WorldWideScience

Sample records for receptor protein interaction

  1. Receptors, G proteins, and their interactions

    NARCIS (Netherlands)

    Hollmann, Markus W.; Strumper, Danja; Herroeder, Susanne; Durieux, Marcel E.

    2005-01-01

    Membrane receptors coupling to intracellular G proteins (G protein-coupled receptors) form one of the major classes of membrane signaling proteins. They are of great importance to the practice of anesthesiology because they are involved in many systems of relevance to the specialty (cardiovascular

  2. VPAC receptors: structure, molecular pharmacology and interaction with accessory proteins.

    Science.gov (United States)

    Couvineau, Alain; Laburthe, Marc

    2012-05-01

    The vasoactive intestinal peptide (VIP) is a neuropeptide with wide distribution in both central and peripheral nervous systems, where it plays important regulatory role in many physiological processes. VIP displays a large biological functions including regulation of exocrine secretions, hormone release, fetal development, immune responses, etc. VIP appears to exert beneficial effect in neuro-degenerative and inflammatory diseases. The mechanism of action of VIP implicates two subtypes of receptors (VPAC1 and VPAC2), which are members of class B receptors belonging to the super-family of GPCR. This article reviews the current knowledge regarding the structure and molecular pharmacology of VPAC receptors. The structure-function relationship of VPAC1 receptor has been extensively studied, allowing to understand the molecular basis for receptor affinity, specificity, desensitization and coupling to adenylyl cyclase. Those studies have clearly demonstrated the crucial role of the N-terminal ectodomain (N-ted) of VPAC1 receptor in VIP recognition. By using different approaches including directed mutagenesis, photoaffinity labelling, NMR, molecular modelling and molecular dynamic simulation, it has been shown that the VIP molecule interacts with the N-ted of VPAC1 receptor, which is itself structured as a 'Sushi' domain. VPAC1 receptor also interacts with a few accessory proteins that play a role in cell signalling of receptors. Recent advances in the structural characterization of VPAC receptor and more generally of class B GPCRs will lead to the design of new molecules, which could have considerable interest for the treatment of inflammatory and neuro-degenerative diseases. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  3. PDZ domain-mediated interactions of G protein-coupled receptors with postsynaptic density protein 95

    DEFF Research Database (Denmark)

    Møller, Thor C; Wirth, Volker F; Roberts, Nina Ingerslev

    2013-01-01

    G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome. Their signaling is regulated by scaffold proteins containing PDZ domains, but although these interactions are important for GPCR function, they are still poorly understood. We here present...... a quantitative characterization of the kinetics and affinity of interactions between GPCRs and one of the best characterized PDZ scaffold proteins, postsynaptic density protein 95 (PSD-95), using fluorescence polarization (FP) and surface plasmon resonance (SPR). By comparing these in vitro findings....... The approach can easily be transferred to other receptors and scaffold proteins and this could help accelerate the discovery and quantitative characterization of GPCR-PDZ interactions....

  4. Treponema pallidum receptor binding proteins interact with fibronectin

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, K.M.; Baseman, J.B.; Alderete, J.F.

    1983-06-01

    Analysis of plasma proteins avidly bound to T. pallidum surfaces revealed the ability of T. pallidum to acquire numerous host macromolecules. No acquisition was evident by the avirulent spirochete, T. phagedenis biotype Reiter. Western blotting technology using hyperimmune antifibronectin serum as a probe revealed the ability of virulent treponemes to avidly bind fibronectin from a complex medium such as plasma. The specificity of the tiplike adherence of motile T. pallidum to fibronectin-coated glass surfaces and to fibronectin on HEp-2 cells was reinforced by the observation that pretreatment of coverslips or cell monolayers with monospecific antiserum against fibronectin substantially reduced T. pallidum attachment. The stoichiometric binding of T. pallidum to fibronectin-coated coverslips and the inability of unlabeled or /sup 35/S-radiolabeled treponemes to interact with glass surfaces treated with other plasma proteins further established the specific nature of the interaction between virulent T. pallidum and fibronectin. The avid association between three outer envelope proteins of T. pallidum and fibronectin was also demonstrated. These treponemal surface proteins have been previously identified as putative receptor-binding proteins responsible for T. pallidum parasitism of host cells. The data suggest that surface fibronectin mediates tip-oriented attachment of T. pallidum to host cells via a receptor-ligand mechanism of recognition.

  5. In silico interaction analysis of cannabinoid receptor interacting protein 1b (CRIP1b) - CB1 cannabinoid receptor.

    Science.gov (United States)

    Singh, Pratishtha; Ganjiwale, Anjali; Howlett, Allyn C; Cowsik, Sudha M

    2017-10-01

    Cannabinoid Receptor Interacting Protein isoform 1b (CRIP1b) is known to interact with the CB1 receptor. Alternative splicing of the CNRIP1 gene produces CRIP1a and CRIP1b with a difference in the third exon only. Exons 1 and 2 encode for a functional domain in both proteins. CRIP1a is involved in regulating CB1 receptor internalization, but the function of CRIP1b is not very well characterized. Since there are significant identities in functional domains of these proteins, CRIP1b is a potential target for drug discovery. We report here predicted structure of CRIP1b followed by its interaction analysis with CB1 receptor by in-silico methods A number of complementary computational techniques, including, homology modeling, ab-initio and protein threading, were applied to generate three-dimensional molecular models for CRIP1b. The computed model of CRIP1b was refined, followed by docking with C terminus of CB1 receptor to generate a model for the CRIP1b- CB1 receptor interaction. The structure of CRIP1b obtained by homology modelling using RHO_GDI-2 as template is a sandwich fold structure having beta sheets connected by loops, similar to predicted CRIP1a structure. The best scoring refined model of CRIP1b in complex with the CB1 receptor C terminus peptide showed favourable polar interactions. The overall binding pocket of CRIP1b was found to be overlapping to that of CRIP1a. The Arg82 and Cys126 of CRIP1b are involved in the majority of hydrogen bond interactions with the CB1 receptor and are possible key residues required for interactions between the CB1 receptor and CRIP1b. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Plant nuclear hormone receptors: a role for small molecules in protein-protein interactions.

    Science.gov (United States)

    Lumba, Shelley; Cutler, Sean; McCourt, Peter

    2010-01-01

    Plant hormones are a group of chemically diverse small molecules that direct processes ranging from growth and development to biotic and abiotic stress responses. Surprisingly, genome analyses suggest that classic animal nuclear hormone receptor homologs do not exist in plants. It now appears that plants have co-opted several protein families to perceive hormones within the nucleus. In one solution to the problem, the hormones auxin and jasmonate (JA) act as “molecular glue” that promotes protein-protein interactions between receptor F-boxes and downstream corepressor targets. In another solution, gibberellins (GAs) bind and elicit a conformational change in a novel soluble receptor family related to hormone-sensitive lipases. Abscisic acid (ABA), like GA, also acts through an allosteric mechanism involving a START-domain protein. The molecular identification of plant nuclear hormone receptors will allow comparisons with animal nuclear receptors and testing of fundamental questions about hormone function in plant development and evolution.

  7. Moonlighting Proteins and Protein–Protein Interactions as Neurotherapeutic Targets in the G Protein-Coupled Receptor Field

    Science.gov (United States)

    Fuxe, Kjell; Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Palkovits, Miklós; Tarakanov, Alexander O; Ciruela, Francisco; Agnati, Luigi F

    2014-01-01

    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. PMID:24105074

  8. Direct interactions between calcitonin-like receptor (CLR) and CGRP-receptor component protein (RCP) regulate CGRP receptor signaling.

    Science.gov (United States)

    Egea, Sophie C; Dickerson, Ian M

    2012-04-01

    Calcitonin gene-related peptide (CGRP) is a neuropeptide with multiple neuroendocrine roles, including vasodilation, migraine, and pain. The receptor for CGRP is a G protein-coupled receptor (GPCR) that requires three proteins for function. CGRP binds to a heterodimer composed of the GPCR calcitonin-like receptor (CLR) and receptor activity-modifying protein (RAMP1), a single transmembrane protein required for pharmacological specificity and trafficking of the CLR/RAMP1 complex to the cell surface. In addition, the CLR/RAMP1 complex requires a third protein named CGRP-receptor component protein (RCP) for signaling. Previous studies have demonstrated that depletion of RCP from cells inhibits CLR signaling, and in vivo studies have demonstrated that expression of RCP correlates with CLR signaling and CGRP efficacy. It is not known whether RCP interacts directly with CLR to exert its effect. The current studies identified a direct interaction between RCP and an intracellular domain of CLR using yeast two-hybrid analysis and coimmunoprecipitation. When this interacting domain of CLR was expressed as a soluble fusion protein, it coimmunoprecipitated with RCP and inhibited signaling from endogenous CLR. Expression of this dominant-negative domain of CLR did not significantly inhibit trafficking of CLR to the cell surface, and thus RCP may not have a chaperone function for CLR. Instead, RCP may regulate CLR signaling in the cell membrane, and direct interaction between RCP and CLR is required for CLR activation. To date, RCP has been found to interact only with CLR and represents a novel neuroendocrine regulatory step in GPCR signaling.

  9. Characterization of G-protein coupled receptor kinase interaction with the neurokinin-1 receptor using bioluminescence resonance energy transfer

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Holliday, Nicholas D; Hansen, Jakob L

    2007-01-01

    To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive muta......To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase...

  10. Receptor recruitment: A mechanism for interactions between G protein-coupled receptors

    OpenAIRE

    Holtbäck, Ulla; Brismar, Hjalmar; DiBona, Gerald F.; Fu, Michael; Greengard, Paul; Aperia, Anita

    1999-01-01

    There is a great deal of evidence for synergistic interactions between G protein-coupled signal transduction pathways in various tissues. As two specific examples, the potent effects of the biogenic amines norepinephrine and dopamine on sodium transporters and natriuresis can be modulated by neuropeptide Y and atrial natriuretic peptide, respectively. Here, we report, using a renal epithelial cell line, that both types of modulation involve recruitment of receptors from the interior of the ce...

  11. Cannabinoid receptor-interacting protein Crip1a modulates CB1 receptor signaling in mouse hippocampus.

    Science.gov (United States)

    Guggenhuber, Stephan; Alpar, Alan; Chen, Rongqing; Schmitz, Nina; Wickert, Melanie; Mattheus, Tobias; Harasta, Anne E; Purrio, Martin; Kaiser, Nadine; Elphick, Maurice R; Monory, Krisztina; Kilb, Werner; Luhmann, Heiko J; Harkany, Tibor; Lutz, Beat; Klugmann, Matthias

    2016-05-01

    The cannabinoid type 1 receptor (Cnr1, CB1R) mediates a plethora of physiological functions in the central nervous system as a presynaptic modulator of neurotransmitter release. The recently identified cannabinoid receptor-interacting protein 1a (Cnrip1a, CRIP1a) binds to the C-terminal domain of CB1R, a region known to be important for receptor desensitization and internalization. Evidence that CRIP1a and CB1R interact in vivo has been reported, but the neuroanatomical distribution of CRIP1a is unknown. Moreover, while alterations of hippocampal CRIP1a levels following limbic seizures indicate a role in controlling excessive neuronal activity, the physiological function of CRIP1a in vivo has not been investigated. In this study, we analyzed the spatial distribution of CRIP1a in the hippocampus and examined CRIP1a as a potential modulator of CB1R signaling. We found that Cnrip1a mRNA is co-expressed with Cnr1 mRNA in pyramidal neurons and interneurons of the hippocampal formation. CRIP1a protein profiles were largely segregated from CB1R profiles in mossy cell terminals but not in hippocampal CA1 region. CB1R activation induced relocalization to close proximity with CRIP1a. Adeno-associated virus-mediated overexpression of CRIP1a specifically in the hippocampus revealed that CRIP1a modulates CB1R activity by enhancing cannabinoid-induced G protein activation. CRIP1a overexpression extended the depression of excitatory currents by cannabinoids in pyramidal neurons of the hippocampus and diminished the severity of chemically induced acute epileptiform seizures. Collectively, our data indicate that CRIP1a enhances hippocampal CB1R signaling in vivo.

  12. Cannabinoid CB1 receptor-interacting proteins: novel targets for central nervous system drug discovery?

    Science.gov (United States)

    Smith, Tricia H; Sim-Selley, Laura J; Selley, Dana E

    2010-01-01

    The main pharmacological effects of marijuana, as well as synthetic and endogenous cannabinoids, are mediated through G-protein-coupled receptors (GPCRs), including CB1 and CB2 receptors. The CB1 receptor is the major cannabinoid receptor in the central nervous system and has gained increasing interest as a target for drug discovery for treatment of nausea, cachexia, obesity, pain, spasticity, neurodegenerative diseases and mood and substance abuse disorders. Evidence has accumulated to suggest that CB1 receptors, like other GPCRs, interact with and are regulated by several other proteins beyond the established role of heterotrimeric G-proteins. These proteins, which include the GPCR kinases, β-arrestins, GPCR-associated sorting proteins, factor associated with neutral sphingomyelinase, other GPCRs (heterodimerization) and the novel cannabinoid receptor-interacting proteins: CRIP1a/b, are thought to play important roles in the regulation of intracellular trafficking, desensitization, down-regulation, signal transduction and constitutive activity of CB1 receptors. This review examines CB1 receptor-interacting proteins, including heterotrimeric G-proteins, but with particular emphasis on non-G-protein entities, that might comprise the CB1 receptosomal complex. The evidence for direct interaction with CB1 receptors and potential functional roles of these interacting proteins is discussed, as are future directions and challenges in this field with an emphasis on the possibility of eventually targeting these proteins for drug discovery. This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x PMID:20590557

  13. Cannabinoid receptor-interacting protein 1a modulates CB1 receptor signaling and regulation.

    Science.gov (United States)

    Smith, Tricia H; Blume, Lawrence C; Straiker, Alex; Cox, Jordan O; David, Bethany G; McVoy, Julie R Secor; Sayers, Katherine W; Poklis, Justin L; Abdullah, Rehab A; Egertová, Michaela; Chen, Ching-Kang; Mackie, Ken; Elphick, Maurice R; Howlett, Allyn C; Selley, Dana E

    2015-04-01

    Cannabinoid CB1 receptors (CB1Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca(2+) channel activity. We now demonstrate cellular colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated CB1R-mediated guanine nucleotide-binding regulatory protein (G-protein) activity. Stable overexpression of CRIP1a in human embryonic kidney (HEK)-293 cells stably expressing CB1Rs (CB1-HEK), or in N18TG2 cells endogenously expressing CB1Rs, decreased CB1R-mediated G-protein activation (measured by agonist-stimulated [(35)S]GTPγS (guanylyl-5'-[O-thio]-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, small-interfering RNA-mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not attributable to differences in CB1R expression or endocannabinoid tone because CB1R levels did not differ between cell lines varying in CRIP1a expression, and endocannabinoid levels were undetectable (CB1-HEK) or unchanged (N18TG2) by CRIP1a overexpression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated [(35)S]GTPγS binding. CRIP1a overexpression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP1a inhibits constitutive CB1R activity and demonstrate that CRIP1a can also inhibit agonist

  14. The Use of FRET/FLIM to Study Proteins Interacting with Plant Receptor Kinases.

    Science.gov (United States)

    Weidtkamp-Peters, Stefanie; Stahl, Yvonne

    2017-01-01

    The investigation of protein interactions in living plant tissue has become of increasing importance in recent years. A high spatial and temporal resolution for the observation of in vivo protein interaction is needed, e.g., in order to follow changes of plant receptor kinase interactions and complex formation over time. In vivo fluorescence or Förster resonance energy transfer (FRET) measurements allow for detailed analyses of interacting proteins in their natural environment at a subcellular level. Especially FRET-FLIM (fluorescence lifetime imaging microscopy) measurements provide an extremely powerful and reliable tool meeting the demands for investigating in vivo protein interaction quantitatively and with high precision. Here, we will describe in detail how to practically perform in vivo FRET measurements of receptor kinases in plants and discuss potential pitfalls and points of consideration.

  15. Identification of a novel protein-protein interaction motif mediating interaction of GPCR-associated sorting proteins with G protein-coupled receptors

    DEFF Research Database (Denmark)

    Bornert, Olivier; Møller, Thor Christian; Boeuf, Julien

    2013-01-01

    GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degra......GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward...... the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure-function relationship analysis of the molecular interaction...... between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence...

  16. Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions

    DEFF Research Database (Denmark)

    Di Roberto, Raphaël B; Chang, Belinda; Trusina, Ala

    2016-01-01

    All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae......, Ste2 is a hub in a network of interactions controlling both signal transduction and signal suppression. Through laboratory evolution, we obtained 21 mutant receptors sensitive to the pheromone of a related yeast species and investigated the molecular mechanisms behind this newfound sensitivity. While...... some mutants show enhanced binding affinity to the foreign pheromone, others only display weakened interactions with the network's negative regulators. Importantly, the latter changes have a limited impact on overall pathway regulation, despite their considerable effect on sensitivity. Our results...

  17. Protein interactions among Fe65, the low-density lipoprotein receptor-related protein, and the amyloid precursor protein.

    Science.gov (United States)

    Mulvihill, Melinda M; Guttman, Miklos; Komives, Elizabeth A

    2011-07-19

    The adapter protein Fe65 has been proposed to be the link between the intracellular domains of the amyloid precursor protein, APP (AICD), and the low-density lipoprotein receptor-related protein (LRP-CT). Functional linkage between these two proteins has been established, and mutations within LRP-CT affect the amount of Aβ produced from APP. Previous work showed that AICD binds to protein interaction domain 2 (PID2) of Fe65. Although the structure of PID1 was determined recently, all attempts to demonstrate LRP-CT binding to this domain failed. We used biophysical experiments and binding studies to investigate the binding among these three proteins. Full-length Fe65 bound more weakly to AICD than did N-terminally truncated forms; however, the intramolecular domain-domain interactions that had been proposed to inhibit binding could not be observed using amide H-D exchange. Surprisingly, when LRP-CT is phosphorylated at Tyr4507, it bound to Fe65 PID1 despite the fact that this domain belongs to the Dab-like subclass of PIDs that are not supposed to be phosphorylation-dependent. Mutation of a critical arginine abolished binding, providing further proof of the phosphorylation dependence. Fe65 PID1 thus provides a link between the Dab-like class and the IRS-like class of PIDs and is the first Dab-like family member to show phosphorylation-dependent binding.

