fluorescence polarization during 30 min in the paracetamol-treated group. The practical advantages of spectral techniques (EEM, SFF, fluorescence polarization) are high sensitivity, reproducibility, minimal quantity of material, and capability to measure the mitochondrial autofluorescence.

Caveolin-3 is a direct molecular partner of the Cav1.1 subunit of the skeletal muscle L-type calcium channel

Caveolin-3 is the striated muscle specific isoform of the scaffolding protein family of caveolins and has been shown to interact with a variety of proteins, including ion channels. Mutations in the human CAV3 gene have been associated with several muscle disorders called caveolinopathies and among these, the P104L mutation (Cav-3P104L) leads to limb girdle muscular dystrophy of type 1C characterized by the loss of sarcolemmal caveolin. There is still no clear-cut explanation as to specifically how caveolin-3 mutations lead to skeletal muscle wasting. Previous results argued in favor of a role for caveolin-3 in dihydropyridine receptor (DHPR) functional regulation and/or T-tubular membrane localization. It appeared worth closely examining such a functional link and investigating if it could result from the direct physical interaction of the two proteins. Transient expression of Cav-3P104L or caveolin-3 specific siRNAs in C2C12 myotubes both led to a significant decrease of the L-type Ca2+ channel maximal conductance. Immunolabeling analysis of adult skeletal muscle fibers revealed the colocalization of a pool of caveolin-3 with the DHPR within the T-tubular membrane. Caveolin-3 was also shown to be present in DHPR-containing triadic membrane preparations from which both proteins co-immunoprecipitated. Using GST-fusion proteins, the I–II loop of Cav1.1 was identified as the domain interacting with caveolin-3, with an apparent affinity of 60nM. The present study thus revealed a direct molecular interaction between caveolin-3 and the DHPR which is likely to underlie their functional link and whose loss might therefore be involved in pathophysiological mechanisms associated to muscle caveolinopathies.

Kefiran can disrupt the cell membrane through induced pore formation

Kefiran, an exopolysaccharide isolated from kefir d’acqua grains, has anti-inflammatory and antimicrobial activities, however, a mechanism of action directly associated with biomembranes is not yet understood. The influence of kefiran on a l-α-Phosphatidylcholine/cholesterol supported bilayer lipid membrane using a Pt electrode was studied by voltammetry and electrochemical impedance spectroscopy. The interaction of the oligosaccharide with s-BLMs promoted the access of Fe(CN)63-/4- probe ions to the electrode surface. Kefiran was found to induce molecular pores at the s-BLM surface within 5min at 11.4μmol/L. The suggested mechanism seems to involve hydrogen bonding between the carbohydrate and the phosphate head group of the phospholipid with a carpet-like model of interaction. The overall results can be contributed to direct molecular interactions between the prebiotic oligosaccharides and the cell surfaces, which can be related to the biological activity of kefiran in several experimental models.

Mix and Match: Heterodimers and Opioid Tolerance

Heterodimerization of G-protein coupled receptors has become increasingly recognized as a valuable mechanism to increase receptor diversity. Heterodimers have been observed in the opioid receptor family, but one of the most intriguing is that formed between mu and delta opioid receptors. In this issue of Neuron He et al. present evidence further implicating these heterodimers in morphine tolerance.

Pairing of (R,R)-2,3-Butanediol Molecules on a Si(001) Surface

We used a combination of scanning tunneling microscopy and DFT calculations to determine the reaction characteristics of (R,R)-2,3-butanediol (BD) molecules on a Si(001)−2 × 1 surface. The BD molecules were adsorbed onto the surface as either single, isolated molecules or paired configurations. As single adsorbed molecules, the BD molecules preferred to bridge adjacent Si dimers through oxygen atoms while preserving their chirality. The adsorption probability depended on the molecular conformation; the CH3-gauche conformer is more stable than the CH3-anti conformer. When BD molecules were adsorbed as paired configurations, BD pairs formed between the CH3-gauche conformers with several types of configurations. Total energy calculations for the pairs revealed that they are stabilized by a direct molecular interaction and/or a Si-mediated interaction, which can be analyzed as the strain energy involved with BD adsorption and the dangling bond reactivity near the adsorbed BD molecule. These results suggest th...

