Well-characterized Interaction Research Articles

The Drosophila Anion Exchanger (DAE) lacks a detectable interaction with the spectrin cytoskeleton

BackgroundCurrent models suggest that the spectrin cytoskeleton stabilizes interacting ion transport proteins at the plasma membrane. The human erythrocyte anion exchanger (AE1) was the first membrane transport protein found to be associated with the spectrin cytoskeleton. Here we evaluated a conserved anion exchanger from Drosophila (DAE) as a marker for studies of the downstream effects of spectrin cytoskeleton mutations.ResultsSequence comparisons established that DAE belongs to the SLC4A1-3 subfamily of anion exchangers that includes human AE1. Striking sequence conservation was observed in the C-terminal membrane transport domain and parts of the N-terminal cytoplasmic domain, but not in the proposed ankyrin-binding site. Using an antibody raised against DAE and a recombinant transgene expressed in Drosophila S2 cells DAE was shown to be a 136 kd plasma membrane protein. A major site of expression was found in the stomach acid-secreting region of the larval midgut. DAE codistributed with an infolded subcompartment of the basal plasma membrane of interstitial cells. However, spectrin did not codistribute with DAE at this site or in anterior midgut cells that abundantly expressed both spectrin and DAE. Ubiquitous knockdown of DAE with dsRNA eliminated antibody staining and was lethal, indicating that DAE is an essential gene product in Drosophila.ConclusionsBased on the lack of colocalization and the lack of sequence conservation at the ankyrin-binding site, it appears that the well-characterized interaction between AE1 and the spectrin cytoskeleton in erythrocytes is not conserved in Drosophila. The results establish a pattern in which most of the known interactions between the spectrin cytoskeleton and the plasma membrane in mammals do not appear to be conserved in Drosophila.

Cyclosporine Blocks Incorporation of HIV-1 Envelope Glycoprotein into Virions

Cyclosporine (CsA) decreases HIV-1 infectivity by blocking HIV-1 capsid (CA) interaction with target cell cyclophilin A (CypA). Yet, HIV-1 virions produced in the presence of CsA also exhibit decreased infectivity that was previously shown to be independent of the well-characterized HIV-1 CA-CypA interaction. Here, we demonstrate that CsA decreases gp120 and gp41 incorporation into HIV-1 virions and that the fusion of these virions with susceptible target cells is impaired. This effect was not observed with HIV-1 virions pseudotyped with the vesicular stomatitis virus glycoprotein or with the amphotropic envelope protein of murine leukemia virus. It was independent of calcineurin signaling, the endoplasmic reticulum luminal protein cyclophilin B, and the long cytoplasmic tail of gp41. Thus, cyclosporine blocks HIV-1 infectivity via two independent mechanisms, the first involving HIV-1 CA in target cells and the second involving HIV-1 Env in producer cells.

Control of granzymes by serpins

Although proteolysis mediated by granzymes has an important role in the immune response to infection or tumours, unrestrained granzyme activity may damage normal cells. In this review, we discuss the role of serpins within the immune system, as specific regulators of granzymes. The well-characterised human granzyme B-SERPINB9 interaction highlights the cytoprotective function that serpins have in safeguarding lymphocytes from granzymes that may leak from granules. We also discuss some of the pitfalls inherent in using rodent models of granzyme-serpin interactions and the ways in which our understanding of serpins can help resolve some of the current, contentious issues in granzyme biology.

An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

Identifying and characterizing protein interactions are fundamental steps toward understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the colocalization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecular tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin α (Impα), and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified colocalization assay is sensitive enough to detect protein interactions with Kd values that span over four orders of magnitude (1nM to 15μM). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris.

FIRST EVIDENCE OF D-HPLC EFFICIENCY FOR AN AUTOMATED CDNA-AFLP IN THE APPLE SCAB RESISTANCE MODEL

Apple scab resistance is one of the most well-characterized plant-pathogen interactions in woody plant species. After the cloning of the apple scab resistance gene HcrVf2, we aimed at identifying the network of genes that are differentially expressed after pathogen challenge by cDNA-AFLP. As electrophoretic analyses are labour-intensive with only limited potential for automation and the recovery of DNA fragments from gels is cumbersome, we investigated the possibility of performing the cDNA-AFLP analysis by d-HPLC (denaturing high performance liquid chromatography) and automating the DNA fragments collection using the Transgenomic WAVE System. Direct sequencing of the collected DNA fragments was also carried out and our preliminary results are reported.