  18. An androgen receptor NH2-terminal conserved motif interacts with the COOH terminus of the Hsp70-interacting protein (CHIP).

    Science.gov (United States)

    He, Bin; Bai, Suxia; Hnat, Andrew T; Kalman, Rebecca I; Minges, John T; Patterson, Cam; Wilson, Elizabeth M

    2004-07-16

    The NH2-terminal sequence of steroid receptors is highly variable between different receptors and in the same receptor from different species. In this study, a primary sequence homology comparison identified a 14-amino acid NH2-terminal motif of the human androgen receptor (AR) that is common to AR from all species reported, including the lower vertebrates. The evolutionarily conserved motif is unique to AR, with the exception of a partial sequence in the glucocorticoid receptor of higher species. The presence of the conserved motif in AR and the glucocorticoid receptor and its absence in other steroid receptors suggests convergent evolution. The function of the AR NH2-terminal conserved motif was suggested from a yeast two-hybrid screen that identified the COOH terminus of the Hsp70-interacting protein (CHIP) as a binding partner. We found that CHIP functions as a negative regulator of AR transcriptional activity by promoting AR degradation. In support of this, two mutations in the AR NH2-terminal conserved motif previously identified in the transgenic adenocarcinoma of mouse prostate model reduced the interaction between CHIP and AR. Our results suggest that the AR NH2-terminal domain contains an evolutionarily conserved motif that functions to limit AR transcriptional activity. Moreover, we demonstrate that the combination of comparative sequence alignment and yeast two-hybrid screening using short conserved peptides as bait provides an effective strategy to probe the structure-function relationships of steroid receptor NH2-terminal domains and other intrinsically unstructured transcriptional regulatory proteins.

  19. Interactions of the NPXY microdomains of the low density lipoprotein receptor-related protein 1.

    Science.gov (United States)

    Guttman, Miklos; Betts, Gina N; Barnes, Helen; Ghassemian, Majid; van der Geer, Peter; Komives, Elizabeth A

    2009-11-01

    The low density lipoprotein receptor-related protein 1 (LRP1) mediates internalization of a large number of proteins and protein-lipid complexes and is widely implicated in Alzheimer's disease. The cytoplasmic domain of LRP1 (LRP1-CT) can be phosphorylated by activated protein-tyrosine kinases at two NPXY motifs in LRP1-CT; Tyr 4507 is readily phosphorylated and must be phosphorylated before phosphorylation of Tyr 4473 occurs. Pull-down experiments from brain lysate revealed numerous proteins binding to LRP1-CT, but the results were highly variable. To separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non-phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC-MS/MS, and confirmed by Western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY(4507) (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine-phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY(4473) (membrane proximal) bound many fewer proteins and only to the phosphorylated form.

  20. Interactions of the NPXY microdomains of the LDL Receptor-Related Protein 1

    Science.gov (United States)

    Guttman, Miklos; Betts, Gina N.; Barnes, Helen; Ghassemian, Majid; van der Geer, Peter; Komives, Elizabeth A.

    2010-01-01

    The LDL receptor-related protein 1 (LRP1) mediates internalization of a large number of proteins and protein-lipid complexes and is widely implicated in Alzheimer's disease. The cytoplasmic domain of LRP1 (LRP1-CT) can be phosphorylated by activated protein-tyrosine kinases at two NPXY motifs in LRP1-CT; Tyr 4507 is readily phosphorylated and must be phosphorylated before phosphorylation of Tyr 4473 occurs. Pull-down experiments from brain lysate revealed numerous proteins binding to LRP1-CT, but the results were highly variable. To separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non-phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC-MS/MS, and confirmed by western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY4507 (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine-phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY4473 (membrane proximal) bound many fewer proteins and only to the phosphorylated form. PMID:19771558

  1. Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET)

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Hansen, Jakob L; Sheikh, Søren P

    2002-01-01

    The calcium-sensing receptor (CaR) belongs to family C of the G-protein coupled receptor superfamily. The receptor is believed to exist as a homodimer due to covalent and non-covalent interactions between the two amino terminal domains (ATDs). It is well established that agonist binding to family C......-induced intermolecular movements in the CaR homodimer using the new bioluminescence resonance energy transfer technique, BRET2, which is based on the transference of energy from Renilla luciferase (Rluc) to the green fluorescent protein mutant GFP2. We tagged CaR with Rluc and GFP2 at different intracellular locations....... Stable and highly receptor-specific BRET signals were obtained in tsA cells transfected with Rluc- and GFP2-tagged CaRs under basal conditions, indicating that CaR is constitutively dimerized. However, the signals were not enhanced by the presence of agonist. These results could indicate that at least...

  2. Factor VIII interacts with the endocytic receptor low-density lipoprotein receptor-related protein 1 via an extended surface comprising "hot-spot" lysine residues

    NARCIS (Netherlands)

    Van Den Biggelaar, Maartje|info:eu-repo/dai/nl/304831433; Madsen, Jesper J.; Faber, Johan H.; Zuurveld, Marleen G.; Van Der Zwaan, Carmen; Olsen, Ole H.; Stennicke, Henning R.; Mertens, Koen|info:eu-repo/dai/nl/070940258; Meijer, Alexander B.

    2015-01-01

    Background: It is unclear how the LDL receptor family binds large protein ligands. Results: HDX and lysine scanning identified factor (F)VIII regions and specific lysine residues binding low-density lipoprotein receptor-related protein 1 (LRP1). Conclusion: FVIII-LRP1 interaction involves multiple

  3. Borna disease virus P protein affects neural transmission through interactions with gamma-aminobutyric acid receptor-associated protein.

    Science.gov (United States)

    Peng, Guiqing; Yan, Yan; Zhu, Chengliang; Wang, Shiqun; Yan, Xiaohong; Lu, Lili; Li, Wei; Hu, Jing; Wei, Wei; Mu, Yongxin; Chen, Yanni; Feng, Yong; Gong, Rui; Wu, Kailang; Zhang, Fengmin; Zhang, Xiaolian; Zhu, Ying; Wu, Jianguo

    2008-12-01

    Borna disease virus (BDV) is one of the infectious agents that causes diseases of the central nervous system in a wide range of vertebrate species and, perhaps, in humans. The phosphoprotein (P) of BDV, an essential cofactor of virus RNA-dependent RNA polymerase, is required for virus replication. In this study, we identified the gamma-aminobutyric acid receptor-associated protein (GABARAP) with functions in neurobiology as one of the viral P protein-interacting cellular factors by using an approach of phage display-based protein-protein interaction analysis. Direct binding between GABARAP and P protein was confirmed by coimmunoprecipitation, protein pull-down, and mammalian two-hybrid analyses. GABARAP originally was identified as a linker between the gamma-aminobutyric acid receptor (GABAR) and the microtubule to regulate receptor trafficking and plays important roles in the regulation of the inhibitory neural transmitter gamma-aminobutyric acid (GABA). We showed that GABARAP colocalizes with P protein in the cells infected with BDV or transfected with the P gene, which resulted in shifting the localization of GABARAP from the cytosol to the nucleus. We further demonstrated that P protein blocks the trafficking of GABAR, a principal GABA-gated ion channel that plays important roles in neural transmission, to the surface of cells infected with BDV or transfected with the P gene. We proposed that during BDV infection, P protein binds to GABARAP, shifts the distribution of GABARAP from the cytoplasm to the nucleus, and disrupts the trafficking of GABARs to the cell membranes, which may result in the inhibition of GABA-induced currents and in the enhancement of hyperactivity and anxiety.

  4. Interaction between G Protein-Coupled Receptor 143 and Tyrosinase: Implications for Understanding Ocular Albinism Type 1.

    Science.gov (United States)

    De Filippo, Elisabetta; Schiedel, Anke C; Manga, Prashiela

    2017-02-01

    Developmental eye defects in X-linked ocular albinism type 1 are caused by G-protein coupled receptor 143 (GPR143) mutations. Mutations result in dysfunctional melanosome biogenesis and macromelanosome formation in pigment cells, including melanocytes and retinal pigment epithelium. GPR143, primarily expressed in pigment cells, localizes exclusively to endolysosomal and melanosomal membranes unlike most G protein-coupled receptors, which localize to the plasma membrane. There is some debate regarding GPR143 function and elucidating the role of this receptor may be instrumental for understanding neurogenesis during eye development and for devising therapies for ocular albinism type I. Many G protein-coupled receptors require association with other proteins to function. These G protein-coupled receptor-interacting proteins also facilitate fine-tuning of receptor activity and tissue specificity. We therefore investigated potential GPR143 interaction partners, with a focus on the melanogenic enzyme tyrosinase. GPR143 coimmunoprecipitated with tyrosinase, while confocal microscopy demonstrated colocalization of the proteins. Furthermore, tyrosinase localized to the plasma membrane when coexpressed with a GPR143 trafficking mutant. The physical interaction between the proteins was confirmed using fluorescence resonance energy transfer. This interaction may be required in order for GPR143 to function as a monitor of melanosome maturation. Identifying tyrosinase as a potential GPR143 binding protein opens new avenues for investigating the mechanisms that regulate pigmentation and neurogenesis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Cannabinoid Receptor–Interacting Protein 1a Modulates CB1 Receptor Signaling and Regulation

    OpenAIRE

    Smith, Tricia H.; Blume, Lawrence C.; Straiker, Alex; Cox, Jordan O.; David, Bethany G.; McVoy, Julie R. Secor; Sayers, Katherine W.; Poklis, Justin L.; Abdullah, Rehab A; Egertová, Michaela; Chen, Ching-Kang; Mackie, Ken; Elphick, Maurice R.; Howlett, Allyn C.; Selley, Dana E.

    2015-01-01

    Cannabinoid CB1 receptors (CB1Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor–interacting protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca2+ channel activity. We now demonstrate cellular colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated ...

  6. Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and β-Arrestins 2.

    Directory of Open Access Journals (Sweden)

    D Malfacini

    Full Text Available Nociceptin/orphanin FQ (N/OFQ controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP. Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells. In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ1 or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.

  7. Interaction of fish aryl hydrocarbon receptor paralogs (AHR1 and AHR2) with the retinoblastoma protein

    Energy Technology Data Exchange (ETDEWEB)

    Merson, Rebeka R., E-mail: rmerson@ric.edu [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Biology Department, Rhode Island College, 500 Mt. Pleasant Ave., Providence, RI 02908 (United States); Karchner, Sibel I.; Hahn, Mark E. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States)

    2009-08-13

    The aryl hydrocarbon receptor (AHR) mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. In some mammalian cell lines, TCDD induces G1 cell cycle arrest, which depends on an interaction between the AHR and the retinoblastoma tumor suppressor (RB). Mammals possess one AHR, whereas fishes possess two or more AHR paralogs that differ in the domains important for AHR-RB interactions in mammals. To test the hypothesis that fish AHR paralogs differ in their ability to interact with RB, we cloned RB cDNA from Atlantic killifish, Fundulus heteroclitus, and studied the interactions of killifish RB protein with killifish AHR1 and AHR2. In coimmunoprecipitation experiments, in vitro-expressed killifish RB coprecipitated with both AHR1 and AHR2. Consistent with these results, both killifish AHR1 and AHR2 interacted with RB in mammalian two-hybrid assays. These results suggest that both fish AHR1 and AHR2 paralogs may have the potential to influence cell proliferation through interactions with RB.

  8. On the G-Protein-Coupled Receptor Heteromers and Their Allosteric Receptor-Receptor Interactions in the Central Nervous System: Focus on Their Role in Pain Modulation

    Directory of Open Access Journals (Sweden)

    Dasiel O. Borroto-Escuela

    2013-01-01

    Full Text Available The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The delta opioid receptor (DOR exerts an antagonistic allosteric influence on the mu opioid receptor (MOR function in a MOR-DOR heteromer. This heteromer contributes to morphine-induced tolerance and dependence, since it becomes abundant and develops a reduced G-protein-coupling with reduced signaling mainly operating via β-arrestin2 upon chronic morphine treatment. A DOR antagonist causes a return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. The gender- and ovarian steroid-dependent recruitment of spinal cord MOR/kappa opioid receptor (KOR heterodimers enhances antinociceptive functions and if impaired could contribute to chronic pain states in women. MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR in the spinal cord, mediating morphine induced itch. Other mechanism for the antinociceptive actions of acupuncture along meridians may be that it enhances the cross-desensitization of the TRPA1 (chemical nociceptor-TRPV1 (capsaicin receptor heteromeric channel complexes within the nociceptor terminals located along these meridians. Selective ionotropic cannabinoids may also produce cross-desensitization of the TRPA1-TRPV1 heteromeric nociceptor channels by being negative allosteric modulators of these channels leading to antinociception and antihyperalgesia.

  9. Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation*

    Science.gov (United States)

    Lalo, Ulyana; Roberts, Jonathan A.; Evans, Richard J.

    2011-01-01

    P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation. PMID:21757694

  10. Challenges predicting ligand-receptor interactions of promiscuous proteins: the nuclear receptor PXR.

    Directory of Open Access Journals (Sweden)

    Sean Ekins

    2009-12-01

    Full Text Available Transcriptional regulation of some genes involved in xenobiotic detoxification and apoptosis is performed via the human pregnane X receptor (PXR which in turn is activated by structurally diverse agonists including steroid hormones. Activation of PXR has the potential to initiate adverse effects, altering drug pharmacokinetics or perturbing physiological processes. Reliable computational prediction of PXR agonists would be valuable for pharmaceutical and toxicological research. There has been limited success with structure-based modeling approaches to predict human PXR activators. Slightly better success has been achieved with ligand-based modeling methods including quantitative structure-activity relationship (QSAR analysis, pharmacophore modeling and machine learning. In this study, we present a comprehensive analysis focused on prediction of 115 steroids for ligand binding activity towards human PXR. Six crystal structures were used as templates for docking and ligand-based modeling approaches (two-, three-, four- and five-dimensional analyses. The best success at external prediction was achieved with 5D-QSAR. Bayesian models with FCFP_6 descriptors were validated after leaving a large percentage of the dataset out and using an external test set. Docking of ligands to the PXR structure co-crystallized with hyperforin had the best statistics for this method. Sulfated steroids (which are activators were consistently predicted as non-activators while, poorly predicted steroids were docked in a reverse mode compared to 5alpha-androstan-3beta-ol. Modeling of human PXR represents a complex challenge by virtue of the large, flexible ligand-binding cavity. This study emphasizes this aspect, illustrating modest success using the largest quantitative data set to date and multiple modeling approaches.

  11. Interaction between the p21ras GTPase activating protein and the insulin receptor

    NARCIS (Netherlands)

    Pronk, G.J.; Medema, R.H.; Burgering, B.M.T.; Clark, R.; McCormick, F.; Bos, J.L.

    1992-01-01

    We investigated the involvement of the p21ras-GTPase activating protein (GAP) in insulin-induced signal transduction. In cells overexpressing the insulin receptor, we did not observe association between GAP and the insulin receptor after insulin treatment nor the phosphorylation of GAP on tyrosine

  12. The identification of novel proteins that interact with the GLP-1 receptor and restrain its activity.

    Science.gov (United States)

    Huang, X; Dai, F F; Gaisano, G; Giglou, K; Han, J; Zhang, M; Kittanakom, S; Wong, V; Wei, L; Showalter, A D; Sloop, K W; Stagljar, I; Wheeler, M B

    2013-09-01

    Glucagon-like peptide 1 receptor (GLP-1R) controls diverse physiological functions in tissues including the pancreatic islets, brain, and heart. To understand the mechanisms that control glucagon-like peptide 1 (GLP-1) signaling better, we sought to identify proteins that interact with the GLP-1R using a membrane-based split ubiquitin yeast two-hybrid (MYTH) assay. A screen of a human fetal brain cDNA prey library with an unliganded human GLP-1R as bait in yeast revealed 38 novel interactor protein candidates. These interactions were confirmed in mammalian Chinese hamster ovarian cells by coimmunoprecipitation. Immunofluorescence was used to show subcellular colocalization of the interactors with GLP-1R. Cluster analysis revealed that the interactors were primarily associated with signal transduction, metabolism, and cell development. When coexpressed with the GLP-1R in Chinese hamster ovarian cells, 15 interactors significantly altered GLP-1-induced cAMP accumulation. Surprisingly, all 15 proteins inhibited GLP-1-activated cAMP. Given GLP-1's prominent role as an incretin, we then focused on 3 novel interactors, SLC15A4, APLP1, and AP2M1, because they are highly expressed and localized to the membrane in mouse insulinoma β-cells. Small interfering RNA-mediated knockdown of each candidate gene significantly enhanced GLP-1-induced insulin secretion. In conclusion, we have generated a novel GLP-1R-protein interactome, identifying several interactors that suppress GLP-1R signaling. We suggest that the inhibition of these interactors may serve as a novel strategy to enhance GLP-1R activity.

