Protein-receptor Interactions Research Articles

Preparation, characterization and molecular modeling of PEGylated human growth hormone with agonist activity

In this study, site-specific PEGylated human growth hormone (hGH) was prepared by microbial transglutaminase, modeled and characterized. To this end, the effects of different reaction parameters including reaction media, PEG:protein ratios, reaction time and pH value were investigated. PEG-hGH was purified by size exclusion chromatography method and analyzed by SDS-PAGE, BCA, peptide mapping, ESI and MALDI-TOF–TOF mass spectroscopy methods. Biophysical and biological properties of PEG-hGH were evaluated. Molecular simulation was utilized to provide molecular insight into the protein–receptor interaction. The optimum conditions that were obtained for PEGylation were phosphate buffer with pH of 7.4, 48h of stirring and PEG:protein ratio of 40:1. By this method, mono-PEG-hGH with high reaction yield was obtained and PEGylation site was at Gln-40 residue. The circular dichroism and fluorescence spectrum indicated that PEGylation did not change the secondary structure while tertiary structure was altered. Upon enzymatic PEGylation, agonistic activity of hGH was preserved; however, Somavert®, which is prepared by chemical PEGylation, is an antagonist form of protein. These data were confirmed by the total energy of affinity obtained by computational protein–receptor interaction. In conclusion, PEGylation of hGH was led to prepare a novel form of hormone with an agonist activity which merits further investigations.

Unraveling the mechanism of cholesterol-mediated regulation of receptor dimerization in plasma membranes in vivo

Membrane cholesterol can alter the signaling pathways of living cells. However, the process that modulates the interaction of receptor proteins is still unclear. We performed single-molecule optical tracking of ligand-induced dimerization of epidermal growth factor receptors (EGFRs) in two cancerous cell lines (HeLa and A431) and one normal endothelial cell line (MCF12A). We discovered that unliganded EGFRs typically reside in non-raft regions of the plasma membrane and can move into raft domains upon ligand binding. This ligand-induced motion could be a common behavior in live cells. We found that the amount of membrane cholesterol significantly affects the stability of EGFR dimers by manipulating the total amount of membrane cholesterol with methyl-β-cyclodextrin and the local concentration of cholesterol with nystatin. The EGFR dimers in the plasma membrane of normal cells are more sensitive to changes in the local concentration of cholesterol compared with the cancer cells. Our methodology can yield useful information for understanding cholesterol-mediated protein-protein interactions in live cells.

Structural Characterization of Modification on the Interface between a Ligand and its Receptor for Biopharmaceuticals

The binding of a protein to its target is a major mode of action for most biopharmaceutical therapies with over 70% of biopharmaceuticals involved in binding between the protein and its target. The interfaces between a biopharmaceutical and its target are key regions for its efficacy. Any modifications to the amino acids at the interfaces invariably affect interactions between the biopharmaceutical and its receptor and may result in lowering therapeutic efficacy. Degradations of biopharmaceuticals by asparagine (Asn) deamidation and/or aspartate (Asp) isomerization have been well characterized and those modifications at the interfaces have resulted in a loss of activity. To characterize modification hot-spots on the interfaces, it is necessary to identify the amino acid residues on the interfaces. We recently addressed a visualization tool for amino acids on the interfaces between a protein ligand and its receptor. This tool was applied to visualize ligand protein-receptor interaction and antigen-antibody interaction. As a model system for ligand protein-receptor interaction, erythropoietin (EPO) and its receptor were selected and amino acids on the interfaces were identified. Modifications on the interfaces were then investigated. Deamidation of Asn was identified at two amino acid residues, Asn47 and Asp147, on Interface 1 of EPO. The relative contents of deamidated residues on the interface of EPO were in the range of 3-5% of the total. As a model system for antigen-antibody interaction, Herceptin and its receptor, HER2, were chosen and amino acids on the interfaces were identified. Then modifications on the interfaces were assessed. Deamidation of the light chain Asn30 and heavy chain Asn55 were identified. The relative contents of the deamidated residues on the interfaces were in the range of 8-9% of the total. Along with deamidation, another modification, isomerization, was identified at the amino acid residue Asp102 of the heavy chain, and the level of oxidation was 13.5% of the total. Our studies provide a targeted method focusing on the interface between a protein and its target that can be coupled with other applications, for example, identification of modified amino acids on the interfaces.