Cholera Toxin Inhibitors Studied with High‐Performance Liquid Affinity Chromatography: A Robust Method to Evaluate Receptor–Ligand Interactions

Anti-adhesion drugs may be an alternative to antibiotics to control infection of micro-organisms. The well-characterized interaction between cholera toxin and the cellular glycolipid GM1 makes it an attractive model for inhibition studies in general. In this report, we demonstrate a high-performance liquid affinity chromatography approach called weak affinity chromatography to evaluate cholera toxin inhibitors. The cholera toxin B-subunit was covalently coupled to porous silica and a (weak) affinity column was produced. The K(D) values of galactose and meta-nitrophenyl alpha-D-galactoside were determined with weak affinity chromatography to be 52 and 1 mM, respectively, which agree well with IC(50) values previously reported. To increase inhibition potency multivalent inhibitors have been developed and the interaction with multivalent glycopolypeptides was also evaluated. The affinity of these compounds was found to correlate with the galactoside content but K(D) values were not obtained because of the inhomogeneous response and slow off-rate from multivalent interactions. Despite the limitations in obtaining direct K(D) values of the multivalent galactopolypeptides, weak affinity chromatography represents an additional and valuable tool in the evaluation of monovalent as well as multivalent cholera toxin inhibitors. It offers multiple advantages, such as a low sample consumption, high reproducibility and short analysis time, which are often not observed in other methods of analysis.

A facile method to screen inhibitors of protein–protein interactions including MDM2–p53 displayed on T7 phage

Protein–protein interactions are essential in many biological processes including cell cycle and apoptosis. It is currently of great medical interest to inhibit specific protein–protein interactions in order to treat a variety of disease states. Here, we describe a facile multiwell plate assay method using T7 phage display to screen for candidate inhibitors of protein–protein interactions. Because T7 phage display is an effective method for detecting protein–protein interactions, we aimed to utilize this technique to screen for small-molecule inhibitors that disrupt these types of interaction. We used the well-characterized interaction between p53 and MDM2 and an inhibitor of this interaction, nutlin 3, as a model system to establish a new screening method. Phage particles displaying p53 interacted with GST–MDM2 immobilized on 96-well plates, and the interaction was inhibited by nutlin 3. Multiwell plate assay was then performed using a natural product library, which identified dehydroaltenusin as a candidate inhibitor of the p53–MDM2 interaction. We discuss the potential applications of this novel T7 phage display methodology, which we propose to call ‘reverse phage display’.

Genetic interactions between the parasitoid wasp Leptopilina boulardi and its Drosophila hosts

Coevolutionary arms races between hosts and parasites would not occur without genetic variation for traits involved in the outcome of parasitism. Genetic variations in resistance and virulence have only rarely been described in pairwise host-parasitoid interactions and have never been analysed in multi-species interactions, in contrast to well-characterized plant-pathogen interactions. This paper reports genetic variation in resistance of Drosophila yakuba to the parasitoid wasp Leptopilina boulardi. The genetic basis and geographic distribution of resistance is analysed. On the basis of these and previous findings, we demonstrate that there are different resistance patterns to the parasitoid species L. boulardi in D. melanogaster and D. yakuba, as well as different specificity levels in the parasitoid species, suggesting complex ecological interactions in the field. This first description of resistance-virulence genetic interactions between a parasitoid and its two host species provides empirical data showing that multi-species interactions may greatly influence coevolutionary processes.

Critical Role for a Single Leucine Residue in Leukemia Induction by E2A-PBX1

In roughly 5% of cases of acute lymphoblastic leukemia, a chromosomal translocation leads to expression of the oncogenic protein E2A-PBX1. The N-terminal portion of E2A-PBX1, encoded by the E2A gene, is identical in sequence to the corresponding portion of the E proteins E12/E47 and includes transcriptional activation domains. The C terminus consists of most of the HOX interacting transcription factor PBX1, including its DNA-binding homeodomain. Structure-function correlative experiments have suggested that oncogenesis by E2A-PBX1 requires an activation domain, called AD1, at the extreme N terminus. We recently demonstrated that a potentially helical portion of AD1 interacts directly with the transcriptional coactivator protein cyclic AMP response element-binding protein (CBP) and that this interaction is essential in the immortalization of primary bone marrow cells in tissue culture. Here we show that a conserved LXXLL motif within AD1 is required in the interaction between E2A-PBX1 and the KIX domain of CBP. We show by circular dichroism spectroscopy that the LXXLL-containing portion of AD1 undergoes a helical transition upon interacting with the KIX domain and that amino acid substitutions that prevent helix formation prevent both the KIX interaction and cell immortalization by E2A-PBX1. Perhaps most strikingly, substitution of a single, conserved leucine residue (L20) within the LXXLL motif impairs leukemia induction in mice after transplantation with E2A-PBX1-expressing bone marrow. The KIX domain of CBP mediates well-characterized interactions with several transcription factors of relevance to leukemia induction. Circumstantial evidence suggests that the side chain of L20 might interact with a deep hydrophobic pocket in the KIX domain. Therefore, our results serve to identify a potential new drug target.