  13. Co-expression of activin receptor-interacting protein 1 and 2 in mouse nerve cells.

    Science.gov (United States)

    Qi, Yan; Ge, Jing-Yan; Wang, Yi-Nan; Liu, Hai-Yan; Li, Yun-Man; Liu, Zhong-Hui; Cui, Xue-Ling

    2013-05-10

    Activin is a neurotrophic and neuroprotective factor in the central nervous system. Activin receptor-interacting protein 1 and 2 (ARIP1 and ARIP2) are identified as activin signal proteins in mouse brain. However, whether ARIP1 and ARIP2 are co-expressed in nerve cells and the differences of their biological activities are not well characterized. In the present study, we found that ARIP1 and ARIP2 mRNA expressions were detectable in mouse brain and their proteins were co-localized at the hypothalamus of cerebrum and granular layers in cerebellum, especially in Purkinje cells. Furthermore, ARIP1 and ARIP2 were co-expressed in mouse Neuro-2a cells, which is similar to the co-localization of ARIP1 and ARIP2 in hypothalamus neurons and Purkinje cells. Overexpression of ARIP1 in Neuro-2a cells inhibited activin signal transduction induced by activin A and Smad3, and activin A-induced voltage-gated Na(+) current (INa), while ARIP2 was only a negative regulator of signal transduction induced by activin A and did not alter activin A-induced INa. Taken together, these data demonstrate that ARIP1 and ARIP2 are co-expressed in some nerve cells and their biological activities are distinct. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  14. Design, synthesis, and validation of a β-turn mimetic library targeting protein-protein and peptide-receptor interactions.

    Science.gov (United States)

    Whitby, Landon R; Ando, Yoshio; Setola, Vincent; Vogt, Peter K; Roth, Bryan L; Boger, Dale L

    2011-07-06

    The design and synthesis of a β-turn mimetic library as a key component of a small-molecule library targeting the major recognition motifs involved in protein-protein interactions is described. Analysis of a geometric characterization of 10,245 β-turns in the protein data bank (PDB) suggested that trans-pyrrolidine-3,4-dicarboxamide could serve as an effective and synthetically accessible library template. This was confirmed by initially screening select compounds against a series of peptide-activated GPCRs that recognize a β-turn structure in their endogenous ligands. This validation study was highlighted by identification of both nonbasic and basic small molecules with high affinities (K(i) = 390 and 23 nM, respectively) for the κ-opioid receptor (KOR). Consistent with the screening capabilities of collaborators and following the design validation, the complete library was assembled as 210 mixtures of 20 compounds, providing a total of 4200 compounds designed to mimic all possible permutations of 3 of the 4 residues in a naturally occurring β-turn. Unique to the design and because of the C(2) symmetry of the template, a typical 20 × 20 × 20-mix (8000 compounds prepared as 400 mixtures of 20 compounds) needed to represent 20 variations in the side chains of three amino acid residues reduces to a 210 × 20-mix, thereby simplifying the library synthesis and subsequent screening. The library was prepared using a solution-phase synthetic protocol with liquid-liquid or liquid-solid extractions for purification and conducted on a scale that insures its long-term availability for screening campaigns. Screening the library against the human opioid receptors (KOR, MOR, and DOR) identified not only the activity of library members expected to mimic the opioid receptor peptide ligands but also additional side-chain combinations that provided enhanced receptor binding selectivities (>100-fold) and affinities (as low as K(i) = 80 nM for KOR). A key insight to emerge from

  15. Interaction of Tomato Spotted Wilt Tospovirus (TSWV) Glycoproteins with a Thrips Midgut Protein, a Potential Cellular Receptor for TSWV.

    Science.gov (United States)

    Bandla, M D; Campbell, L R; Ullman, D E; Sherwood, J L

    1998-02-01

    ABSTRACT Interactions between viral and cellular membrane fusion proteins mediate virus penetration of cells for many arthropod-borne viruses. Electron microscope observations and circumstantial evidence indicate insect acquisition of tomato spotted wilt virus (TSWV) (genus Tospovirus, family Bunyaviridae) is receptor mediated, and TSWV membrane glycoproteins (GP1 and GP2) serve as virus attachment proteins. The tospoviruses are plant-infecting members of the family Bunyaviridae and are transmitted by several thrips species, including Frankliniella occidentalis. Gel overlay assays and immunolabeling were used to investigate the putative role of TSWV GPs as viral attachment proteins and deter mine whether a corresponding cellular receptor may be present in F. occidentalis. A single band in the 50-kDa region was detected with murine monoclonal antibodies (MAbs) to the TSWV-GPs when isolated TSWV or TSWV-GPs were used to overlay separated thrips proteins. This band was not detected when blots were probed with antibody to the non-structural protein encoded by the small RNA of TSWV or the TSWV nucleocapsid protein, nor were proteins from nonvector insects labeled. Anti-idiotype antibodies prepared to murine MAbs against GP1 or GP2 specifically labeled a single band at 50 kDa in Western blots and the plasmalemma of larval thrips midguts. These results support the putative role of the TSWV GPs as viral attachment proteins and identified potential cellular receptor(s) in thrips.

  16. Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

    NARCIS (Netherlands)

    Kasheverov, I.E.; Zhmak, M.N.; Fish, A.; Rucktooa, P.; Khruschov, A.Y.; Osipov, A.V.; Ziganshin, R.H.; D'Hoedt, D.; Bertrand, D.; Sixma, T.K.; Smit, A.B.; Tsetlin, V.I.

    2009-01-01

    α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7

  17. Coronavirus spike-receptor interactions

    NARCIS (Netherlands)

    Mou, H.

    2015-01-01

    Coronaviruses cause important diseases in humans and animals. Coronavirus infection starts with the virus binding with its spike proteins to molecules present on the surface of host cells that act as receptors. This spike-receptor interaction is highly specific and determines the virus’ cell, tissue

  18. Fluorescent Approaches for Understanding Interactions of Ligands with G Protein Coupled Receptors

    Science.gov (United States)

    Sridharan, Rajashri; Zuber, Jeffrey; Connelly, Sara M.; Mathew, Elizabeth; Dumont, Mark E.

    2014-01-01

    G Protein Coupled Receptors (GPCRs) are responsible for a wide variety of signaling responses in diverse cell types. Despite major advances in the determination of structures of this class of receptors, the underlying mechanisms by which binding of different types of ligands specifically elicits particular signaling responses remains unclear. The use of fluorescence spectroscopy can provide important information about the process of ligand binding and ligand dependent conformational changes in receptors, especially kinetic aspects of these processes, that can be difficult to extract from x-ray structures. We present an overview of the extensive array of fluorescent ligands that have been used in studies of GPCRs and describe spectroscopic approaches for assaying binding and probing the environment of receptor-bound ligands with particular attention to examples involving yeast pheromone receptors. In addition, we discuss the use of fluorescence spectroscopy for detecting and characterizing conformational changes in receptors induced by the binding of ligands. Such studies have provided strong evidence for diversity of receptor conformations elicited by different ligands, consistent with the idea that GPCRs are not simple on and off switches. This diversity of states constitutes an underlying mechanistic basis for biased agonism, the observation that different stimuli can produce different responses from a single receptor. It is likely that continued technical advances will allow fluorescence spectroscopy to play an important role in continued probing of structural transitions in GPCRs. PMID:24055822

  19. Signal transduction of Helicobacter pylori during interaction with host cell protein receptors of epithelial and immune cells

    Science.gov (United States)

    Pachathundikandi, Suneesh Kumar; Tegtmeyer, Nicole; Backert, Steffen

    2013-01-01

    Helicobacter pylori infections can induce pathologies ranging from chronic gastritis, peptic ulceration to gastric cancer. Bacterial isolates harbor numerous well-known adhesins, vacuolating cytotoxin VacA, protease HtrA, urease, peptidoglycan, and type IV secretion systems (T4SS). It appears that H. pylori targets more than 40 known host protein receptors on epithelial or immune cells. A series of T4SS components such as CagL, CagI, CagY, and CagA can bind to the integrin α5β1 receptor. Other targeted membrane-based receptors include the integrins αvβ3, αvβ5, and β2 (CD18), RPTP-α/β, GP130, E-cadherin, fibronectin, laminin, CD46, CD74, ICAM1/LFA1, T-cell receptor, Toll-like receptors, and receptor tyrosine kinases EGFR, ErbB2, ErbB3, and c-Met. In addition, H. pylori is able to activate the intracellular receptors NOD1, NOD2, and NLRP3 with important roles in innate immunity. Here we review the interplay of various bacterial factors with host protein receptors. The contribution of these interactions to signal transduction and pathogenesis is discussed. PMID:24280762

  20. Signal transduction of Helicobacter pylori during interaction with host cell protein receptors of epithelial and immune cells.

    Science.gov (United States)

    Pachathundikandi, Suneesh Kumar; Tegtmeyer, Nicole; Backert, Steffen

    2013-01-01

    Helicobacter pylori infections can induce pathologies ranging from chronic gastritis, peptic ulceration to gastric cancer. Bacterial isolates harbor numerous well-known adhesins, vacuolating cytotoxin VacA, protease HtrA, urease, peptidoglycan, and type IV secretion systems (T4SS). It appears that H. pylori targets more than 40 known host protein receptors on epithelial or immune cells. A series of T4SS components such as CagL, CagI, CagY, and CagA can bind to the integrin α 5β 1 receptor. Other targeted membrane-based receptors include the integrins αvβ 3, αvβ 5, and β 2 (CD18), RPTP-α/β, GP130, E-cadherin, fibronectin, laminin, CD46, CD74, ICAM1/LFA1, T-cell receptor, Toll-like receptors, and receptor tyrosine kinases EGFR, ErbB2, ErbB3, and c-Met. In addition, H. pylori is able to activate the intracellular receptors NOD1, NOD2, and NLRP3 with important roles in innate immunity. Here we review the interplay of various bacterial factors with host protein receptors. The contribution of these interactions to signal transduction and pathogenesis is discussed.

  1. Cannabinoid Receptor–Interacting Protein 1a Modulates CB1 Receptor Signaling and Regulation

    Science.gov (United States)

    Smith, Tricia H.; Blume, Lawrence C.; Straiker, Alex; Cox, Jordan O.; David, Bethany G.; McVoy, Julie R. Secor; Sayers, Katherine W.; Poklis, Justin L.; Abdullah, Rehab A.; Egertová, Michaela; Chen, Ching-Kang; Mackie, Ken; Elphick, Maurice R.; Howlett, Allyn C.

    2015-01-01

    Cannabinoid CB1 receptors (CB1Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor–interacting protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca2+ channel activity. We now demonstrate cellular colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated CB1R-mediated guanine nucleotide–binding regulatory protein (G-protein) activity. Stable overexpression of CRIP1a in human embryonic kidney (HEK)-293 cells stably expressing CB1Rs (CB1-HEK), or in N18TG2 cells endogenously expressing CB1Rs, decreased CB1R-mediated G-protein activation (measured by agonist-stimulated [35S]GTPγS (guanylyl-5′-[O-thio]-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, small-interfering RNA–mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not attributable to differences in CB1R expression or endocannabinoid tone because CB1R levels did not differ between cell lines varying in CRIP1a expression, and endocannabinoid levels were undetectable (CB1-HEK) or unchanged (N18TG2) by CRIP1a overexpression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated [35S]GTPγS binding. CRIP1a overexpression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP1a inhibits constitutive CB1R activity and demonstrate that CRIP1a can also inhibit agonist

  2. [Programmed necrosis mediated by receptor-interacting protein 3: a new target for liver disease research].

    Science.gov (United States)

    Zhang, J; Jing, Y; Li, Y N; Zhou, L; Wang, B M

    2016-09-20

    Hepatocyte death mainly includes apoptosis and necrosis and is a critical process in the pathophysiological mechanism of liver injury caused by various reasons. Recent studies have shown that key regulatory molecules in the inhibition of apoptosis such as caspase cannot be used as targets for inhibiting disease progression in clinical practice. In recent years, programmed necrosis mediated by receptor-interacting protein 3(RIP3)becomes a new hot research topic. It not only plays an important role in inducing inflammatory response, but also is closely regulated by intracellular signal factors, and it is a type of active cell death which can be interfered with. Compared with apoptosis, programmed necrosis is accompanied by the release of various inflammatory factors, which significantly affects local immune microenvironment. RIP3-mediated programmed necrosis has been taken seriously in many diseases. Although its mechanism of action in liver disease remains unclear, the results of recent studies confirmed its important role in the development of liver disease. This article reviews the research advances in the role of RIP3-mediated programmed necrosis signaling pathway in liver disease of various causes and investigates the possibility of RIP3-mediated programmed necrosis as a new target in the treatment of liver disease.

  3. Identification of interacting proteins of retinoid-related orphan nuclear receptor gamma in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Ze-Min Huang1,#, Jun Wu2,#, Zheng-Cai Jia1, Yi Tian1, Jun Tang3, Yan Tang1, Ying Wang2, Yu-Zhang Wu1,* & Bing Ni1,*

    2012-06-01

    Full Text Available The retinoid-related orphan nuclear receptor gamma (RORγplays critical roles in regulation of development, immunity andmetabolism. As transcription factor usually forms a proteincomplex to function, thus capturing and dissecting of theRORγ protein complex will be helpful for exploring themechanisms underlying those functions. After construction ofthe recombinant tandem affinity purification (TAP plasmid,pMSCVpuro RORγ-CTAP(SG, the nuclear localization ofRORγ-CTAP(SG fusion protein was verified. Followingisolation of RORγ protein complex by TAP strategy, sevencandidate interacting proteins were identified. Finally, the heatshock protein 90 (HSP90 and receptor-interacting protein 140(RIP140 were confirmed to interplay with RORγ byco-immunoprecipitation. Interference of HSP90 or/and RIP140genes resulted in dramatically decreased expression ofCYP2C8 gene, the RORγ target gene. Data from this studydemonstrate that HSP90 and RIP140 proteins interact withRORγ protein in a complex format and function asco-activators in the RORγ-mediated regulatory processes ofHepG2 cells.

  4. Role of Receptor-Interacting Protein 140 in human fat cells

    Directory of Open Access Journals (Sweden)

    Stenson Britta M

    2010-01-01

    Full Text Available Abstract Background Mice lacking Receptor-interacting protein 140 (RIP140 have reduced body fat which at least partly is mediated through increased lipid and glucose metabolism in adipose tissue. In humans, RIP140 is lower expressed in visceral white adipose tissue (WAT of obese versus lean subjects. We investigated the role of RIP140 in human subcutaneous WAT, which is the major fat depot of the body. Methods Messenger RNA levels of RIP140 were measured in samples of subcutaneous WAT from women with a wide variation in BMI and in different human WAT preparations. RIP140 mRNA was knocked down with siRNA in in vitro differentiated adipocytes and the impact on glucose transport and mRNA levels of target genes determined. Results RIP140 mRNA levels in subcutaneous WAT were decreased among obese compared to lean women and increased by weight-loss, but did not associate with mitochondrial DNA copy number. RIP140 expression increased during adipocyte differentiation in vitro and was higher in isolated adipocytes compared to corresponding pieces of WAT. Knock down of RIP140 increased basal glucose transport and mRNA levels of glucose transporter 4 and uncoupling protein-1. Conclusions Human RIP140 inhibits glucose uptake and the expression of genes promoting energy expenditure in the same fashion as the murine orthologue. Increased levels of human RIP140 in subcutaneous WAT of lean subjects may contribute to economize on energy stores. By contrast, the function and expression pattern does not support that RIP140 regulate human obesity.

  5. Neuroendocrine-immune interaction: regulation of inflammation via G-protein coupled receptors

    NARCIS (Netherlands)

    Verburg-van Kemenade, B.M.L.; Aa, van der L.M.; Chadzinska, M.K.

    2013-01-01

    Neuroendocrine- and immune systems interact in a bi-directional fashion to communicate the status of pathogen recognition to the brain and the immune response is influenced by physiological changes. The network of ligands and their receptors involved includes cytokines and chemokines,

  6. Osteopontin interaction with integrin receptors

    DEFF Research Database (Denmark)

    Kläning, Eva

    Osteopontin is a negatively charged and unstructured protein that is found in many types of tissue in humans. Osteopontin can interact with many different types of cells via interaction with integrins, which are a particular class of receptors expressed on the cellular surface. Via contact...... with integrins, osteopontin can affect cellular behaviour in both normal and disease-related contexts....

  7. G protein coupled receptor interactions with cholesterol deep in the membrane.

    Science.gov (United States)

    Genheden, Samuel; Essex, Jonathan W; Lee, Anthony G

    2017-02-01

    G protein coupled receptors (GPCRs) are located in membranes rich in cholesterol. The membrane spanning surfaces of GPCRs contain exposed backbone carbonyl groups and residue side chains potentially capable of forming hydrogen bonds to cholesterol molecules buried deep within the hydrophobic core of the lipid bilayer. Coarse-grained molecular dynamics (CGMD) simulations allow the observation of GPCRs in cholesterol-containing lipid bilayers for long times (50μs), sufficient to ensure equilibration of the system. We have detected a number of deep cholesterol binding sites on β2 adrenergic and A2A adenosine receptors, and shown changes in these sites on agonist binding. The requirements for binding are modest, just a potential hydrogen bond partner close to a cleft or hole in the surface. This makes it likely that similar binding sites for cholesterol will exist on other classes of membrane protein. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Interaction between the P1 protein of Mycoplasma pneumoniae and receptors on HEp-2 cells

    DEFF Research Database (Denmark)

    Drasbek, Mette; Christiansen, Gunna; Drasbek, Kim Ryun

    2007-01-01

    The human pathogen Mycoplasma pneumoniae can cause atypical pneumonia through adherence to epithelial cells in the respiratory tract. The major immunogenic protein, P1, participates in the attachment of the bacteria to the host cells. To investigate the adhesion properties of P1, a recombinant...... protein (rP1-II) covering amino acids 1107-1518 of the P1 protein was produced. This protein inhibited the adhesion of M. pneumoniae to human HEp-2 cells, as visualized in a competitive-binding assay using immunofluorescence microscopy. Previous studies have shown that mAbs that recognize two epitopes...... in this region of P1 also reduce M. pneumoniae adhesion. Therefore, peptides covering these epitopes, of 8 and 13 aa, respectively, were synthesized to further investigate the adhesion region. None of these synthetic peptides reduced the binding of M. pneumoniae to the receptors on the host cells. Instead, 10...