Interactions with M-band Titin and Calpain 3 Link Myospryn (CMYA5) to Tibial and Limb-girdle Muscular Dystrophies

Mutations in the C terminus of titin, situated at the M-band of the striated muscle sarcomere, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy (LGMD) type 2J. Mutations in the protease calpain 3 (CAPN3), in turn, lead to LGMD2A, and secondary CAPN3 deficiency in LGMD2J suggests that the pathomechanisms of the diseases are linked. Yeast two-hybrid screens carried out to elucidate the molecular pathways of TMD/LGMD2J and LGMD2A resulted in the identification of myospryn (CMYA5, cardiomyopathy-associated 5) as a binding partner for both M-band titin and CAPN3. Additional yeast two-hybrid and coimmunoprecipitation studies confirmed both interactions. The interaction of myospryn and M-band titin was supported by localization of endogenous and transfected myospryn at the M-band level. Coexpression studies showed that myospryn is a proteolytic substrate for CAPN3 and suggested that myospryn may protect CAPN3 from autolysis. Myospryn is a muscle-specific protein of the tripartite motif superfamily, reported to function in vesicular trafficking and protein kinase A signaling and implicated in the pathogenesis of Duchenne muscular dystrophy. The novel interactions indicate a role for myospryn in the sarcomeric M-band and may be relevant for the molecular pathomechanisms of TMD/LGMD2J and LGMD2A.

Molecular interactions between cereal soluble dietary fibre polymers and a model bile salt deduced from 13C NMR titration

Soluble dietary fibres (SDF) such as (1,3:1,4)-beta- d-glucan (βG) and arabinoxylan (AX) have been reported to lower plasma cholesterol levels in the human body, at least in part by preventing bile salts (BS) from being reabsorbed into the enterohepatic circulation. The mechanism(s) by which SDF interact with BS in the digestive tract is not known. This report describes investigations of molecular interactions between a model bile salt (taurochenodeoxycholate – TCDC) micelle with each of βG and AX using 13C NMR. In the presence of βG, chemical shift changes were observed for many bile salt resonances, but not βG resonances, without any apparent change in line widths. In contrast, in the presence of AX, no consistent chemical shift changes were observed for either TCDC or AX resonances, but TCDC signal intensities were reduced. This was not due to simple viscosity effects as the viscosity of the βG used was greater than that of the AX. The results suggest two different mechanisms of interaction between cereal non-starch polysaccharides and TCDC micelles: βG interacts directly on a molecular length scale with the micelles, whilst AX changes the local environment, resulting in reduced micellar mobility without direct molecular interaction.

Automatic reconstruction of the mouse segmentation network from an experimental evidence database

Mammalian vertebrae, ribs, body wall musculature and back skin develop from repetitive embryonic tissues called somites. The development of somites depends on the molecular oscillations of the products of so-called cyclic genes. The underlying network involves the Wnt, Fgf/Mapk, Notch signaling pathways and the T-box genes. The discovery of this network is based on genetic interactions. Because of regulatory feedbacks and cross-regulation between pathways, it is often difficult to intuitively identify direct molecular interactions underlying genetic interactions. To address this problem, we developed a method based on a database and graph theory algorithms. We first encoded genetic and non-genetic experiments in a relational database. Next, we built a reference network with the data from non-genetic experiments and the KEGG pathway database. Then, we computed the shortest path between the nodes for each genetic interaction in the reference network to propose direct molecular interactions. The resulting network is the largest computational representation of the mammalian segmentation network to date with 36 nodes and 57 interactions. In some instances, a number of genetic interactions could be explained by adding a single link to the reference network, which leads to experimentally testable hypotheses. Two examples of such predictions are the direct transcriptional regulation of Dll3 and Fgf8 genes by the Rbpj and Ctnnb1 products, respectively. Furthermore, the computed shortest paths suggest that cross-talks from the Wnt to the Fgf/Mapk and Notch pathways might be mediated by the Dvl genes. This method can be applied in any system where gene expression changes are observed as a response to some gene perturbation, for instance in cancer cells.