Probing homodimer formation of epidermal growth factor receptor by selective crosslinking.

Ligand binding promotes conformational rearrangement of the epidermal growth factor receptor (EGFR) leading to receptor autophosphorylation and downstream signaling. However, transient interactions between unstimulated EGFR molecules on the cell surface are not fully understood. In this report, we describe the investigation of homodimer formation of EGFR by means of an SNAP-tag based selective crosslinking approach (S-CROSS). EGFR homodimers were selectively captured in living cells and utilized for analysis of protein receptor interactions on the plasma membrane and ligand-induced activation. We showed that EGFR forms homodimers in unstimulated cells with efficiencies similar to those seen in cells treated with the epidermal growth factor ligand (EGF) supporting the existence of constitutive transient receptor-receptor interactions. EGFR crosslinked homodimers displayed a substantially increase in kinase activation upon ligand stimulation. Interestingly, in unstimulated cells the levels of spontaneous phosphorylation were found to correlate with the yields of the crosslinked homodimers species. In addition, we demonstrated that this crosslinking approach can be applied to interrogate the effect of small molecule inhibitors on receptor dimerization and kinase activity. Our crosslinking assay provides a new tool to dissect ligand-independent dimerization and activation mechanisms of receptor tyrosine kinases, many of which are important anticancer drug targets.

The NMDA receptor complex: a multifunctional machine at the glutamatergic synapse.

The N-methyl-D-aspartate receptors (NMDARs) are part of a large multiprotein complex at the glutamatergic synapse. The assembly of NMDARs with synaptic proteins offers a means to regulate NMDAR channel properties and receptor trafficking, and couples NMDAR activation to distinct intracellular signaling pathways, thus contributing to the versatility of NMDAR functions. Receptor-protein interactions at the synapse provide a dynamic and powerful mechanism for regulating synaptic efficacy, but can also contribute to NMDAR overactivation-induced excitotoxicity and cellular damage under pathological conditions. An emerging concept is that by understanding the mechanisms and functions of disease-specific protein-protein interactions in the NMDAR complex, we may be able to develop novel therapies based on protein-NMDAR interactions for the treatment of brain diseases in which NMDAR dysfunction is at the root of their pathogenesis.

A Rapid and Efficient Immunoenzymatic Assay to Detect Receptor Protein Interactions: G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors, and are the target of at least one-third of the current therapeutic drugs on the market. Along their life cycle, GPCRs are accompanied by a range of specialized GPCR-interacting proteins (GIPs), which take part in receptor proper folding, targeting to the appropriate subcellular compartments and in receptor signaling tasks, and also in receptor regulation processes, such as desensitization and internalization. The direction of protein-protein interactions and multi-protein complexes formation is crucial in understanding protein function and their implication in pathological events. Although several methods have been already developed to assay protein complexes, some of them are quite laborious, expensive, and, more important, they do not generate fully quantitative results. Herein, we show a rapid immunoenzymatic assay to quantify GPCR interactionswith its signaling proteins. The recently de-orphanized GPCR, GPR17, was chosen as a GPCR prototype to optimize the assay. In a GPR17 transfected cell line and primary oligodendrocyte precursor cells, GPR17 interaction with proteins involved in the typical GPCR regulation, such as desensitization and internalization machinery, was investigated. The obtained results were validated by co-immunoprecipitation experiments, confirming this new method as a rapid and quantitative assay to study protein-protein interactions.