  9. A protein interaction atlas for the nuclear receptors: properties and quality of a hub-based dimerisation network

    Directory of Open Access Journals (Sweden)

    De Graaf David

    2007-07-01

    Full Text Available Abstract Background The nuclear receptors are a large family of eukaryotic transcription factors that constitute major pharmacological targets. They exert their combinatorial control through homotypic heterodimerisation. Elucidation of this dimerisation network is vital in order to understand the complex dynamics and potential cross-talk involved. Results Phylogeny, protein-protein interactions, protein-DNA interactions and gene expression data have been integrated to provide a comprehensive and up-to-date description of the topology and properties of the nuclear receptor interaction network in humans. We discriminate between DNA-binding and non-DNA-binding dimers, and provide a comprehensive interaction map, that identifies potential cross-talk between the various pathways of nuclear receptors. Conclusion We infer that the topology of this network is hub-based, and much more connected than previously thought. The hub-based topology of the network and the wide tissue expression pattern of NRs create a highly competitive environment for the common heterodimerising partners. Furthermore, a significant number of negative feedback loops is present, with the hub protein SHP [NR0B2] playing a major role. We also compare the evolution, topology and properties of the nuclear receptor network with the hub-based dimerisation network of the bHLH transcription factors in order to identify both unique themes and ubiquitous properties in gene regulation. In terms of methodology, we conclude that such a comprehensive picture can only be assembled by semi-automated text-mining, manual curation and integration of data from various sources.

  10. Receptor-Interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing.

    Directory of Open Access Journals (Sweden)

    Andrew Godwin

    Full Text Available Wound healing consists of a complex, dynamic and overlapping process involving inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular level is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3 controls programmed necrosis in response to TNF-α during inflammation and has been shown to be highly induced during cutaneous wound repair. However, its role in wound healing remains to be demonstrated. To study this, we created dorsal cutaneous wounds on male wild-type (WT and RIPK3-deficient (Ripk3-/- mice. Wound area was measured daily until day 14 post-wound and skin tissues were collected from wound sites at various days for analysis. The wound healing rate in Ripk3-/- mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in Ripk3-/- mice was 53% larger than that of WT mice. H&E and Masson-Trichrome staining analysis showed impaired quality of wound closure in Ripk3-/- wounds with delayed re-epithelialization and angiogenesis and defected granulation tissue formation and collagen deposition compared to WT. The neutrophil infiltration pattern was altered in Ripk3-/- wounds with less neutrophils at day 1 and more neutrophils at day 3. This altered pattern was also reflected in the differential expression of IL-6, KC, IL-1β and TNF-α between WT and Ripk3-/- wounds. MMP-9 protein expression was decreased with increased Timp-1 mRNA in the Ripk3-/- wounds compared to WT. The microvascular density along with the intensity and timing of induction of proangiogenic growth factors VEGF and TGF-β1 were also decreased or delayed in the Ripk3-/- wounds. Furthermore, mouse embryonic fibroblasts (MEFs from Ripk3-/- mice migrated less towards chemoattractants TGF-β1 and PDGF than MEFs from WT mice. These results clearly demonstrate that RIPK3 is an essential molecule to maintain the temporal manner of the

  11. Protein-protein interactions

    DEFF Research Database (Denmark)

    Byron, Olwyn; Vestergaard, Bente

    2015-01-01

    Responsive formation of protein:protein interaction (PPI) upon diverse stimuli is a fundament of cellular function. As a consequence, PPIs are complex, adaptive entities, and exist in structurally heterogeneous interplays defined by the energetic states of the free and complexed protomers. The bi...

  12. Signal transduction by the formyl peptide receptor. Studies using chimeric receptors and site-directed mutagenesis define a novel domain for interaction with G-proteins.

    Science.gov (United States)

    Amatruda, T T; Dragas-Graonic, S; Holmes, R; Perez, H D

    1995-11-24

    The binding of small peptide ligands to high affinity chemoattractant receptors on the surface of neutrophils and monocytes leads to activation of heterotrimeric G-proteins, stimulation of phosphatidylinositol-phospholipase C (PI-PLC), and subsequently to the inflammatory response. It was recently shown (Amatruda, T. T., Gerard, N. P., Gerard, C., and Simon, M. I. (1993) J. Biol. Chem. 268, 10139-10144) that the receptor for the chemoattractant peptide C5a specifically interacts with G alpha 16, a G-protein alpha subunit of the Gq class, to trigger ligand-dependent stimulation of PI-PLC in transfected cells. In order to further characterize this chemoattractant peptide signal transduction pathway, we transfected cDNAs encoding the formylmethionylleucylphenylalanine receptor (fMLPR) into COS cells and measured the production of inositol phosphates. Ligand-dependent activation of PI-PLC was seen in COS cells transfected with the fMLPR and G alpha 16 and stimulated with fMLP but not in cells transfected with receptor alone or with receptor plus G alpha q. Chimeric receptors in which the N-terminal extracellular domain, the second intracellular domain, or the intracellular C-terminal tail of the fMLP receptor was replaced with C5a receptor domains (Perez, H. D., Holmes, R., Vilander, L. R., Adams, R. R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295) were capable of ligand-dependent activation of PI-PLC when co-transfected with G alpha 16. A chimeric receptor exchanging the first intracellular domain of the fMLPR was constitutively activated, stimulating PI-PLC in the absence of ligand. Constitutive activation of PI-PLC, to a level 233% of that seen in cells transfected with wild-type fMLP receptors, was dependent on G alpha 16. Site-directed mutagenesis of the first intracellular domain of the fMLPR (amino acids 54-62) reveals this to be a domain necessary for ligand-dependent activation of G alpha 16. These results suggest that

  13. Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Knaap, E. van der; Sauter, M.; Kende, H. (Michigan State Univ., East Lansing, MI (United States). DOE Plant Research Lab.); Song, W.Y.; Ruan, D.L.; Ronald, P.C. (Univ. of California, Davis, CA (United States). Dept. of Plant Pathology)

    1999-06-01

    The authors identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

  14. Sorting Nexin 27 Protein Regulates Trafficking of a p21-activated Kinase (PAK) Interacting Exchange Factor (β-Pix)-G Protein-coupled Receptor Kinase Interacting Protein (GIT) Complex via a PDZ Domain Interaction*

    Science.gov (United States)

    Valdes, Julie L.; Tang, Jingrong; McDermott, Mark I.; Kuo, Jean-Cheng; Zimmerman, Seth P.; Wincovitch, Stephen M.; Waterman, Clare M.; Milgram, Sharon L.; Playford, Martin P.

    2011-01-01

    Sorting nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27-PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells using a GST-SNX27 fusion construct as bait. We found that β-Pix (p21-activated kinase-interactive exchange factor), a guanine nucleotide exchange factor for the Rho family of small GTPases known to regulate cell motility directly interacted with SNX27. The association of β-Pix and SNX27 is specific for β-Pix isoforms terminating in the type-1 PDZ binding motif (ETNL). In the same screen we also identified Git1/2 as a potential SNX27 interacting protein. The interaction between SNX27 and Git1/2 is indirect and mediated by β-Pix. Furthermore, we show recruitment of the β-Pix·Git complex to endosomal sites in a SNX27-dependent manner. Finally, migration assays revealed that depletion of SNX27 from HeLa and mouse principal kidney cortical collecting duct cells significantly decreases cell motility. We propose a model by which SNX27 regulates trafficking of β-Pix to focal adhesions and thereby influences cell motility. PMID:21926430

  15. A library of 7TM receptor C-terminal tails - Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

    DEFF Research Database (Denmark)

    Heydorn, A.; Sondergaard, B.P.; Ersbøll, Bjarne Kjær

    2004-01-01

    sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor...... only a single receptor tail, i.e. the beta(2)-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein......-coupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the delta-opioid receptor, confirmed...

  16. Natural Compounds Interacting with Nicotinic Acetylcholine Receptors: From Low-Molecular Weight Ones to Peptides and Proteins

    Directory of Open Access Journals (Sweden)

    Denis Kudryavtsev

    2015-05-01

    Full Text Available Nicotinic acetylcholine receptors (nAChRs fulfill a variety of functions making identification and analysis of nAChR subtypes a challenging task. Traditional instruments for nAChR research are d-tubocurarine, snake venom protein α-bungarotoxin (α-Bgt, and α-conotoxins, neurotoxic peptides from Conus snails. Various new compounds of different structural classes also interacting with nAChRs have been recently identified. Among the low-molecular weight compounds are alkaloids pibocin, varacin and makaluvamines C and G. 6-Bromohypaphorine from the mollusk Hermissenda crassicornis does not bind to Torpedo nAChR but behaves as an agonist on human α7 nAChR. To get more selective α-conotoxins, computer modeling of their complexes with acetylcholine-binding proteins and distinct nAChRs was used. Several novel three-finger neurotoxins targeting nAChRs were described and α-Bgt inhibition of GABA-A receptors was discovered. Information on the mechanisms of nAChR interactions with the three-finger proteins of the Ly6 family was found. Snake venom phospholipases A2 were recently found to inhibit different nAChR subtypes. Blocking of nAChRs in Lymnaea stagnalis neurons was shown for venom C-type lectin-like proteins, appearing to be the largest molecules capable to interact with the receptor. A huge nAChR molecule sensible to conformational rearrangements accommodates diverse binding sites recognizable by structurally very different compounds.

  17. Interaction of Agouti protein with the melanocortin 1 receptor in vitro and in vivo

    Science.gov (United States)

    Ollmann, Michael M.; Lamoreux, M. Lynn; Wilson, Brent D.; Barsh, Gregory S.

    1998-01-01

    Agouti protein and Agouti-related protein (Agrp) are paracrine-signaling molecules that normally regulate pigmentation and body weight, respectively. These proteins antagonize the effects of α-melanocyte-stimulating hormone (α-MSH) and other melanocortins, and several alternatives have been proposed to explain their biochemical mechanisms of action. We have used a sensitive bioassay based on Xenopus melanophores to characterize pharmacologic properties of recombinant Agouti protein, and have directly measured its cell-surface binding to mammalian cells by use of an epitope-tagged form (HA–Agouti) that retains biologic activity. In melanophores, Agouti protein has no effect in the absence of α-MSH, but its action cannot be explained solely by inhibition of α-MSH binding. In 293T cells, expression of the Mc1r confers a specific, high-affinity binding site for HA-Agouti. Binding is inhibited by α-MSH, or by Agrp, which indicates that α-MSH and Agouti protein bind in a mutually exclusive way to the Mc1r, and that the similarity between Agouti protein and Agrp includes their binding sites. The effects of Agouti and the Mc1r in vivo have been examined in a sensitized background provided by the chinchilla (Tyrc-ch) mutation, which uncovers a phenotypic difference between overexpression of Agouti in lethal yellow (Ay/a) mice and loss of Mc1r function in recessive yellow (Mc1re/Mc1re) mice. Double and triple mutant studies indicate that a functional Mc1r is required for the pigmentary effects of Agouti, and suggest that Agouti protein can act as an agonist of the Mc1r in a way that differs from α-MSH stimulation. These results resolve questions regarding the biochemical mechanism of Agouti protein action, and provide evidence of a novel signaling mechanism whereby α-MSH and Agouti protein or Agrp function as independent ligands that inhibit each other’s binding and transduce opposite signals through a single receptor. PMID:9450927

  18. In situ proximity ligation assays indicate that hemochromatosis proteins Hfe and transferrin receptor 2 (Tfr2) do not interact.

    Science.gov (United States)

    Rishi, Gautam; Crampton, Emily M; Wallace, Daniel F; Subramaniam, V Nathan

    2013-01-01

    The hemochromatosis associated proteins HFE and Transferrin Receptor 2 (TFR2) have been shown to be important for the proper regulation of hepcidin. A number of in vitro studies using transient overexpression systems have suggested that an interaction between HFE and TFR2 is required for the regulation of hepcidin. This model of iron sensing which centers upon the requirement for an interaction between HFE and TFR2 has recently been questioned with in vivo studies in mice from our laboratory and others which suggest that Hfe and Tfr2 can regulate hepcidin independently of each other. To re-examine the postulated interaction between Hfe and Tfr2 we developed a novel expression system in which both proteins are stably co-expressed and used the proximity ligation assay to examine the interactions between Hfe, Tfr1 and Tfr2 at a cellular level. We were able to detect the previously described interaction between Hfe and Tfr1, and heterodimers between Tfr1 and Tfr2; however no interaction between Hfe and Tfr2 was observed in our system. The results from this study indicate that Hfe and Tfr2 do not interact with each other when they are stably expressed at similar levels. Furthermore, these results support in vivo studies which suggest that Hfe and Tfr2 can independently regulate hepcidin.

  19. In situ proximity ligation assays indicate that hemochromatosis proteins Hfe and transferrin receptor 2 (Tfr2 do not interact.

    Directory of Open Access Journals (Sweden)

    Gautam Rishi

    Full Text Available The hemochromatosis associated proteins HFE and Transferrin Receptor 2 (TFR2 have been shown to be important for the proper regulation of hepcidin. A number of in vitro studies using transient overexpression systems have suggested that an interaction between HFE and TFR2 is required for the regulation of hepcidin. This model of iron sensing which centers upon the requirement for an interaction between HFE and TFR2 has recently been questioned with in vivo studies in mice from our laboratory and others which suggest that Hfe and Tfr2 can regulate hepcidin independently of each other. To re-examine the postulated interaction between Hfe and Tfr2 we developed a novel expression system in which both proteins are stably co-expressed and used the proximity ligation assay to examine the interactions between Hfe, Tfr1 and Tfr2 at a cellular level. We were able to detect the previously described interaction between Hfe and Tfr1, and heterodimers between Tfr1 and Tfr2; however no interaction between Hfe and Tfr2 was observed in our system. The results from this study indicate that Hfe and Tfr2 do not interact with each other when they are stably expressed at similar levels. Furthermore, these results support in vivo studies which suggest that Hfe and Tfr2 can independently regulate hepcidin.

  20. Cannabinoid CB1 receptor-interacting proteins: novel targets for central nervous system drug discovery?

    National Research Council Canada - National Science Library

    Smith, Tricia H; Sim-Selley, Laura J; Selley, Dana E

    2010-01-01

    ...), including CB1 and CB2 receptors. The CB1 receptor is the major cannabinoid receptor in the central nervous system and has gained increasing interest as a target for drug discovery for treatment of nausea, cachexia, obesity, pain...

  1. The PDZ3 domain of the cellular scaffolding protein MAGI-1 interacts with the Coxsackievirus and adenovirus receptor (CAR).

    Science.gov (United States)

    Yan, Ran; Sharma, Priyanka; Kolawole, Abimbola O; Martin, Sterling C T; Readler, James M; Kotha, Poornima L N; Hostetler, Heather A; Excoffon, Katherine J D A

    2015-04-01

    The Coxsackievirus and adenovirus receptor (CAR) is an essential cellular protein that is involved in cell-cell adhesion, protein trafficking, and viral infection. The major isoform of CAR is selectively sorted to the basolateral membrane of polarized epithelial cells where it co-localizes with the cellular scaffolding protein membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1). Previously, we demonstrated CAR interacts with MAGI-1 through a PDZ-domain dependent interaction. Here, we show that the PDZ3 domain of MAGI-1 is exclusively responsible for the high affinity interaction between the seven exon isoform of CAR and MAGI-1 using yeast-two-hybrid analysis and confirming this interaction biochemically and in cellular lysates by in vitro pull down assay and co-immunoprecipitation. The high affinity interaction between the PDZ3 domain and CAR C-terminus was measured by fluorescence resonance energy transfer. Further, we investigated the biological relevance of this high affinity interaction between CAR and the PDZ3 domain of MAGI-1 and found that it does not alter CAR-mediated adenovirus infection. By contrast, interruption of this high affinity interaction altered the localization of MAGI-1 indicating that CAR is able to traffic MAGI-1 to cell junctions. These data deepen the molecular understanding of the interaction between CAR and MAGI-1 and indicate that although CAR plays a role in trafficking PDZ-based scaffolding proteins to cellular junctions, association with a high affinity intracellular binding partner does not significantly alter adenovirus binding and entry via CAR. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. A physiologically required G protein-coupled receptor (GPCR)-regulator of G protein signaling (RGS) interaction that compartmentalizes RGS activity.

    Science.gov (United States)

    Croft, Wayne; Hill, Claire; McCann, Eilish; Bond, Michael; Esparza-Franco, Manuel; Bennett, Jeannette; Rand, David; Davey, John; Ladds, Graham

    2013-09-20

    G protein-coupled receptors (GPCRs) can interact with regulator of G protein signaling (RGS) proteins. However, the effects of such interactions on signal transduction and their physiological relevance have been largely undetermined. Ligand-bound GPCRs initiate by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins. Signaling is terminated by hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, a reaction catalyzed by RGS proteins. Using yeast as a tool to study GPCR signaling in isolation, we define an interaction between the cognate GPCR (Mam2) and RGS (Rgs1), mapping the interaction domains. This reaction tethers Rgs1 at the plasma membrane and is essential for physiological signaling response. In vivo quantitative data inform the development of a kinetic model of the GTPase cycle, which extends previous attempts by including GPCR-RGS interactions. In vivo and in silico data confirm that GPCR-RGS interactions can impose an additional layer of regulation through mediating RGS subcellular localization to compartmentalize RGS activity within a cell, thus highlighting their importance as potential targets to modulate GPCR signaling pathways.

  3. Pancreatic Beta Cell G-Protein Coupled Receptors and Second Messenger Interactions: A Systems Biology Computational Analysis.