Beyond co-localization: inferring spatial interactions between sub-cellular structures from microscopy images

BackgroundSub-cellular structures interact in numerous direct and indirect ways in order to fulfill cellular functions. While direct molecular interactions crucially depend on spatial proximity, other interactions typically result in spatial correlations between the interacting structures. Such correlations are the target of microscopy-based co-localization analysis, which can provide hints of potential interactions. Two complementary approaches to co-localization analysis can be distinguished: intensity correlation methods capitalize on pattern discovery, whereas object-based methods emphasize detection power.ResultsWe first reinvestigate the classical co-localization measure in the context of spatial point pattern analysis. This allows us to unravel the set of implicit assumptions inherent to this measure and to identify potential confounding factors commonly ignored. We generalize object-based co-localization analysis to a statistical framework involving spatial point processes. In this framework, interactions are understood as position co-dependencies in the observed localization patterns. The framework is based on a model of effective pairwise interaction potentials and the specification of a null hypothesis for the expected pattern in the absence of interaction. Inferred interaction potentials thus reflect all significant effects that are not explained by the null hypothesis. Our model enables the use of a wealth of well-known statistical methods for analyzing experimental data, as demonstrated on synthetic data and in a case study considering virus entry into live cells. We show that the classical co-localization measure typically under-exploits the information contained in our data.ConclusionsWe establish a connection between co-localization and spatial interaction of sub-cellular structures by formulating the object-based interaction analysis problem in a spatial statistics framework based on nearest-neighbor distance distributions. We provide generic procedures for inferring interaction strengths and quantifying their relative statistical significance from sets of discrete objects as provided by image analysis methods. Within our framework, an interaction potential can either refer to a phenomenological or a mechanistic model of a physico-chemical interaction process. This increased flexibility in designing and testing different hypothetical interaction models can be used to quantify the parameters of a specific interaction model or may catalyze the discovery of functional relations.

Method for In-Vitro Studies of Cellular Interactions at the Interface of Two Tissues

We describe a simple and reliable experimental technique that enables one to create a high fidelity linear interface between two opposing cell layers. The method employs a custom designed lid that fits a standard 3cm cell culture dish. During cell plating, the dish is divided by a 200 micron thick separator that is part of the lid. The separator is covered in a thin layer of parafilm that forms a hermetic seal with the underlying coverslip and creates a temporary gap between the two cell plating environments. After cells attach, the custom lid is replaced with a standard lid and cells are allowed to grow under standard cell culture conditions. When expanding cell layers fill the gap, a linear interface is formed between the two opposing fields. Paracrinal factors released from an approaching cell front as well as direct physical and molecular interactions between two cell types affect intercellular orientation, individual cell morphology, and the degree of cells invasion into the opposing layer. The local interface appearance thus depends on a specific cell pair and may vary dramatically. We describe several types of such interfaces for different cell pairs, including cardiomyocytes, fibroblasts, melanocytes, endothelial cells and colon carcinoma cell lines. The method serves as a practical in vitro tool to study cell growth and invasion that occur on the interface of two neighboring tissues.

The co-chaperone SGT of Leishmania donovani is essential for the parasite's viability

Molecular chaperone proteins play a pivotal role in the protozoan parasite Leishmania donovani, controlling cell fate and ensuring intracellular survival. In higher eukaryotes, the so-called co-chaperone proteins are required for client protein recognition and proper function of chaperones, among them the small glutamine-rich tetratricopeptide repeat proteins (SGT) which interact with both HSP70 and HSP90 chaperones. An atypical SGT homolog is found in the L. donovani genome, encoding a protein lacking the C-terminal glutamine-rich region, normally typical for SGT family members. The gene is expressed constitutively during the life cycle and is essential for survival and/or growth of the parasites. LdSGT forms large, stable complexes that also include another putative co-chaperone, HSC70 interacting protein (HIP). The gene product forms cytoplasmic clusters, matching the subcellular distribution of HIP and partly that of the major cytoplasmic chaperones, HSP70 and HSP90, reflecting a direct molecular interaction with both chaperones.