Chemical crosslinkers enhance detection of receptor interactomes

Receptor function is dependent on interaction with various intracellular proteins that ensure the localization and signaling of the receptor. While a number of approaches have been optimized for the isolation, purification, and proteomic characterization of receptor–protein interaction networks (interactomes) in cells, the capture of receptor interactomes and their dynamic properties remains a challenge. In particular, the study of interactome components that bind to the receptor with low affinity or can rapidly dissociate from the macromolecular complex is difficult. Here we describe how chemical crosslinking (CC) can aid in the isolation and proteomic analysis of receptor–protein interactions. The addition of CC to standard affinity purification and mass spectrometry protocols boosts the power of protein capture within the proteomic assay and enables the identification of specific binding partners under various cellular and receptor states. The utility of CC in receptor interactome studies is highlighted for the nicotinic acetylcholine receptor as well as several other receptor types. A better understanding of receptors and their interactions with proteins spearheads molecular biology, informs an integral part of bench medicine which helps in drug development, drug action, and understanding the pathophysiology of disease.

GABAB receptor trafficking and interacting proteins: Targets for the development of highly specific therapeutic strategies to treat neurological disorders?

GABAB receptors mediate slow inhibitory neurotransmission throughout the central nervous system thereby controlling the excitability of neurons. They have been implicated in numerous neurological disorders making them an attractive drug target. However, due to considerable side effects, the agonist baclofen is so far the only drug on the market targeting GABAB receptors, primarily for the treatment of spasticity. Because GABAB receptors are involved in a variety of brain functions it is rather unlikely to avoid unwanted effects with systemically administered drugs directly addressing ligand binding sites of the receptor. To minimize side effects, it would be desirable to target only those receptors involved in a given pathological state. This commentary discusses the idea that restoring impaired GABAB receptor function in diseased neurons by interfering with receptor–protein interactions may be an approach to specifically target only those receptors involved in the pathological state. Two recently discovered mechanisms that down-regulate the level of functional GABAB receptors most likely contribute to cerebral ischemia and neuropathic pain, respectively. In both mechanisms, small interfering peptides disrupting protein–protein interactions may offer a highly specific means to restore normal receptor function selectively at the site of malfunction. If restored functional GABAB receptor expression in these diseases has beneficial effects, this may serve as a starting point for the development of a highly specific therapeutic interventions. Such an approach is expected to minimize side effects because it promises to leave those GABAB receptors unaffected which are not involved in the dysfunction.

Moonlighting proteins and protein-protein interactions as neurotherapeutic targets in the G protein-coupled receptor field.

There is serious interest in understanding the dynamics of the receptor-receptor and receptor-protein interactions in space and time and their integration in GPCR heteroreceptor complexes of the CNS. Moonlighting proteins are special multifunctional proteins because they perform multiple autonomous, often unrelated, functions without partitioning into different protein domains. Moonlighting through receptor oligomerization can be operationally defined as an allosteric receptor-receptor interaction, which leads to novel functions of at least one receptor protomer. GPCR-mediated signaling is a more complicated process than previously described as every GPCR and GPCR heteroreceptor complex requires a set of G protein interacting proteins, which interacts with the receptor in an orchestrated spatio-temporal fashion. GPCR heteroreceptor complexes with allosteric receptor-receptor interactions operating through the receptor interface have become major integrative centers at the molecular level and their receptor protomers act as moonlighting proteins. The GPCR heteroreceptor complexes in the CNS have become exciting new targets for neurotherapeutics in Parkinson's disease, schizophrenia, drug addiction, and anxiety and depression opening a new field in neuropsychopharmacology.

Search for invisible binding sites of low-molecular-weight compounds on protein molecules and prediction of inhibitory activity

Current computational methods have not been able to discover an unknown site for low-molecular-weight ligands on a protein receptor and predict parameters of their interaction when the binding site is not distinguished by energy of binding or structural features. The authors propose a method to find an unknown, structurally undefined site for binding low-molecular-weight inhibitors to a protein, as well as to predict kinetic parameters for new compounds using the X-ray structure of a protein receptor and experimental interaction constants of a training set of inhibitors. The developed method was applied to discover the structural and kinetic parameters of the binding between C1q, a protein of the first component of complement system, and low-molecular-weight ligands that inhibit the C1q interaction with immune complexes. Authors suggest that these ligands bind to a region of C1q globular head near residues Arg150 of chain B and Lys160 and His167 of chain C, which supposedly inhibits the classical pathway of complement activation. Ligands that inhibit the interaction of C1q with immune complexes can be used in the therapy of pathological conditions that are related to the activation of unwanted complements, i.e., allergic reactions, xenograft rejection, etc.