    Directory of Open Access Journals (Sweden)

    Leonid E Fridlyand

    Full Text Available Insulin secretory in pancreatic beta-cells responses to nutrient stimuli and hormonal modulators include multiple messengers and signaling pathways with complex interdependencies. Here we present a computational model that incorporates recent data on glucose metabolism, plasma membrane potential, G-protein-coupled-receptors (GPCR, cytoplasmic and endoplasmic reticulum calcium dynamics, cAMP and phospholipase C pathways that regulate interactions between second messengers in pancreatic beta-cells. The values of key model parameters were inferred from published experimental data. The model gives a reasonable fit to important aspects of experimentally measured metabolic and second messenger concentrations and provides a framework for analyzing the role of metabolic, hormones and neurotransmitters changes on insulin secretion. Our analysis of the dynamic data provides support for the hypothesis that activation of Ca2+-dependent adenylyl cyclases play a critical role in modulating the effects of glucagon-like peptide 1 (GLP-1, glucose-dependent insulinotropic polypeptide (GIP and catecholamines. The regulatory properties of adenylyl cyclase isoforms determine fluctuations in cytoplasmic cAMP concentration and reveal a synergistic action of glucose, GLP-1 and GIP on insulin secretion. On the other hand, the regulatory properties of phospholipase C isoforms determine the interaction of glucose, acetylcholine and free fatty acids (FFA (that act through the FFA receptors on insulin secretion. We found that a combination of GPCR agonists activating different messenger pathways can stimulate insulin secretion more effectively than a combination of GPCR agonists for a single pathway. This analysis also suggests that the activators of GLP-1, GIP and FFA receptors may have a relatively low risk of hypoglycemia in fasting conditions whereas an activator of muscarinic receptors can increase this risk. This computational analysis demonstrates that study of

  4. The interaction of lipopolysaccharide-coated polystyrene particle with membrane receptor proteins on macrophage measured by optical tweezers

    Science.gov (United States)

    Wei, Ming-Tzo; Hua, Kuo-Feng; Hsu, Jowey; Karmenyan, Artashes; Hsu, Hsien-Yeh; Chiou, Arthur

    2006-08-01

    Lipopolysaccharide (LPS) is one of the cell wall components of Gram-positive bacteria recognized by and interacted with receptor proteins such as CD14 on macrophage cells. Such a process plays an important role in our innate immune system. In this paper, we report the application of optical tweezers (λ = 1064nm Gaussian beam focused by a water-immersed objective lens with N.A. = 1.0) to the study of the dynamics of the binding of a LPS-coated polystyrene particle (diameter = 1.5μm) onto the plasma membrane of a macrophage cell. We demonstrated that the binding rate increased significantly when the macrophage cell was pre-treated with the extract of Reishi polysaccharides (EORP) which has been shown to enhance the cell surface expression of CD14 (receptor of LPS) on macrophage cells.

  5. LINGO-1 Protein Interacts with the p75 Neurotrophin Receptor in Intracellular Membrane Compartments*

    Science.gov (United States)

    Meabon, James S.; De Laat, Rian; Ieguchi, Katsuaki; Wiley, Jesse C.; Hudson, Mark P.; Bothwell, Mark

    2015-01-01

    Axon outgrowth inhibition in response to trauma is thought to be mediated via the binding of myelin-associated inhibitory factors (e.g. Nogo-66, myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, and myelin basic protein) to a putative tripartite LINGO-1·p75NTR·Nogo-66 receptor (NgR) complex at the cell surface. We found that endogenous LINGO-1 expression in neurons in the cortex and cerebellum is intracellular. Mutation or truncation of the highly conserved LINGO-1 C terminus altered this intracellular localization, causing poor intracellular retention and increased plasma membrane expression. p75NTR associated predominantly with natively expressed LINGO-1 containing immature N-glycans, characteristic of protein that has not completed trans-Golgi-mediated processing, whereas mutant forms of LINGO-1 with enhanced plasma membrane expression did not associate with p75NTR. Co-immunoprecipitation experiments demonstrated that LINGO-1 and NgR competed for binding to p75NTR in a manner that is difficult to reconcile with the existence of a LINGO-1·p75NTR·NgR ternary complex. These findings contradict models postulating functional LINGO-1·p75NTR·NgR complexes in the plasma membrane. PMID:25666623

  6. LINGO-1 protein interacts with the p75 neurotrophin receptor in intracellular membrane compartments.

    Science.gov (United States)

    Meabon, James S; De Laat, Rian; Ieguchi, Katsuaki; Wiley, Jesse C; Hudson, Mark P; Bothwell, Mark

    2015-04-10

    Axon outgrowth inhibition in response to trauma is thought to be mediated via the binding of myelin-associated inhibitory factors (e.g. Nogo-66, myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, and myelin basic protein) to a putative tripartite LINGO-1·p75(NTR)·Nogo-66 receptor (NgR) complex at the cell surface. We found that endogenous LINGO-1 expression in neurons in the cortex and cerebellum is intracellular. Mutation or truncation of the highly conserved LINGO-1 C terminus altered this intracellular localization, causing poor intracellular retention and increased plasma membrane expression. p75(NTR) associated predominantly with natively expressed LINGO-1 containing immature N-glycans, characteristic of protein that has not completed trans-Golgi-mediated processing, whereas mutant forms of LINGO-1 with enhanced plasma membrane expression did not associate with p75(NTR). Co-immunoprecipitation experiments demonstrated that LINGO-1 and NgR competed for binding to p75(NTR) in a manner that is difficult to reconcile with the existence of a LINGO-1·p75(NTR)·NgR ternary complex. These findings contradict models postulating functional LINGO-1·p75(NTR)·NgR complexes in the plasma membrane. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Structural model for the interaction of a designed Ankyrin Repeat Protein with the human epidermal growth factor receptor 2.

    Directory of Open Access Journals (Sweden)

    V Chandana Epa

    Full Text Available Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2. HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84-1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions.

  8. G protein-coupled receptors mediate coronary flow- and agonist-induced responses via lectin-oligosaccharide interactions.

    Science.gov (United States)

    Perez-Aguilar, Sandra; Torres-Tirado, David; Martell-Gallegos, Guadalupe; Velarde-Salcedo, Jimena; Barba-de la Rosa, Ana Paulina; Knabb, Maureen; Rubio, Rafael

    2014-03-01

    Blood flow acts parallel to the coronary luminal endothelial surface layer (LESL) and modulates multiple parenchymal functions via the release of paracrine agents. Evidence indicates that the LESL may be a flow-sensing organelle and that perhaps through flow-induced lectin (L)·oligosaccharide (O) complex formation (L·O) participates in this process. LESL integrins and selectins are both lectinic and flow sensitive, but the L properties of flow-sensitive G protein-coupled receptors (GPCRs) are unknown. Therefore, we investigated the presence of L in the LESL and hypothesized that if flow-sensitive GPCRs are L, flow and O will determine their response to receptor activation. The LESL protein fraction isolated from guinea pig hearts was passed through an affinity chromatography column made of three sugars, mannose, galactose, and N-acetylglucosamine, and the lectinic fraction was eluted. Immune dot blot was used to identify L proteins in the LESL fraction. Our results indicate the following. 1) Two-dimensional SDS-PAGE (2D-SDS-PAGE) of the LESL lectinic fraction revealed at least 167 Ls. 2) Among these Ls, we identified three selectins and the GPCRs: angiotensin II, bradykinin (B2-R), adenosine A1 and A2, prolactin, endothelin, α1-adrenergic (α1A-R), thromboxane A2, β1-adrenergic, β3-adrenergic, and insulin receptors; the first six GPCRs are known to be flow sensitive. 3) The amplitude of receptor-induced vascular responses by α1A-R and B2-R activation (phenylephrine or bradykinin, respectively) was a function of flow and O (hyaluronidate). Our results support a novel mechanism of GPCR-mediated responses to flow via L·O interaction.

  9. Protocadherin 19 (PCDH19) interacts with paraspeckle protein NONO to co-regulate gene expression with estrogen receptor alpha (ERα).

    Science.gov (United States)

    Pham, Duyen H; Tan, Chuan C; Homan, Claire C; Kolc, Kristy L; Corbett, Mark A; McAninch, Dale; Fox, Archa H; Thomas, Paul Q; Kumar, Raman; Gecz, Jozef

    2017-06-01

    De novo and inherited mutations of X-chromosome cell adhesion molecule protocadherin 19 (PCDH19) cause frequent, highly variable epilepsy, autism, cognitive decline and behavioural problems syndrome. Intriguingly, hemizygous null males are not affected while heterozygous females are, contradicting established X-chromosome inheritance. The disease mechanism is not known. Cellular mosaicism is the likely driver. We have identified p54nrb/NONO, a multifunctional nuclear paraspeckle protein with known roles in nuclear hormone receptor gene regulation, as a PCDH19 protein interacting partner. Using breast cancer cells we show that PCDH19-NONO complex is a positive co-regulator of ERα-mediated gene expression. Expression of mutant PCDH19 affects at least a subset of known ERα-regulated genes. These data are consistent with our findings that genes regulated by nuclear hormone receptors and those involved in the metabolism of neurosteroids in particular are dysregulated in PCDH19-epilepsy girls and affected mosaic males. Overall we define and characterize a novel mechanism of gene regulation driven by PCDH19, which is mediated by paraspeckle constituent NONO and is ERα-dependent. This PCDH19-NONO-ERα axis is of relevance not only to PCDH19-epilepsy and its comorbidities but likely also to ERα and generally nuclear hormone receptor-associated cancers. © The Author 2017. Published by Oxford University Press.

  10. Interaction of human laminin receptor with Sup35, the [PSI⁺] prion-forming protein from S. cerevisiae: a yeast model for studies of LamR interactions with amyloidogenic proteins.

    Directory of Open Access Journals (Sweden)

    Christine Pampeno

    Full Text Available The laminin receptor (LamR is a cell surface receptor for extracellular matrix laminin, whereas the same protein within the cell interacts with ribosomes, nuclear proteins and cytoskeletal fibers. LamR has been shown to be a receptor for several bacteria and viruses. Furthermore, LamR interacts with both cellular and infectious forms of the prion protein, PrP(C and PrP(Sc. Indeed, LamR is a receptor for PrP(C. Whether LamR interacts with PrP(Sc exclusively in a capacity of the PrP receptor, or LamR specifically recognizes prion determinants of PrP(Sc, is unclear. In order to explore whether LamR has a propensity to interact with prions and amyloids, we examined LamR interaction with the yeast prion-forming protein, Sup35. Sup35 is a translation termination factor with no homology or functional relationship to PrP. Plasmids expressing LamR or LamR fused with the green fluorescent protein (GFP were transformed into yeast strain variants differing by the presence or absence of the prion conformation of Sup35, respectively [PSI⁺] and [psi⁻]. Analyses by immunoprecipitation, centrifugal fractionation and fluorescent microscopy reveal interaction between LamR and Sup35 in [PSI⁺] strains. The presence of [PSI⁺] promotes LamR co-precipitation with Sup35 as well as LamR aggregation. In [PSI⁺] cells, LamR tagged with GFP or mCherry forms bright fluorescent aggregates that co-localize with visible [PSI⁺] foci. The yeast prion model will facilitate studying the interaction of LamR with amyloidogenic prions in a safe and easily manipulated system that may lead to a better understanding and treatment of amyloid diseases.

  11. Defining the molecular basis of interaction between R3 receptor-type protein tyrosine phosphatases and VE-cadherin.

    Directory of Open Access Journals (Sweden)

    Olga Dorofejeva

    Full Text Available Receptor-type protein tyrosine phosphatases (RPTPs of the R3 subgroup play key roles in the immune, vascular and nervous systems. They are characterised by a large ectodomain comprising multiple FNIII-like repeats, a transmembrane domain, and a single intracellular phosphatase domain. The functional role of the extracellular region has not been clearly defined and potential roles in ligand interaction, dimerization, and regulation of cell-cell contacts have been reported. Here bimolecular fluorescence complementation (BiFC in live cells was used to examine the molecular basis for the interaction of VE-PTP with VE-cadherin, two proteins involved in endothelial cell contact and maintenance of vascular integrity. The potential of other R3-PTPs to interact with VE-cadherin was also explored using this method. Quantitative BiFC analysis, using a VE-PTP construct expressing only the ectodomain and transmembrane domain, revealed a specific interaction with VE-cadherin, when compared with controls. Controls were sialophorin, an unrelated membrane protein with a large ectodomain, and a membrane anchored C-terminal Venus-YFP fragment, lacking both ectodomain and transmembrane domains. Truncation of the first 16 FNIII-like repeats from the ectodomain of VE-PTP indicated that removal of this region is not sufficient to disrupt the interaction with VE-cadherin, although it occurs predominantly in an intracellular location. A construct with a deletion of only the 17th domain of VE-PTP was, in contrast to previous studies, still able to interact with VE-cadherin, although this also was predominantly intracellular. Other members of the R3-PTP family (DEP-1, GLEPP1 and SAP-1 also exhibited the potential to interact with VE-cadherin. The direct interaction of DEP-1 with VE-cadherin is likely to be of physiological relevance since both proteins are expressed in endothelial cells. Together the data presented in the study suggest a role for both the ectodomain and

  12. Interaction of HIV-1 Nef protein with the host protein Alix promotes lysosomal targeting of CD4 receptor.

    Science.gov (United States)

    Amorim, Nathaly A; da Silva, Eulália M L; de Castro, Rodrigo O; da Silva-Januário, Mara E; Mendonça, Luiza M; Bonifacino, Juan S; da Costa, Luciana J; daSilva, Luis L P

    2014-10-03

    Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. Here, we report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We show that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. We also show that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Interaction of HIV-1 Nef Protein with the Host Protein Alix Promotes Lysosomal Targeting of CD4 Receptor*

    Science.gov (United States)

    Amorim, Nathaly A.; da Silva, Eulália M. L.; de Castro, Rodrigo O.; da Silva-Januário, Mara E.; Mendonça, Luiza M.; Bonifacino, Juan S.; da Costa, Luciana J.; daSilva, Luis L. P.

    2014-01-01

    Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. Here, we report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We show that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. We also show that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef. PMID:25118280

  14. Interaction of Protease-Activated Receptor 2 with G Proteins and Beta-Arrestin 1 Studied by Bioluminescence Resonance Energy Transfer

    Directory of Open Access Journals (Sweden)

    Mohammed Akli eAyoub

    2013-12-01

    Full Text Available G protein-coupled receptors (GPCRs are well recognized as being able to activate several signaling pathways through the activation of different G proteins as well as other signaling proteins such as beta-arrestins. Therefore, understanding how such multiple GPCR-mediated signaling can be integrated constitute an important aspect. Here, we applied bioluminescence resonance energy transfer (BRET to shed more light on the G protein coupling profile of trypsin receptor, or protease-activated receptor 2 (PAR2, and its interaction with beta-arrestin1. Using YFP and Rluc fusion constructs expressed in COS-7 cells, BRET data revealed a pre-assembly of PAR2 with both Galphai1 and Galphao and a rapid and transient activation of these G proteins upon receptor activation. In contrast, no preassembly of PAR2 with Galpha12 could be detected and their physical association can be measured with a very slow and sustained kinetics similar to that of beta-arrestin1 recruitment. These data demonstrate the coupling of PAR2 with Galphai1, Galphao and Galpha12 in COS-7 cells with differences in the kinetics of GPCR-G protein coupling, a parameter that very likely influences the cellular response. Moreover, this further illustrates that preassembly or agonist-induced G protein interaction depends on receptor-G protein pairs indicating another level of complexity and regulation of the signaling of GPCR-G protein complexes and its multiplicity.

  15. Protein O-fucosyltransferase 1 (Pofut1) regulates lymphoid and myeloid homeostasis through modulation of Notch receptor ligand interactions

    Science.gov (United States)

    Yao, David; Huang, Yuanshuai; Huang, Xiaoran; Wang, Weihuan; Yan, Quanjian; Wei, Lebing; Xin, Wei; Gerson, Stanton; Stanley, Pamela; Lowe, John B.

    2011-01-01

    Notch signaling is essential for lymphocyte development and is also implicated in myelopoiesis. Notch receptors are modified by O-fucosylation catalyzed by protein O-fucosyltransferase 1 (Pofut1). Fringe enzymes add N-acetylglucosamine to O-fucose and modify Notch signaling by altering the sensitivity of Notch receptors to Notch ligands. To address physiologic functions in hematopoiesis of Notch modified by O-fucose glycans, we examined mice with inducible inactivation of Pofut1 using Mx-Cre. These mice exhibited a reduction in T lymphopoiesis and in the production of marginal-zone B cells, in addition to myeloid hyperplasia. Restoration of Notch1 signaling rescued T lymphopoiesis and the marrow myeloid hyperplasia. After marrow transfer, both cell-autonomous and environmental cues were found to contribute to lymphoid developmental defects and myeloid hyperplasia in Pofut1-deleted mice. Although Pofut1 deficiency slightly decreased cell surface expression of Notch1 and Notch2, it completely abrogated the binding of Notch receptors with Delta-like Notch ligands and suppressed downstream Notch target activation, indicating that O-fucose glycans are critical for efficient Notch-ligand binding that transduce Notch signals. The combined data support a key role for the O-fucose glycans generated by Pofut1 in Notch regulation of hematopoietic homeostasis through modulation of Notch-ligand interactions. PMID:21464368

  16. Identification of cytoplasmic proteins interacting with unliganded estrogen receptor α and β in human breast cancer cells.

    Science.gov (United States)

    Stellato, Claudia; Nassa, Giovanni; Tarallo, Roberta; Giurato, Giorgio; Ravo, Maria; Rizzo, Francesca; Marchese, Giovanna; Alexandrova, Elena; Cordella, Angela; Baumann, Marc; Nyman, Tuula A; Weisz, Alessandro; Ambrosino, Concetta

    2015-06-01

    Estrogen receptor subtypes (ERα and ERβ) are transcription factors sharing a similar structure but exerting opposite roles in breast cancer cells. Besides the well-characterized genomic actions of nuclear ERs upon ligand binding, specific actions of ligand-free ERs in the cytoplasm also affect cellular functions. The identification of cytoplasmic interaction partners of unliganded ERα and ERβ may help characterize the molecular basis of the extra-nuclear mechanism of action of these receptors, revealing novel mechanisms to explain their role in breast cancer response or resistance to endocrine therapy. To this aim, cytoplasmic extracts from human breast cancer MCF-7 cells stably expressing tandem affinity purification-tagged ERα and ERβ and maintained in estrogen-free medium were subject to affinity-purification and MS analysis, leading to the identification of 84 and 142 proteins associated with unliganded ERα and ERβ, respectively. Functional analyses of ER subtype-specific interactomes revealed significant differences in the molecular pathways targeted by each receptor in the cytoplasm. This work, reporting the first identification of the unliganded ERα and ERβ cytoplasmic interactomes in breast cancer cells, provides novel experimental evidence on the nongenomic effects of ERs in the absence of hormonal stimulus. All MS data have been deposited in the ProteomeXchange with identifier PXD001202 (http://proteomecentral.proteomexchange.org/dataset/PXD001202). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Intracellular calcium levels determine differential modulation of allosteric interactions within G protein-coupled receptor heteromers

    NARCIS (Netherlands)

    Navarro, G.; Aguinaga, D.; Hradsky, J.; Moreno, E.; Reddy, P.P.; Cortés, A.; Mallol, J.; Casadó, V.; Mikhaylova, Marina; Kreutz, M.R.; Lluís, C.; Canela, E.I.; McCormick, P.J.; Ferreira, S.; Ferré, S.