Protein‐protein interactions in fixed cells and glial tumors revealed by antibody‐based fluorescence resonance energy transfer (FRET)

The overall objective of our research is to quantify protein-protein interactions in fixed pathological archival specimens. Our data demonstrate proof-of-principle for the use of antibody-based fluorescence resonance energy transfer (FRET) to evaluate colocalization of components of the MEK/ERK intracellular signaling pathway, first in cultured cells and then archival tumor samples. We first validated the use of FRET in this context by measuring the well-characterized interaction between MEK1 and ERK2 in tissue culture. Cos-1 cells transfected with epitope-tagged wild type or mutant (non-MEK binding) MEK1 and ERK2 constructs were fixed in formalin and processed for indirect immunofluorescence using epitope tag antibodies and fluorophore-conjugated secondary antibodies. FRET analysis utilizing the three filter method and the PFRET software package revealed significant FRET signal for wild type ERK2, but not the mutant. FRET between endogenous MEK1 and ERK2 was revealed in cultured U251 glioma cells, and responded to serum starvation/stimulation paradigms as predicted (low ERK1/MEK2 FRET signal in serum conditions; increased with serum starvation; see figure). Current work is aimed at analyzing FRET in archival astrocytic tumor specimens and non-neoplastic control tissue. Figure 1Open in figure viewerPowerPoint This work was supported by a Farrow fellowship grant from the University of Virginia Cancer Center.

Health-related Interactions of Phytochemicals

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Screening for enzyme inhibitors by surface plasmon resonance combined with mass spectrometry.

We have developed a novel strategy to identify enzyme inhibitors that interact directly with their enzyme targets. In the approach, an enzyme is immobilized on a sensor chip, and it is determined whether the immobilized enzyme is still active by incubation with model substrates and mass spectrometric analysis of the products. Putative inhibitors or mixtures containing putative inhibitors are then injected over the sensor chip for binding analysis with surface plasmon resonance. It is then tested whether the bound compounds inhibit the enzymatic activity by subsequent incubation with the model substrate and mass spectrometric analysis. If the bound compound inhibits the enzyme, the inhibitor is eluted from the enzyme and characterized by mass spectrometry. To test the strategy, it has been applied to the well-characterized interaction between trypsin and pure bovine pancreas trypsin inhibitor. Furthermore, fractions of plant extracts were screened for binding to and inhibition of carboxypeptidase B.

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

To gain insights into complex biological processes, such as transcription and replication, the analysis of protein-DNA interactions and the determination of their sequence requirements are of central importance. In this study, we probed protein microarray technology and ultraviolet crosslinking combined with mass spectrometry (MS) for their practicability to study protein–DNA interactions. We chose as a model system the well-characterized interaction of bacterial replication initiator DnaA with its cognate binding site, the DnaA box. Interactions of DnaA domain 4 with a high-affinity DnaA box (R4) and with a low-affinity DnaA box (R3) were compared. A mutant DnaA domain 4, A440V, was included in the study. DnaA domain 4, wt, spotted onto FAST slides, revealed a strong signal only with a Cy5-labeled, double-stranded, 21-mer oligonucleotide containing DnaA box R4. No signals were obtained when applying the mutant protein. Ultraviolet crosslinking combined with nanoLC/MALDI-TOF MS located the site of interaction to a peptide spanning amino acids 433– 442 of Escherichia coli DnaA. This fragment contains six residues that were identified as being involved in DNA binding by recently published crystal structure and nuclear magnetic resonance (NMR) analysis. In the future, the technologies applied in this study will become important tools for studying protein–DNA interactions.

A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis.

A molecular platform for protein detection and quantification is reported in which recognition has been integrated with direct monitoring of target-protein binding. The platform is based on a versatile 42-residue helix-loop-helix polypeptide that dimerizes to form four-helix bundles and allows site-selective modification with recognition and reporter elements on the side chains of individually addressable lysine residues. The well-characterized interaction between the model target-protein carbonic anhydrase and its inhibitor benzenesulfonamide was used for a proof-of-concept demonstration. An affinity array was designed where benzenesulfonamide derivatives with aliphatic or oligoglycine spacers and a fluorescent dansyl reporter group were introduced into the scaffold. The affinities of the array members for human carbonic anhydrase II (HCAII) were determined by titration with the target protein and were found to be highly affected by the properties of the spacers (dissociation constant Kd=0.02-3 microM). The affinity of HCAII for acetazolamide (Kd=4 nM) was determined in a competition experiment with one of the benzenesulfonamide array members to address the possibility of screening substance libraries for new target-protein binders. Also, successful affinity discrimination between different carbonic anhydrase isozymes highlighted the possibility of performing future isoform-expression profiling. Our platform is predicted to become a flexible tool for a variety of biosensor and protein-microarray applications within biochemistry, diagnostics and pharmaceutical chemistry.