Defining a Key Receptor–CheA Kinase Contact and Elucidating Its Function in the Membrane-Bound Bacterial Chemosensory Array: A Disulfide Mapping and TAM-IDS Study

The three core components of the ubiquitous bacterial chemosensory array - the transmembrane chemoreceptor, the histidine kinase CheA, and the adaptor protein CheW - assemble to form a membrane-bound, hexagonal lattice in which receptor transmembrane signals regulate kinase activity. Both the regulatory domain of the kinase and the adaptor protein bind to overlapping sites on the cytoplasmic tip of the receptor (termed the protein interaction region). Notably, the kinase regulatory domain and the adaptor protein share the same fold constructed of two SH3-like domains. The present study focuses on the structural interface between the receptor and the kinase regulatory domain. Two models have been proposed for this interface: Model 1 is based on the crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheW adaptor protein. This model has been used in current models of chemosensory array architecture to build the receptor-CheA kinase interface. Model 2 is based on a newly determined crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheA kinase regulatory domain. Both models present unique strengths and weaknesses, and current evidence is unable to resolve which model best describes contacts in the native chemosensory arrays of Escherichia coli, Salmonella typhimurium, and other bacteria. Here we employ disulfide mapping and tryptophan and alanine mutation to identify docking sites (TAM-IDS) to test Models 1 and 2 in well-characterized membrane-bound arrays formed from E. coli and S. typhimurium components. The results reveal that the native array interface between the receptor protein interaction region and the kinase regulatory domain is accurately described by Model 2, but not by Model 1. In addition, the results show that the interface possesses both a structural function that contributes to stable CheA kinase binding in the array and a regulatory function central to transmission of the activation signal from receptor to CheA kinase. On-off switching alters the disulfide formation rates of specific Cys pairs at the interface, but not most Cys pairs, indicating that signaling perturbs localized regions of the interface. The findings suggest a simple model for the rearrangement of the interface triggered by the attractant signal and for longer range transmission of the signal in the chemosensory array.

Do low-affinity ErbB receptor protein interactions represent the base of a cell signaling iceberg?

We recently described one of the most comprehensive and quantitative assessments of human ErbB receptor recruitment potential for SH2 domains to date using a high-throughput fluorescence polarizati...

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Current computational methods have not been able to discover an unknown binding site for low-molecular ligands on a protein receptor and predict parameters of their interaction when this binding site is not distinguished by energy of binding or structural features. Authors propose a method to find an unknown, structurally undefined site for binding low-molecular inhibitors with a protein, as well as to predict kinetic parameters for new compounds using x-ray structure of a protein receptor and experimental interaction constants of a training set of inhibitors. The developed method is applied to discover structural and kinetic parameters of binding C1q, a protein from the first component of complement system, to low-molecular ligands that inhibit its interactions with immune complexes. Authors have suggested that these ligands bind to a region of C1q globular head near residues Arg150 of chain B, and Lys160 and His67 of chain C, supposedly inhibiting the classical pathway of complement activation. Ligands that inhibit interaction of C1q with immune complexes can be used in the therapy of pathological conditions that are related to unwanted complement activation: allergic reactions, xenograft rejection, etc.

The Aryl-Hydrocarbon Receptor Protein Interaction Network (AHR-PIN) as Identified by Tandem Affinity Purification (TAP) and Mass Spectrometry.