    2014-01-01

    The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson’s disease and other neuropsychiatric disorders. However, the physiological factors that

  18. Bypassing Protein Corona Issue on Active Targeting: Zwitterionic Coatings Dictate Specific Interactions of Targeting Moieties and Cell Receptors.

    Science.gov (United States)

    Safavi-Sohi, Reihaneh; Maghari, Shokoofeh; Raoufi, Mohammad; Jalali, Seyed Amir; Hajipour, Mohammad J; Ghassempour, Alireza; Mahmoudi, Morteza

    2016-09-07

    Surface functionalization strategies for targeting nanoparticles (NP) to specific organs, cells, or organelles, is the foundation for new applications of nanomedicine to drug delivery and biomedical imaging. Interaction of NPs with biological media leads to the formation of a biomolecular layer at the surface of NPs so-called as "protein corona". This corona layer can shield active molecules at the surface of NPs and cause mistargeting or unintended scavenging by the liver, kidney, or spleen. To overcome this corona issue, we have designed biotin-cysteine conjugated silica NPs (biotin was employed as a targeting molecule and cysteine was used as a zwitterionic ligand) to inhibit corona-induced mistargeting and thus significantly enhance the active targeting capability of NPs in complex biological media. To probe the targeting yield of our engineered NPs, we employed both modified silicon wafer substrates with streptavidin (i.e., biotin receptor) to simulate a target and a cell-based model platform using tumor cell lines that overexpress biotin receptors. In both cases, after incubation with human plasma (thus forming a protein corona), cellular uptake/substrate attachment of the targeted NPs with zwitterionic coatings were significantly higher than the same NPs without zwitterionic coating. Our results demonstrated that NPs with a zwitterionic surface can considerably facilitate targeting yield of NPs and provide a promising new type of nanocarriers in biological applications.

  19. Investigation of the vitamin D receptor gene (VDR) and its interaction with protein tyrosine phosphatase, non-receptor type 2 gene (PTPN2) on risk of islet autoimmunity and type 1 diabetes : The Diabetes Autoimmunity Study in the Young (DAISY)

    NARCIS (Netherlands)

    Frederiksen, B.; Liu, E.; Romanos, J.; Steck, A. K.; Yin, X.; Kroehl, M.; Fingerlin, T. E.; Erlich, H.; Eisenbarth, G. S.; Rewers, M.; Norris, J. M.

    The present study investigated the association between variants in the vitamin D receptor gene (VDR) and protein tyrosine phosphatase, non-receptor type 2 gene (PTPN2), as well as an interaction between VDR and PTPN2 and the risk of islet autoimmunity (IA) and progression to type 1 diabetes (T1D).

  20. Interaction of LDL receptor-related protein 4 (LRP4) with postsynaptic scaffold proteins via its C-terminal PDZ domain-binding motif, and its regulation by Ca/calmodulin-dependent protein kinase II.

    Science.gov (United States)

    Tian, Qing-Bao; Suzuki, Tatsuo; Yamauchi, Takashi; Sakagami, Hiroyuki; Yoshimura, Yoshiyuki; Miyazawa, Shoko; Nakayama, Kohzo; Saitoh, Fuminori; Zhang, Jing-Ping; Lu, Yonghao; Kondo, Hisatake; Endo, Shogo

    2006-06-01

    We cloned here a full-length cDNA of Dem26[Tian et al. (1999)Mol. Brain Res., 72, 147-157], a member of the low-density lipoprotein (LDL) receptor gene family from the rat brain. We originally named the corresponding protein synaptic LDL receptor-related protein (synLRP) [Tian et al. (2002) Soc. Neurosci. Abstr., 28, 405] and have renamed it LRP4 to accord it systematic nomenclature (GenBank(TM) accession no. AB073317). LRP4 protein interacted with postsynaptic scaffold proteins such as postsynaptic density (PSD)-95 via its C-terminal tail sequence, and associated with N-methyl-D-aspartate (NMDA)-type glutamate receptor subunit. The mRNA of LRP4 was localized to dendrites, as well as somas, of neuronal cells, and the full-length protein of 250 kDa was highly concentrated in the brain and localized to various subcellular compartments in the brain, including synaptic fractions. Immunocytochemical study using cultured cortical neurons suggested surface localization in the neuronal cells both in somas and dendrites. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylated the C-terminal cytoplasmic region of LRP4 at Ser1887 and Ser1900, and the phosphorylation at the latter site suppressed the interaction of the protein with PSD-95 and synapse-associated protein 97 (SAP97). These findings suggest a postsynaptic role for LRP4, a putative endocytic multiligand receptor, and a mechanism in which CaMKII regulates PDZ-dependent protein-protein interactions and receptor dynamics.

  1. A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells.

    Science.gov (United States)

    Dai, Feihan F; Bhattacharjee, Alpana; Liu, Ying; Batchuluun, Battsetseg; Zhang, Ming; Wang, Xinye Serena; Huang, Xinyi; Luu, Lemieux; Zhu, Dan; Gaisano, Herbert; Wheeler, Michael B

    2015-10-09

    GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H(+)-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca(2+) influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. An unusual case of an ACTH-secreting macroadenoma with a germline variant in the aryl hydrocarbon receptor-interacting protein (AIP) gene

    DEFF Research Database (Denmark)

    Dinesen, Pia T; Dal, Jakob; Gabrovska, Plamena

    2015-01-01

    UNLABELLED: A patient of Cushing's disease (CD) characterized by a large tumor and only subtle symptoms of hormonal hypersecretion was examined. The patient had a germline variant in the aryl hydrocarbon receptor-interacting protein (AIP) gene. A 50-year-old male presenting with headache was diag......UNLABELLED: A patient of Cushing's disease (CD) characterized by a large tumor and only subtle symptoms of hormonal hypersecretion was examined. The patient had a germline variant in the aryl hydrocarbon receptor-interacting protein (AIP) gene. A 50-year-old male presenting with headache...

  3. Effect of angiotensin II type 2 receptor-interacting protein on adipose tissue function via modulation of macrophage polarization.

    Directory of Open Access Journals (Sweden)

    Fei Jing

    Full Text Available We demonstrated that angiotensin II type 2 (AT2 receptor-interacting protein (ATIP 1 ameliorates inflammation-mediated vascular remodeling independent of the AT2 receptor, leading us to explore the possibility of whether ATIP1 could exert anti-inflammatory effects and play a role in other pathophysiological conditions. We examined the possible anti-inflammatory effects of ATIP1 in adipose tissue associated with amelioration of insulin resistance. In mice fed a high-cholesterol diet, adipose tissue macrophage (ATM infiltration and M1-to-M2 ratio were decreased in ATIP1 transgenic mice (ATIP1-Tg compared with wild-type mice (WT, with decreased expression of inflammatory cytokines such as tumor necrosis factor-α and monocyte chemoattractant protein-1 in white adipose tissue (WAT, but an increase in interleukin-10, an anti-inflammatory cytokine. Moreover, 2-[(3H]deoxy-d-glucose (2-[(3H]DG uptake was significantly increased in ATIP1-Tg compared with WT. Next, we examined the roles of ATIP1 in BM-derived hematopoietic cells, employing chimeric mice produced by BM transplantation into irradiated type 2 diabetic mice with obesity, KKAy, as recipients. ATM infiltration and M1-to-M2 ratio were decreased in ATIP1 chimera (ATIP1-tg as BM donor, with improvement of insulin-mediated 2-[(3H]DG uptake and amelioration of inflammation in WAT. Moreover, serum adiponectin concentration in ATIP1 chimera was significantly higher than that in WT chimera (WT as BM donor and KKAy chimera (KKAy as BM donor. These results indicate that ATIP1 could exert anti-inflammatory effects in adipose tissue via macrophage polarization associated with improvement of insulin resistance, and ATIP1 in hematopoietic cells may contribute to these beneficial effects on adipose tissue functions in type 2 diabetes.

  4. Identification of the subunit of cAMP receptor protein (CRP) that functionally interacts with CytR in CRP-CytR-mediated transcriptional repression

    DEFF Research Database (Denmark)

    Meibom, K L; Kallipolitis, B H; Ebright, R H

    2000-01-01

    At promoters of the Escherichia coli CytR regulon, the cAMP receptor protein (CRP) interacts with the repressor CytR to form transcriptionally inactive CRP-CytR-promoter or (CRP)(2)-CytR-promoter complexes. Here, using "oriented heterodimer" analysis, we show that only one subunit of the CRP dime...

  5. A library of 7TM receptor C-terminal tails. Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

    DEFF Research Database (Denmark)

    Heydorn, Arne; Søndergaard, Birgitte P; Ersbøll, Bjarne

    2004-01-01

    Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor...... sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor...... that the tail library provides useful information on the general importance of certain adaptor proteins, for example, in this case, ruling out EBP50 as being a broad spectrum-recycling adaptor....

  6. Lupin Peptides Modulate the Protein-Protein Interaction of PCSK9 with the Low Density Lipoprotein Receptor in HepG2 Cells

    Science.gov (United States)

    Lammi, Carmen; Zanoni, Chiara; Aiello, Gilda; Arnoldi, Anna; Grazioso, Giovanni

    2016-07-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been recently identified as a new useful target for hypercholesterolemia treatment. This work demonstrates that natural peptides, deriving from the hydrolysis of lupin protein and absorbable at intestinal level, are able to inhibit the protein-protein interaction between PCSK9 and the low density lipoprotein receptor (LDLR). In order to sort out the best potential inhibitors among these peptides, a refined in silico model of the PCSK9/LDLR interaction was developed. Docking, molecular dynamics (MD) simulations and peptide binding energy estimations, by MM-GBSA approach, permitted to select the two best candidates among tested peptides that were synthesized and evaluated for their inhibitory activity. The most active was P5 that induced a concentration dependent inhibition of the PCSK9-LDLR binding, with an IC50 value equal to 1.6 ± 0.33 μM. Tested at a 10 μM concentration, this peptide increased by 66 ± 21.4% the ability of HepG2 cells to take up LDL from the extracellular environment.

  7. Alteration of Receptor/G-protein Interaction by Putative Endogenous Protein Kinase Activity in Dictyostelium discoideum Membranes

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1987-01-01

    Membranes of Dictyostelium discoideum cells were incubated under phosphorylation conditions and washed, and the effects on cAMP binding to chemotactic receptors in the absence and presence of guanosine 5’-O-(3-thiotriphosphate) (GTPγS) were investigated. Most experiments were done with adenosine

  8. Interactions of purified bovine brain A1-adenosine receptors with G-proteins. Reciprocal modulation of agonist and antagonist binding.

    Science.gov (United States)

    Freissmuth, M; Selzer, E; Schütz, W

    1991-05-01

    The bovine brain A1-adenosine receptor was purified 8000-fold by affinity chromatography on xanthine-amine-congener (XAC)-Sepharose. Addition of a 120-fold molar excess of a purified bovine brain G-protein preparation (Go,i a mixture of Go and Gi, containing predominantly Go) decreases the Bmax of the binding of the antagonist radioligand [3H]XAC to the receptor. This decrease is observed not only after insertion into phospholipid vesicles but also in detergent solution, and is reversed by GTP analogues. In the presence of Go,i, about 20 and 40% of the receptors display guanine-nucleotide-sensitive high-affinity binding of the agonist radioligand (-)-N6-3-([125I]iodo-4-hydroxyphenylisopropyl)adenosine after reconstitution into lipid vesicles and in detergent solution, respectively. The ability of Go,i to enhance agonist binding and decrease antagonist binding is concentration-dependent, with a half-maximal effect occurring at approximately 10-fold molar excess of G-proteins over A1-adenosine receptors. In the presence of the receptor, the rate of guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]) binding to Go,i is accelerated. This rate is further enhanced if the receptor is activated by the agonist (-)(R)-N6-phenylisopropyladenosine, whereas the antagonist XAC decreases the association rate of GTP[35S] to levels observed in the absence of receptor. These results show (1) that detergent removal is not a prerequisite for the observation of coupling between the A1-adenosine receptor and Go,i, and (2) that the regulatory effect of G-proteins on antagonist binding to the A1-adenosine receptor can be reconstituted by using purified components.

  9. Brain-specific interaction of a 91-kDa membrane-bound protein with the cytoplasmic tail of the 300-kDa mannose 6-phosphate receptor

    DEFF Research Database (Denmark)

    Rosorius, O; Issinger, O G; Braulke, T

    1996-01-01

    The cytoplasmic tail of the 300 kDa mannose 6-phosphate receptor (MPR 300-CT) is thought to play an important role in sorting and targeting of lysosomal enzymes and the insulin-like growth factor II along the biosynthetic and endocytic pathway. In this study a brain specific 91 kDa protein and a 35...... kDa protein salt-washed from membranes (referred as TIP 91-M and TIP 35-M) were found to interact with the cytoplasmic receptor tail as assayed by cross-linkage with recombinant [32P] labeled MPR 300-CT. Subcellular fractionation revealed a distinct pattern of distribution of TIP 35-M and TIP 91-M...... in microsomal and synaptosomal fractions. Furthermore, the formation of cross-link complexes with membrane proteins appeared to be developmentally and regionally regulated in the brain and inhibited upon ATP hydrolysis. The data suggest the requirement of specific protein interactions for MPR 300 functions...

  10. Receptor Interacting Protein 3-Mediated Necroptosis Promotes Lipopolysaccharide-Induced Inflammation and Acute Respiratory Distress Syndrome in Mice.

    Directory of Open Access Journals (Sweden)

    Linlin Wang

    Full Text Available Necrosis amplifies inflammation and plays important roles in acute respiratory distress syndrome (ARDS. Necroptosis is a newly identified programmed necrosis that is mediated by receptor interacting protein 3 (RIP3. However, the potential involvement and impact of necroptosis in lipopolysaccharide (LPS-induced ARDS remains unknown. We therefore explored the role and mechanism of RIP3-mediated necroptosis in LPS-induced ARDS. Mice were instilled with increasing doses of LPS intratracheally to induce different degrees of ARDS. Lung tissues were harvested for histological and TUNEL staining and western blot for RIP3, p-RIP3, X-linked inhibitor of apoptosis protein (XIAP, mixed lineage kinase domain-like protein (MLKL, total and cleaved caspases-3/8. Then, wild-type and RIP3 knock-out mice were induced ARDS with 30 mg/kg LPS. Pulmonary cellular necrosis was labeled by the propidium Iodide (PI staining. Levels of TNF-a, Interleukin (IL-1β, IL-6, IL-1α, IL-10 and HMGB1, tissue myeloperoxidase (MPO activity, neutrophil counts and total protein concentration were measured. Results showed that in high dose LPS (30mg/kg and 40mg/kg -induced severe ARDS, RIP3 protein was increased significantly, accompanied by increases of p-RIP3 and MLKL, while in low dose LPS (10mg/kg and 20mg/kg -induced mild ARDS, apoptosis was remarkably increased. In LPS-induced severe ARDS, RIP3 knock-out alleviated the hypothermia symptom, increased survival rate and ameliorated the lung tissue injury RIP3 depletion also attenuated LPS-induced increase in IL-1α/β, IL-6 and HMGB1 release, decreased tissue MPO activity, and reduced neutrophil influx and total protein concentration in BALF in severe ARDS. Further, RIP3 depletion reduced the necrotic cells in the lung and decreased the expression of MLKL, but had no impact on cleaved caspase-3 in LPS-induced ARDS. It is concluded that RIP3-mediated necroptosis is a major mechanism of enhanced inflammation and lung tissue injury in

  11. A highly conserved glycine within linker I and the extreme C terminus of G protein alpha subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity

    DEFF Research Database (Denmark)

    Kostenis, Evi; Martini, Lene; Ellis, James

    2004-01-01

    recognition by Galpha(q) proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant Galpha(q) proteins in which glycine was replaced with various amino acids...... on GPCR-to-effector specificity. Dually modified Galpha proteins were also superior in conferring high-affinity agonist sites onto a coexpressed GPCR in the absence, but not in the presence, of guanine nucleotides. Together, our data suggest that receptor-G protein coupling selectivity involves...

  12. Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis

    NARCIS (Netherlands)

    Gouget, A.; Senchou, V.; Govers, F.; Sanson, A.; Barre, A.; Rougé, P.; Pont-Lezica, R.; Canut, H.

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis

  13. Interaction of the Hereditary Hemochromatosis Protein, HFE, with Transferrin Receptor 2 Is Required for Transferrin-Induced Hepcidin Expression

    Science.gov (United States)

    Gao, Junwei; Chen, Juxing; Kramer, Maxwell; Tsukamoto, Hidekazu; Zhang, An-Sheng; Enns, Caroline A.