A polymer scaffold for protein oligomerization.

We report the design and synthesis of well-defined polymers for the noncovalent oligomerization of proteins. The reported scaffolds, which were generated by atom-transfer radical polymerization (ATRP), take advantage of the well-characterized interaction between a Ni2+ complex and an oligohistidine sequence (His tag). Thus, our polymers are designed to facilitate the oligomerization of any protein possessing a His tag. We demonstrate that they can oligomerize fibroblast growth factor-8b (FGF-8b) and promote FGF-8b-mediated cell proliferation in the absence of heparin.

Study of G-protein-coupled receptor-protein interactions using gel overlay assays and glutathione-S-transferase-fusion protein pull-downs.

Numerous recent studies have suggested that the predicted cytosolic domains of G-protein-coupled receptors (GPCRs) represent a surface for association with proteins that may serve multiple roles in receptor localization, turnover, and signaling beyond the well-characterized interactions of these receptors with heterotrimeric G-proteins. This chapter describes two in vitro methods for ascertaining interactions between GPCRs and various binding partners: gel overlay strategies and GST-fusion protein pull-downs.

Quantifying structural changes and stoichiometry of protein interactions using size and density profiling

The use of Dual Polarisation Interferometry, an emerging analytical biophysical technique, is described for the determination of the optogeometrical properties (thickness and density) at high resolution of adsorbed protein layers at the solid–liquid interface. The technique has been used to quantify, in real time and at subatomic resolution, the structural changes occurring in two well-characterised protein interaction systems, an antibody–antigen interaction and the biotin–streptavidin interaction. The real-time data obtained on structural changes during the interactions is in excellent agreement with previously reported X-ray crystallography and neutron reflection data. The precision of the measurements taken was of the order of 0.01 nm with respect to protein size. The dual-parameter approach also allowed the stoichiometry of both of these interactions to be calculated, giving values that confirm the current understanding of the interactions. This approach provides detailed insights into the inherent and subtle link between structural change and function in proteins, to a degree not previously possible through mass change measurements alone. The technique is expected to find utility in the increasingly important study of protein structure and function.

Electronic neuron within a ganglion of a leech (Hirudo medicinalis)

We report the construction of an electronic device that models and replaces a neuron in a midbody ganglion of the leech Hirudo medicinalis. In order to test the behavior of our device, we used a well-characterized synaptic interaction between the mechanosensory, sensitive to pressure, (P) cell and the anteropagoda (because of the action potential shape) (AP) neuron. We alternatively stimulated a P neuron and our device connected to the AP neuron, and studied the response of the latter. The number and timing of the AP spikes were the same when the electronic parameters were properly adjusted. Moreover, after changes in the depolarization of the AP cell, the responses under the stimulation of both the biological neuron and the electronic device vary in a similar manner.

A functional genetic approach suggests a novel interaction between the human immunodeficiency virus type 1 (HIV-1) Tat protein and HIV-1 TAR RNA in vivo.

Human immunodeficiency virus type 1 (HIV-1) Tat and human Cyclin T1 form a complex and together recognize the viral TAR RNA element with specificity. Using HIV-1/equine infectious anaemia virus TAR chimeras, we show that in addition to the well-characterized interaction with the bulge, Tat recognizes the distal stem and the loop of TAR. These data support previously proposed, but unproven, molecular models.

Characterization of the human beta-globin downstream promoter region.

The human beta-globin gene is abundantly expressed specifically in adult erythroid cells. Stage-specific transcription is regulated principally by promoter proximal cis-regulatory elements. The basal promoter contains a non-canonical TATA-like motif as well as an initiator element. These two elements have been shown to interact with the TFII-D complex. Here we show that in addition to the TATA and initiator elements, conserved E-box motifs are located in the beta-globin downstream promoter. One of the E-box motifs overlaps the initiator and this composite element interacts with USF1 and TFII-I in vitro. Another E-box, located 60 bp 3' to the transcription initiation site, interacts with USF1 and USF2. Mutations of either the initiator or the downstream E-box impair transcription of the beta-globin gene in vitro. Mutations of a putative NF-E2-binding site in the downstream promoter region do not affect transcription in vitro. USF1, USF2, TFII-I and p45 can be crosslinked to a beta-globin promoter fragment in MEL cells in vivo, whereas only TFII-I and USF2 crosslink to the beta-globin gene in K562 cells. The summary data demonstrate that in addition to the well-characterized interactions of the TFII-D complex with the basal promoter, E-box motifs contribute to the efficient formation of transcription complexes on the adult beta-globin gene.