The aryl-hydrocarbon receptor (AHR), a ligand activated PAS superfamily transcription factor, mediates most, if not all, of the toxicity induced upon exposure to various dioxins, dibenzofurans, and planar polyhalogenated biphenyls. While AHR-mediated gene regulation plays a central role in the toxic response to dioxin exposure, a comprehensive understanding of AHR biology remains elusive. AHR-mediated signaling starts in the cytoplasm, where the receptor can be found in a complex with the heat shock protein of 90 kDa (Hsp90) and the immunophilin-like protein, aryl-hydrocarbon receptor-interacting protein (AIP). The role these chaperones and other putative interactors of the AHR play in the toxic response is not known. To more comprehensively define the AHR-protein interaction network (AHR-PIN) and identify other potential pathways involved in the toxic response, a proteomic approach was undertaken. Using tandem affinity purification (TAP) and mass spectrometry we have identified several novel protein interactions with the AHR. These interactions physically link the AHR to proteins involved in the immune and cellular stress responses, gene regulation not mediated directly via the traditional AHR:ARNT heterodimer, and mitochondrial function. This new insight into the AHR signaling network identifies possible secondary signaling pathways involved in xenobiotic-induced toxicity.

BRET Biosensor Analysis of Receptor Tyrosine Kinase Functionality

Bioluminescence resonance energy transfer (BRET) is an improved version of earlier resonance energy transfer technologies used for the analysis of biomolecular protein interaction. BRET analysis can be applied to many transmembrane receptor classes, however the majority of the early published literature on BRET has focused on G protein-coupled receptor (GPCR) research. In contrast, there is limited scientific literature using BRET to investigate receptor tyrosine kinase (RTK) activity. This limited investigation is surprising as RTKs often employ dimerization as a key factor in their activation, as well as being important therapeutic targets in medicine, especially in the cases of cancer, diabetes, neurodegenerative, and respiratory conditions. In this review, we consider an array of studies pertinent to RTKs and other non-GPCR receptor protein–protein signaling interactions; more specifically we discuss receptor-protein interactions involved in the transmission of signaling communication. We have provided an overview of functional BRET studies associated with the RTK superfamily involving: neurotrophic receptors [e.g., tropomyosin-related kinase (Trk) and p75 neurotrophin receptor (p75NTR)]; insulinotropic receptors [e.g., insulin receptor (IR) and insulin-like growth factor receptor (IGFR)] and growth factor receptors [e.g., ErbB receptors including the EGFR, the fibroblast growth factor receptor (FGFR), the vascular endothelial growth factor receptor (VEGFR) and the c-kit and platelet-derived growth factor receptor (PDGFR)]. In addition, we review BRET-mediated studies of other tyrosine kinase-associated receptors including cytokine receptors, i.e., leptin receptor (OB-R) and the growth hormone receptor (GHR). It is clear even from the relatively sparse experimental RTK BRET evidence that there is tremendous potential for this technological application for the functional investigation of RTK biology.

Controlling Molecular Recognition with Lipid/Polymer Domains in Vesicle Membranes

The molecular recognition between cholera toxin B and GM1-functionalized phospholipid/block copolymer hybrid membranes can be controlled by varying the lipid/block copolymer composition. Confocal laser scanning microscopy and fluorescence correlation spectroscopy were used to study the protein–receptor interaction and dynamic processes in the membrane.

High-Contrast Imaging of Biomolecular Interactions Using Liquid Crystals Supported on Roller Printed Protein Surfaces

In this study, we report a new method for the high contrast imaging of biomolecular interactions at roller printed protein surfaces using thermotropic liquid crystals (LCs). Avidin was roller printed and covalently immobilized onto the obliquely deposited gold surface that was decorated with carboxylic acid-terminated self-assembled monolayers (SAMs). The optical response of LCs on the roller printed film of avidin contrasted sharply with that on the obliquely deposited gold surface. The binding of biotin-peroxidase to the roller printed avidin was then investigated on the obliquely deposited gold substrate. LCs exhibited a non-uniform and random orientation on the roller printed area decorated with the complex of avidin and biotin-peroxidase, while LCs displayed a uniform and planar orientation on the area without roller printed proteins. The orientational transition of LCs from uniform to non-uniform state was triggered by the erasion of nanometer-scale topographies on the roller printed surface after the binding of biotin-peroxidase to the surface-immobilized avidin. The specific binding events of protein-receptor interactions were also confirmed by atomic force microscopy and ellipsometry. These results demonstrate that the roller printing of proteins on obliquely deposited gold substrates could provide a high contrast signal for imaging biomolecular interactions using LC-based sensors.