    2009-01-01

    SUMMARY The mechanisms that allow the body to sense iron levels in order to maintain iron homeostasis are unknown. Patients with the most common form of hereditary iron overload have mutations in the hereditary hemochromatosis protein, HFE. They have lower levels of hepcidin, than unaffected individuals. Hepcidin, a hepatic peptide hormone, negatively regulates iron efflux from the intestines into the blood. We report two hepatic cell lines, WIF-B cells and HepG2 cells transfected with HFE, where hepcidin expression responded to iron-loaded transferrin. The response was abolished when endogenous transferrin receptor 2 (TfR2) was suppressed or in primary hepatocytes lacking either functional TfR2 or HFE. Furthermore, transferrin-treated HepG2 cells transfected with HFE chimeras containing only the α3 and cytoplasmic domains could upregulate hepcidin expression. Since the HFE α3 domain interacts with TfR2, these results supported our finding that TfR2/HFE complex is required for transcriptional regulation of hepcidin by holo-Tf. PMID:19254567

  14. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    of research are explored. Here we present an overview of the most widely used protein-protein interaction databases and the methods they employ to gather, combine, and predict interactions. We also point out the trade-off between comprehensiveness and accuracy and the main pitfall scientists have to be aware...

  15. Molecular dynamics simulations and structure-based network analysis reveal structural and functional aspects of G-protein coupled receptor dimer interactions

    Science.gov (United States)

    Baltoumas, Fotis A.; Theodoropoulou, Margarita C.; Hamodrakas, Stavros J.

    2016-06-01

    A significant amount of experimental evidence suggests that G-protein coupled receptors (GPCRs) do not act exclusively as monomers but also form biologically relevant dimers and oligomers. However, the structural determinants, stoichiometry and functional importance of GPCR oligomerization remain topics of intense speculation. In this study we attempted to evaluate the nature and dynamics of GPCR oligomeric interactions. A representative set of GPCR homodimers were studied through Coarse-Grained Molecular Dynamics simulations, combined with interface analysis and concepts from network theory for the construction and analysis of dynamic structural networks. Our results highlight important structural determinants that seem to govern receptor dimer interactions. A conserved dynamic behavior was observed among different GPCRs, including receptors belonging in different GPCR classes. Specific GPCR regions were highlighted as the core of the interfaces. Finally, correlations of motion were observed between parts of the dimer interface and GPCR segments participating in ligand binding and receptor activation, suggesting the existence of mechanisms through which dimer formation may affect GPCR function. The results of this study can be used to drive experiments aimed at exploring GPCR oligomerization, as well as in the study of transmembrane protein-protein interactions in general.

  16. The Arabidopsis thaliana AAA protein CDC48A interacts in vivo with the somatic embryogenesis receptor-like kinase 1 receptor at the plasma membrane.

    NARCIS (Netherlands)

    Aker, J.C.M.; Borst, J.W.; Karlova, R.B.; Vries, de S.C.

    2006-01-01

    Fluorescent cell division cycle (CDC)48 proteins were studied in living plant protoplasts. CDC48A and somatic embryogenesis receptor like kinase 1 (SERK1) were found to co-localize in the endoplasmatic reticulum (ER) and at the plasma membrane (PM), but not in endosomal compartments. Fluorescent

  17. Interactions of purified bovine brain A1-adenosine receptors with G-proteins. Reciprocal modulation of agonist and antagonist binding.

    OpenAIRE

    Freissmuth, M.; Selzer, E.; Schütz, W.

    1991-01-01

    The bovine brain A1-adenosine receptor was purified 8000-fold by affinity chromatography on xanthine-amine-congener (XAC)-Sepharose. Addition of a 120-fold molar excess of a purified bovine brain G-protein preparation (Go,i a mixture of Go and Gi, containing predominantly Go) decreases the Bmax of the binding of the antagonist radioligand [3H]XAC to the receptor. This decrease is observed not only after insertion into phospholipid vesicles but also in detergent solution, and is reversed by GT...

  18. Xanthomonas citri subsp. citri surface proteome by 2D-DIGE: Ferric enterobactin receptor and other outer membrane proteins potentially involved in citric host interaction.

    Science.gov (United States)

    Carnielli, Carolina Moretto; Artier, Juliana; de Oliveira, Julio Cezar Franco; Novo-Mansur, Maria Teresa Marques

    2017-01-16

    Xanthomonas citri subsp. citri (XAC) is the causative agent of citrus canker, a disease of great economic impact around the world. Understanding the role of proteins on XAC cellular surface can provide new insights on pathogen-plant interaction. Surface proteome was performed in XAC grown in vivo (infectious) and in vitro (non-infectious) conditions, by labeling intact cells followed by cellular lysis and direct 2D-DIGE analysis. Seventy-nine differential spots were analyzed by mass spectrometry. Highest relative abundance for in vivo condition was observed for spots containing DnaK protein, 60kDa chaperonin, conserved hypothetical proteins, malate dehydrogenase, phosphomannose isomerase, and ferric enterobactin receptors. Elongation factor Tu, OmpA-related proteins, Oar proteins and some Ton-B dependent receptors were found in spots decreased in vivo. Some proteins identified on XAC's surface in infectious condition and predicted to be cytoplasmic, such as DnaK and 60KDa chaperonin, have also been previously found at cellular surface in other microorganisms. This is the first study on XAC surface proteome and results point to mediation of molecular chaperones in XAC-citrus interaction. The approach utilized here can be applied to other pathogen-host interaction systems and help to achieve new insights in bacterial pathogenicity toward promising targets of biotechnological interest. This research provides new insights for current knowledge of the Xanthomonas sp. pathogenicity. For the first time the 2D-DIGE approach was applied on intact cells to find surface proteins involved in the pathogen-plant interaction. Results point to the involvement of new surface/outer membrane proteins in the interaction between XAC and its citrus host and can provide potential targets of biotechnological interest for citrus canker control. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Protein Connectivity in Chemotaxis Receptor Complexes.

    Directory of Open Access Journals (Sweden)

    Stephan Eismann

    2015-12-01

    Full Text Available The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures.

  20. Human papillomavirus 16 E2 interacts with neuregulin receptor degradation protein 1 affecting ErbB-3 expression in vitro and in clinical samples of cervical lesions.

    Science.gov (United States)

    Paolini, Francesca; Curzio, Gianfranca; Melucci, Elisa; Terrenato, Irene; Antoniani, Barbara; Carosi, Mariantonia; Mottolese, Marcella; Vici, Patrizia; Mariani, Luciano; Venuti, Aldo

    2016-05-01

    The ErbB tyrosine kinase receptors play a key role in regulating many cellular functions and human papillomaviruses (HPVs) may interact with transductional pathway of different growth factor receptors. Here, these interactions were analysed in W12 cell line carrying HPV 16 genome and in clinical samples. W12 cells, in which HPV16 becomes integrated during passages, were utilised to detect viral and ErbB family expression at early (W12E) and late passages (W12G). Interestingly, a strong reduction of ErbB-3 expression was observed in W12G. Loss of the E2 and E5 viral genes occurs in W12G and this may affect ErbB-3 receptor expression. E2 and E5 rescue experiments demonstrated that only E2 gene was able to restore ErbB-3 expression. E2 is a transcriptional factor but the expression levels of ErbB3 were unaffected and ErbB-3 promoter did not show any consensus sequence for E2, thus E2 may interact in another way with ErbB3. Indeed, HPV 16 E2 can modulate ErbB-3 by interacting with the ubiquitin ligase neuregulin receptor degradation protein 1 (Nrdp-1) that is involved in the regulation of this receptor, via ubiquitination and degradation. E2 co-immunoprecipitated in a complex with Nrdp-1 leading to hypothesise an involvement of this interaction in ErbB-3 regulation. In addition, 90% of the clinical samples with integrated virus and E2 loss showed no or low ErbB-3 positivity, confirming in vitro results. In conclusion, the new discovered interaction of HPV-16 E2 with Nrdp-1 may affect ErbB-3 expression. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. DEHP reduces thyroid hormones via interacting with hormone synthesis-related proteins, deiodinases, transthyretin, receptors, and hepatic enzymes in rats.

    Science.gov (United States)

    Liu, Changjiang; Zhao, Letian; Wei, Li; Li, Lianbing

    2015-08-01

    Di-(2-ethylhexyl) phthalate (DEHP) is used extensively in many personal care and consumer products, resulting in widespread nonoccupational human exposure through multiple routes and media. Limited studies suggest that exposure to DEHP may be associated with altered thyroid function, but detailed mechanisms are unclear. In order to elucidate potential mechanisms by which DEHP disturbs thyroid hormone homeostasis, Sprague-Dawley (SD) rats were dosed with DEHP by gavage at 0, 250, 500, and 750 mg/kg/day for 30 days and sacrificed within 24 h after the last dose. Gene expressions of thyroid hormone receptors, deiodinases, transthyretin, and hepatic enzymes were measured by RT-PCR; protein levels of transthyretin were also analyzed by Western blot. Results showed that DEHP caused histological changes in the thyroid and follicular epithelial cell hypertrophy and hyperplasia were observed. DEHP significantly reduced thyroid hormones (T3, T4) and thyrotropin releasing hormone (TRH) levels, whereas thyroid stimulating hormone (TSH) was not affected. After exposure to DEHP, biosynthesis of thyroid hormones was suppressed, and sodium iodide symporter (NIS) and thyroid peroxidase (TPO) levels were significantly reduced. Additionally, levels of deiodinases and transthyretin were also affected. TSH receptor (TSHr) level was downregulated, while TRH receptor (TRHr) level was upregulated. Metabolism of thyroid hormones was accelerated due to elevated gene expression of hepatic enzymes (UDPGTs and CYP2B1) by DEHP. Taken together, observed findings indicate that DEHP could reduce thyroid hormones through influencing biosynthesis, biotransformation, biotransport, receptor levels, and metabolism of thyroid hormones.

  2. Maize and Arabidopsis ARGOS Proteins Interact with Ethylene Receptor Signaling Complex, Supporting a Regulatory Role for ARGOS in Ethylene Signal Transduction[OPEN

    Science.gov (United States)

    Shi, Jinrui; Wang, Hongyu; Habben, Jeffrey E.

    2016-01-01

    The phytohormone ethylene regulates plant growth and development as well as plant response to environmental cues. ARGOS genes reduce plant sensitivity to ethylene when overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). A previous genetic study suggested that the endoplasmic reticulum and Golgi-localized maize ARGOS1 targets the ethylene signal transduction components at or upstream of CONSTITUTIVE TRIPLE RESPONSE1, but the mechanism of ARGOS modulating ethylene signaling is unknown. Here, we demonstrate in Arabidopsis that ZmARGOS1, as well as the Arabidopsis ARGOS homolog ORGAN SIZE RELATED1, physically interacts with Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), an ethylene receptor interacting protein that regulates the activity of ETHYLENE RESPONSE1. The protein-protein interaction was also detected with the yeast split-ubiquitin two-hybrid system. Using the same yeast assay, we found that maize RTE1 homolog REVERSION-TO-ETHYLENE SENSITIVITY1 LIKE4 (ZmRTL4) and ZmRTL2 also interact with maize and Arabidopsis ARGOS proteins. Like AtRTE1 in Arabidopsis, ZmRTL4 and ZmRTL2 reduce ethylene responses when overexpressed in maize, indicating a similar mechanism for ARGOS regulating ethylene signaling in maize. A polypeptide fragment derived from ZmARGOS8, consisting of a Pro-rich motif flanked by two transmembrane helices that are conserved among members of the ARGOS family, can interact with AtRTE1 and maize RTL proteins in Arabidopsis. The conserved domain is necessary and sufficient to reduce ethylene sensitivity in Arabidopsis and maize. Overall, these results suggest a physical association between ARGOS and the ethylene receptor signaling complex via AtRTE1 and maize RTL proteins, supporting a role for ARGOS in regulating ethylene perception and the early steps of signal transduction in Arabidopsis and maize. PMID:27268962

  3. Non-classical testosterone signaling is mediated by a G-protein-coupled receptor interacting with Gnα11.

    Science.gov (United States)

    Shihan, Mazen; Bulldan, Ahmed; Scheiner-Bobis, Georgios

    2014-06-01

    Testosterone is known to mediate its effects by two different mechanisms of action. In the so-called "classical" pathway testosterone binds to cytosolic androgen receptors (AR), which essentially function as ligand-activated transcription factors. Once activated, these receptors bind to DNA and activate the expression of target genes. In the "non-classical" pathway, the steroid hormone binds to receptors associated with the plasma membrane and induces signaling cascades mediated through activation of Erk1/2. The precise nature of the membrane-associated AR, however, remains controversial. Although some assume that the membrane and cytosolic AR are identical, others propose that the AR of the membrane is a G-protein-coupled receptor (GPCR). To evaluate these two possibilities we first searched for testosterone-induced signaling cascades in the spermatogenic cell line GC-2. Testosterone was found to cause phosphorylation (activation) of Erk1/2, CREB, and ATF-1, consistent with its non-classical mechanism of action. Silencing of AR expression by means of siRNA did not influence testosterone-induced activation of Erk1/2, CREB, or ATF-1, indicating that this pathway is not activated by the classical cytosolic/nuclear AR. In contrast, when the expression of the G-protein Gnα11 is suppressed, the activation of these signaling molecules is abolished, suggesting that these responses are elicited through a membrane-bound GPCR. The results presented here and the identification of the testosterone-specific GPCR in future investigations will help to reveal and characterize new testosterone-mediated mechanisms associated not only with fertility and reproduction but perhaps also with other physiological processes. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Interaction between estrogen receptor and retinol-binding protein-4 polymorphisms as a tool for the selection of prolific pigs

    Directory of Open Access Journals (Sweden)

    Iara Denise Vasconcellos Gonçalves

    2008-01-01

    Full Text Available The aim of the present study was to investigate the association of the estrogen receptor (ER-PvuII and retinol-binding protein 4 (RBP4-MspI gene polymorphisms and their interactions with prolificacy in a commercial synthetic pig line reared in Brazil. A total of 10,374 piglet records from 218 sows and 817 litters were used for litter size analysis. Only females with three or four farrowings were included in the analysis. The mean litter size ranged from 5.0 to 19.5 piglets. DNA was extracted from leukocytes by a standard method, and ER-PvuII and RBP4-MspI polymorphisms were characterized by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP technique. The association between alleles or genotypes and reproductive performance was analyzed using a general linear model including the interaction between the ER-PvuII and RBP4-MspI genotypes. For the ER-PvuII gene, the allele frequencies of allele A and allele B were 0.56 and 0.44, respectively. For the RBP4-MspI gene, the frequencies of alleles A1 and A2 were 0.29 and 0.71, respectively. The total number of piglets born (TNB, born alive (NBA, or number of mummies and stillborn piglets (NMUM and NSB per litter did not differ between the various ER-PvuII and RBP4-MspI genotypes. However, when the ER-PvuII and RBP4-MspI genotypes were considered together in each sow, TNB and NBA were 1.4 (p = 0.0026 and 0.9 (p = 0.019 higher in AA/A1 and AB/A1 animals, respectively, than in AA/A2 and BB/A1 animals. Likewise, TNB and NBA were 0.9 (p = 0.0258 and 0.8 (p = 0.0168 higher in BB/A2 and AB/A2 sows, respectively, than in AA/A2 and BB/A1 animals, but no difference was observed compared to AA/A1 and AB/A1 animals. The results showed larger litter sizes (TNB and NBA for sows carrying the ER-PvuII allele A and the RBP4-MspI genotype A1, and for animals carrying the ER-PvuII allele B and the RBP4-MspI genotype A2. In conclusion, the interaction between genotypes ER-PvuII and RBP4-MspI is

  5. Aquaporin Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Jennifer Virginia Roche

    2017-10-01

    Full Text Available Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein–protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein–protein interactions; dividing the interactions into three types: (1 interactions between aquaporin tetramers; (2 interactions between aquaporin monomers within a tetramer (hetero-tetramerization; and (3 transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.

  6. Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor.

    Science.gov (United States)

    Kasheverov, Igor E; Zhmak, Maxim N; Fish, Alexander; Rucktooa, Prakash; Khruschov, Alexey Yu; Osipov, Alexey V; Ziganshin, Rustam H; D'hoedt, Dieter; Bertrand, Daniel; Sixma, Titia K; Smit, August B; Tsetlin, Victor I

    2009-11-01

    alpha-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. alpha-Conotoxins blocking muscle-type or alpha7 nAChRs compete with alpha-bungarotoxin. However, alpha-conotoxin ImII, a close homolog of the alpha7 nAChR-targeting alpha-conotoxin ImI, blocked alpha7 and muscle nAChRs without displacing alpha-bungarotoxin (Ellison et al. 2003, 2004), suggesting binding at a different site. We synthesized alpha-conotoxin ImII, its ribbon isomer (ImIIiso), 'mutant' ImII(W10Y) and found similar potencies in blocking human alpha7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [(125)I]-alpha-bungarotoxin from human alpha7 nAChRs in the cell line GH(4)C(1) (IC(50) 17 and 23 microM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC(50) 2.0-9.0 microM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized alpha-bungarotoxin (K(d) and IC(50) 2.5-8.2 microM). On Torpedo nAChR, alpha-conotoxin [(125)I]-ImII(W10Y) revealed specific binding (K(d) 1.5-6.1 microM) and could be displaced by alpha-conotoxin ImII, ImIIiso and ImII(W10Y) with IC(50) 2.7, 2.2 and 3.1 microM, respectively. As alpha-cobratoxin and alpha-conotoxin ImI displaced [(125)I]-ImII(W10Y) only at higher concentrations (IC(50)> or = 90 microM), our results indicate that alpha-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.