Light-Induced Plasticity of Synaptic AMPA Receptor Composition in Retinal Ganglion Cells

Light-evoked responses of all three major classes of retinal ganglion cells (RGCs) are mediated by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Although synaptic activity at RGC synapses is highly dynamic, synaptic plasticity has not been observed in adult RGCs. Here, using patch-clamp recordings in dark-adapted mouse retina, we report a retina-specific form of AMPAR plasticity. Both chemical and light activation of NMDARs caused the selective endocytosis of GluA2-containing, Ca(2+)-impermeable AMPARs on RGCs and replacement with GluA2-lacking, Ca(2+)-permeable AMPARs. The plasticity was expressed in ON but not OFF RGCs and was restricted solely to the ON responses in ON-OFF RGCs. Finally, the plasticity resulted in a shift in the light responsiveness of ON RGCs. Thus, physiologically relevant light stimuli can induce a change in synaptic receptor composition of ON RGCs, providing a mechanism by which the sensitivity of RGC responses may be modified under scotopic conditions.

Abstract 2844: Inhibiting androgen receptor and JunD protein interaction to prevent prostate cancer progression

Abstract Development of an effective therapy to prevent prostate cancer (PCa) progression to castrate-resistant PCa (CRPCa) remains an unmet medical need, mainly due to a poor understanding of the mechanism of PCa progression. Reactive oxygen species (ROS) are produced in high amounts in PCa cells. ROS play a major role in the development and progression of PCa. We have published that the activated androgen receptor (AR)-JunD complex induces spermidine/spermine N1-acetyl transferase (SSAT). SSAT is the first, and a regulatory, enzyme in a major polyamine catabolism pathway that leads to an over-production of ROS, specifically in the polyamine-rich PCa cells. A focus of our current research is to identify compounds that specifically target and block steps in this newly discovered ROS production pathway downstream to AR activation. Such compounds have the potential to become new targeted therapeutic agents offering minimal side effects for preventing progression to CRPCa in patients with early stage progressing PCa. Here we present data on drug-like small molecule inhibitors of the AR-JunD interaction that initiates this ROS-generating pathway in PCa. A novel high throughput assay based on Gaussia Luciferase enzyme reconstitution via protein-protein interaction was used to screen NCI Diversity Set and Life Chemicals libraries of drug-like small molecules to identify inhibitors of the AR-JunD interaction. The 27 selected hits were categorized as antiandrogens or non-antiandrogens through the use of a published fluorescence polarization assay. Sixteen non-antiandrogens, acting downstream of AR activation, were chosen for further study. In vitro studies entailed the measurement of the ability of the compounds to inhibit androgen-independent and dependent growth as well as androgen-induced ROS in human PCa cells. The top four compounds exhibited significant inhibition of growth and androgen-induced ROS at concentrations of 5 uM or less. Immunocytochemistry (ICC) studies were performed to determine the ability of the selected compounds to specifically inhibit the AR-JunD co-translocation to the nuclei. Based on the cell culture and ICC data, lead compounds were selected for pharmacokinetics studies to determine oral bioavailability. Detailed pharmacokinetic data of selected top compounds showing their bioavailability after intra venous and oral administration will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2844. doi:1538-7445.AM2012-2844

Molecular docking studies of protein-nucleotide complexes using MOLSDOCK (mutually orthogonal Latin squares DOCK)

Understanding the principles of protein receptor recognition, interaction, and association with molecular substrates and inhibitors is of principal importance in the drug discovery process. MOLSDOCK is a molecular docking method that we have recently developed. It uses mutually orthogonal Latin square sampling (together with a variant of the mean field technique) to identify the optimal docking conformation and pose of a small molecule ligand in the appropriate receptor site. Here we report the application of this method to simultaneously identify both the low energy conformation and the one with the best pose in the case of 62 protein-bound nucleotide ligands. The experimental structures of all these complexes are known. We have compared our results with those obtained from two other well-known molecular docking software, viz. AutoDock 4.2.3 and GOLD 5.1. The results show that the MOLSDOCK method was able to sample a wide range of binding modes for these ligands and also scores them well.