  7. Neto1 is a novel CUB-domain NMDA receptor-interacting protein required for synaptic plasticity and learning.

    Science.gov (United States)

    Ng, David; Pitcher, Graham M; Szilard, Rachel K; Sertié, Andréa; Kanisek, Marijana; Clapcote, Steven J; Lipina, Tatiana; Kalia, Lorraine V; Joo, Daisy; McKerlie, Colin; Cortez, Miguel; Roder, John C; Salter, Michael W; McInnes, Roderick R

    2009-02-24

    The N-methyl-D-aspartate receptor (NMDAR), a major excitatory ligand-gated ion channel in the central nervous system (CNS), is a principal mediator of synaptic plasticity. Here we report that neuropilin tolloid-like 1 (Neto1), a complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing transmembrane protein, is a novel component of the NMDAR complex critical for maintaining the abundance of NR2A-containing NMDARs in the postsynaptic density. Neto1-null mice have depressed long-term potentiation (LTP) at Schaffer collateral-CA1 synapses, with the subunit dependency of LTP induction switching from the normal predominance of NR2A- to NR2B-NMDARs. NMDAR-dependent spatial learning and memory is depressed in Neto1-null mice, indicating that Neto1 regulates NMDA receptor-dependent synaptic plasticity and cognition. Remarkably, we also found that the deficits in LTP, learning, and memory in Neto1-null mice were rescued by the ampakine CX546 at doses without effect in wild-type. Together, our results establish the principle that auxiliary proteins are required for the normal abundance of NMDAR subunits at synapses, and demonstrate that an inherited learning defect can be rescued pharmacologically, a finding with therapeutic implications for humans.

  8. Neto1 is a novel CUB-domain NMDA receptor-interacting protein required for synaptic plasticity and learning.

    Directory of Open Access Journals (Sweden)

    David Ng

    2009-02-01

    Full Text Available The N-methyl-D-aspartate receptor (NMDAR, a major excitatory ligand-gated ion channel in the central nervous system (CNS, is a principal mediator of synaptic plasticity. Here we report that neuropilin tolloid-like 1 (Neto1, a complement C1r/C1s, Uegf, Bmp1 (CUB domain-containing transmembrane protein, is a novel component of the NMDAR complex critical for maintaining the abundance of NR2A-containing NMDARs in the postsynaptic density. Neto1-null mice have depressed long-term potentiation (LTP at Schaffer collateral-CA1 synapses, with the subunit dependency of LTP induction switching from the normal predominance of NR2A- to NR2B-NMDARs. NMDAR-dependent spatial learning and memory is depressed in Neto1-null mice, indicating that Neto1 regulates NMDA receptor-dependent synaptic plasticity and cognition. Remarkably, we also found that the deficits in LTP, learning, and memory in Neto1-null mice were rescued by the ampakine CX546 at doses without effect in wild-type. Together, our results establish the principle that auxiliary proteins are required for the normal abundance of NMDAR subunits at synapses, and demonstrate that an inherited learning defect can be rescued pharmacologically, a finding with therapeutic implications for humans.

  9. The human cytomegalovirus UL11 protein interacts with the receptor tyrosine phosphatase CD45, resulting in functional paralysis of T cells.

    Directory of Open Access Journals (Sweden)

    Ildar Gabaev

    2011-12-01

    Full Text Available Human cytomegalovirus (CMV exerts diverse and complex effects on the immune system, not all of which have been attributed to viral genes. Acute CMV infection results in transient restrictions in T cell proliferative ability, which can impair the control of the virus and increase the risk of secondary infections in patients with weakened or immature immune systems. In a search for new immunomodulatory proteins, we investigated the UL11 protein, a member of the CMV RL11 family. This protein family is defined by the RL11 domain, which has homology to immunoglobulin domains and adenoviral immunomodulatory proteins. We show that pUL11 is expressed on the cell surface and induces intercellular interactions with leukocytes. This was demonstrated to be due to the interaction of pUL11 with the receptor tyrosine phosphatase CD45, identified by mass spectrometry analysis of pUL11-associated proteins. CD45 expression is sufficient to mediate the interaction with pUL11 and is required for pUL11 binding to T cells, indicating that pUL11 is a specific CD45 ligand. CD45 has a pivotal function regulating T cell signaling thresholds; in its absence, the Src family kinase Lck is inactive and signaling through the T cell receptor (TCR is therefore shut off. In the presence of pUL11, several CD45-mediated functions were inhibited. The induction of tyrosine phosphorylation of multiple signaling proteins upon TCR stimulation was reduced and T cell proliferation was impaired. We therefore conclude that pUL11 has immunosuppressive properties, and that disruption of T cell function via inhibition of CD45 is a previously unknown immunomodulatory strategy of CMV.

  10. Down syndrome candidate region-1 protein interacts with Tollip and positively modulates interleukin-1 receptor-mediated signaling.

    Science.gov (United States)

    Lee, Jae Youn; Lee, Hyun Jung; Lee, Eun Jung; Jang, Sung Hee; Kim, Hyeyoung; Yoon, Joo-Heon; Chung, Kwang Chul

    2009-12-01

    The Down syndrome candidate region-1 gene (DSCR1, also known as RCAN1) is situated close to the Down Syndrome Critical Region (DSCR), which contains genes responsible for many features of Down syndrome. DSCR1 modulates calcineurin phosphatase activity, though its functional role is incompletely understood. Here we investigated the role of DSCR1-1S isoform in IL-1 receptor (IL-1R)-mediated signaling by analyzing interaction between DSCR1-1S and the IL-1R pathway components Tollip, IRAK-1, and TRAF6. Co-immunoprecipitation analyses of HEK293 cells revealed that DSCR1-1S interacted with Tollip, an IRAK-1 inhibitor, leading to the dissociation of IRAK-1 from Tollip. Similarly, both DSCR1-1S and Tollip interacted with TRAF6, with DSCR1 reducing interaction between Tollip and TRAF6. DSCR1-1S also stimulated IL-1R-mediated signaling pathways, TAK1 activation, NF-kappaB transactivation, and IL-8 production, all downstream consequences of IL-1R activation. Together, these results suggest that DSCR1-1S isoform positively modulates IL-1R-mediated signaling pathways by regulating Tollip/IRAK-1/TRAF6 complex formation.

  11. Alix (AIP1) is a vasopressin receptor (V2R)-interacting protein that increases lysosomal degradation of the V2R.

    Science.gov (United States)

    Yi, Xianhua; Bouley, Richard; Lin, Herbert Y; Bechoua, Shaliha; Sun, Tian-Xiao; Del Re, Elisabetta; Shioda, Toshi; Raychowdhury, Malay K; Lu, Hua A J; Abou-Samra, Abdul B; Brown, Dennis; Ausiello, Dennis A

    2007-05-01

    The vasopressin type 2 receptor (V2R) is a G protein-coupled receptor that plays a central role in renal water reabsorption. Termination of ligand (vasopressin) stimulation is an important physiological regulatory event, but few proteins that interact with the V2R during downregulation after vasopressin (VP) binding have been identified. Using yeast two-hybrid screening of a human kidney cDNA library, we show that a 100-kDa protein called ALG-2-interacting protein X (Alix) interacts with the last 29 amino acids of the V2R COOH terminus. This was confirmed by pull-down assays using a GST-V2R-COOH-tail fusion protein. Alix was immunolocalized in principal cells of the kidney, which also express the V2R. The function of the Alix-V2R interaction was studied by transfecting Alix into LLC-PK(1) epithelial cells expressing V2R-green fluorescent protein (GFP). Under basal conditions, V2R-GFP localized mainly at the plasma membrane. On VP treatment, V2R-GFP was internalized into perinuclear vesicles in the nontransfected cells. In contrast, V2R-GFP fluorescence was virtually undetectable 2 h after exposure to VP in cells that coexpressed Alix. Western blotting using an anti-GFP antibody showed marked degradation of the V2R after 2 h in the presence of VP and Alix, a time point at which little or no degradation was detected in the absence of Alix. In contrast, little or no degradation of the parathyroid hormone receptor was detectable in the presence or absence of Alix and/or the PTH ligand. The VP-induced disappearance of V2R-GFP was abolished by chloroquine, a lysosomal degradation inhibitor, but not by MG132, a proteosome inhibitor. These data suggest that Alix increases the rate of lysosomal degradation of V2R and may play an important regulatory role in the VP response by modulating V2R downregulation.

  12. Glucocorticoids curtail stimuli-induced CREB phosphorylation in TRH neurons through interaction of the glucocorticoid receptor with the catalytic subunit of protein kinase A.

    Science.gov (United States)

    Sotelo-Rivera, Israim; Cote-Vélez, Antonieta; Uribe, Rosa-María; Charli, Jean-Louis; Joseph-Bravo, Patricia

    2017-03-01

    Corticosterone prevents cold-induced stimulation of thyrotropin-releasing hormone (Trh) expression in rats, and the stimulatory effect of dibutyryl cyclic-adenosine monophosphate (dB-cAMP) on Trh transcription in hypothalamic cultures. We searched for the mechanism of this interference. Immunohistochemical analyses of phosphorylated cAMP-response element binding protein (pCREB) were performed in the paraventricular nucleus (PVN) of Wistar rats, and in cell cultures of 17-day old rat hypothalami, or neuroblastoma SH-SY5Y cells. Cultures were incubated 1h with dB-cAMP, dexamethasone and both drugs combined; their nuclear extracts were used for chromatin immunoprecipitation; cytosolic or nuclear extracts for coimmunoprecipitation analyses of catalytic subunit of protein kinase A (PKAc) and of glucocorticoid receptor (GR); their subcellular distribution was analyzed by immunocytochemistry. Cold exposure increased pCREB in TRH neurons of rats PVN, effect blunted by corticosterone previous injection. Dexamethasone interfered with forskolin increase in nuclear pCREB and its binding to Trh promoter; antibodies against histone deacetylase-3 precipitated chromatin from nuclear extracts of hypothalamic cells treated with tri-iodothyronine but not with dB-cAMP + dexamethasone, discarding chromatin compaction as responsible mechanism. Co-immunoprecipitation analyses of cytosolic or nuclear extracts showed protein:protein interactions between activated GR and PKAc. Immunocytochemical analyses of hypothalamic or SH-SY5Y cells revealed diminished nuclear translocation of PKAc and GR in cells incubated with forskolin + dexamethasone, compared to either forskolin or dexamethasone alone. Glucocorticoids and cAMP exert mutual inhibition of Trh transcription through interaction of activated glucocorticoid receptor with protein kinase A catalytic subunit, reducing their nuclear translocation, limiting cAMP-response element binding protein phosphorylation and its binding to Trh promoter.

  13. Interaction between the inner nuclear membrane lamin B receptor and the heterochromatic methyl binding protein, MeCP2

    Energy Technology Data Exchange (ETDEWEB)

    Guarda, Alessia, E-mail: alessiaguarda@libero.it [Department of Structural and Functional Biology, University of Insubria, via Alberto da Giussano 12, Busto Arsizio (Italy); Bolognese, Fabrizio, E-mail: fabrizio.bolognese@uninsubria.it [Department of Structural and Functional Biology, University of Insubria, via Alberto da Giussano 12, Busto Arsizio (Italy); Bonapace, Ian Marc, E-mail: ian.bonapace@uninsubria.it [Department of Structural and Functional Biology, University of Insubria, via Alberto da Giussano 12, Busto Arsizio (Italy); Badaracco, Gianfranco, E-mail: gianfranco.badaracco@uninsubria.it [Department of Structural and Functional Biology, University of Insubria, via Alberto da Giussano 12, Busto Arsizio (Italy)

    2009-07-01

    The nuclear membrane has an important role for the dynamic regulation of the genome, besides the well-established cytoskeletal function. The nuclear lamina is emerging as an important player in the organization of the position and functional state of interphase chromosomes. Epigenetic modifications such as DNA methylation and histone modifications are required for genome reprogramming during development, tissue-specific gene expression and global gene silencing. The Methyl-CpG binding protein MeCP2 binds methyl-CpG dinucleotides in the mammalian genome and functions as a transcriptional repressor in vivo by interacting with Sin3A, thereby recruiting histone deacetylases (HDAC). MeCP2 also mediates the formation of higher-order chromatin structures contributing to determine the architectural organization of the nucleus. In this paper, we show that MeCP2 interacts in vitro and in vivo with the inner nuclear membrane protein LBR and that the unstructured aminoacidic sequence linking the MBD and TRD domains of MeCP2 is responsible for this association. The formation of an LBR-MeCP2 protein complex might help providing a molecular explanation to the distribution of part of the heterochromatin at the nuclear periphery linked to inner membrane.

  14. Interaction between the inner nuclear membrane lamin B receptor and the heterochromatic methyl binding protein, MeCP2.

    Science.gov (United States)

    Guarda, Alessia; Bolognese, Fabrizio; Bonapace, Ian Marc; Badaracco, Gianfranco

    2009-07-01

    The nuclear membrane has an important role for the dynamic regulation of the genome, besides the well-established cytoskeletal function. The nuclear lamina is emerging as an important player in the organization of the position and functional state of interphase chromosomes. Epigenetic modifications such as DNA methylation and histone modifications are required for genome reprogramming during development, tissue-specific gene expression and global gene silencing. The Methyl-CpG binding protein MeCP2 binds methyl-CpG dinucleotides in the mammalian genome and functions as a transcriptional repressor in vivo by interacting with Sin3A, thereby recruiting histone deacetylases (HDAC). MeCP2 also mediates the formation of higher-order chromatin structures contributing to determine the architectural organization of the nucleus. In this paper, we show that MeCP2 interacts in vitro and in vivo with the inner nuclear membrane protein LBR and that the unstructured aminoacidic sequence linking the MBD and TRD domains of MeCP2 is responsible for this association. The formation of an LBR-MeCP2 protein complex might help providing a molecular explanation to the distribution of part of the heterochromatin at the nuclear periphery linked to inner membrane.

  15. Our interests in protein-protein interactions

    Indian Academy of Sciences (India)

    protein interactions. Evolution of P-P partnerships. Evolution of P-P structures. Evolutionary dynamics of P-P interactions. Dynamics of P-P interaction network. Host-pathogen interactions. CryoEM mapping of gigantic protein assemblies.

  16. Possible Relevance of Receptor-Receptor Interactions between Viral- and Host-Coded Receptors for Viral-Induced Disease

    Directory of Open Access Journals (Sweden)

    Luigi F. Agnati

    2007-01-01

    Full Text Available It has been demonstrated that some viruses, such as the cytomegalovirus, code for G-protein coupled receptors not only to elude the immune system, but also to redirect cellular signaling in the receptor networks of the host cells. In view of the existence of receptor-receptor interactions, the hypothesis is introduced that these viral-coded receptors not only operate as constitutively active monomers, but also can affect other receptor function by interacting with receptors of the host cell. Furthermore, it is suggested that viruses could also insert not single receptors (monomers, but clusters of receptors (receptor mosaics, altering the cell metabolism in a profound way. The prevention of viral receptor-induced changes in host receptor networks may give rise to novel antiviral drugs that counteract viral-induced disease.

  17. Interaction of purified bovine brain A1-adenosine receptors with guanine nucleotide-binding proteins of human platelet membranes following reconstitution.

    Science.gov (United States)

    Munshi, R; Linden, J

    1990-08-01

    A1-adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) have been co-purified from bovine cerebral cortex by agonist affinity chromatography [J. Biol. Chem. 264:14853-14859 (1989)]. In this study we have reconstituted purified bovine brain A1 receptors into human platelet membranes that contain A2- but no detectable A1-adenosine receptors. The recovery of reconstituted receptors was assessed from the binding of the antagonist radioligand [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentyl-xanthine and ranged from 32 to 84%. Coupling of reconstituted A1 receptors to platelet G proteins was evaluated by measurement of the high affinity binding of an agonist radioligand, 125I-aminobenzyladenosine, to receptor-G protein complexes and by stereospecific photoaffinity labeling of a 35,000-Da receptor polypeptide with the agonist photoaffinity label 125I-azidobenzyladenosine. Fifty percent of receptors reconstituted into platelet membranes bound agonists with high affinity, indicative of coupling to platelet G proteins. Reconstituted A1 receptors bound various ligands with affinities characteristic of A1 receptors of bovine brain. Although platelets contain both pertussis toxin-sensitive and -insensitive G proteins, reconstituted high affinity agonist binding was almost completely abolished by treatment of platelet membranes with guanosine 5'-3-O-(thio)triphosphate, pertussis toxin, N-ethylmaleimide, or heparin. Following reconstitution, A1 receptors could be resolubilized in complexes with platelet G proteins. The data suggest that marked species differences in the binding affinity of ligands to adenosine receptors result from differences in the receptors rather than membrane structure or G proteins and, further, that A1 receptors couple selectively and tightly to pertussis toxin-sensitive G proteins.

  18. Binding interactions of human interleukin 5 with its receptor alpha subunit. Large scale production, structural, and functional studies of Drosophila-expressed recombinant proteins.

    Science.gov (United States)

    Johanson, K; Appelbaum, E; Doyle, M; Hensley, P; Zhao, B; Abdel-Meguid, S S; Young, P; Cook, R; Carr, S; Matico, R

    1995-04-21

    Human interleukin 5 (hIL5) and soluble forms of its receptor alpha subunit were expressed in Drosophila cells and purified to homogeneity, allowing a detailed structural and functional analysis. B cell proliferation confirmed that the hIL5 was biologically active. Deglycosylated hIL5 remained active, while similarly deglycosylated receptor alpha subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-A resolution and found to be similar to that of the protein produced in Escherichia coli. Human IL5 was shown by analytical ultracentrifugation to form a 1:1 complex with the soluble domain of the hIL5 receptor alpha subunit (shIL5R alpha). Additionally, the relative abundance of ligand and receptor in the hIL5.shIL5R alpha complex was determined to be 1:1 by both titration calorimetry and SDS-polyacrylamide gel electrophoresis analysis of dissolved cocrystals of the complex. Titration microcalorimetry yielded equilibrium dissociation constants of 3.1 and 2.0 nM, respectively, for the binding of hIL5 to shIL5R alpha and to a chimeric form of the receptor containing shIL5R alpha fused to the immunoglobulin Fc domain (shIL5R alpha-Fc). Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction. Kinetic analysis using surface plasmon resonance yielded data consistent with the Kd values from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor alpha subunit. Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5R alpha in Drosophila membranes was found to be 6 nM, in accord with the affinities measured for the soluble receptor forms. Hence, most of the binding energy of the alpha receptor is supplied by the soluble domain. Taken with other aspects of hIL5 structure and biological activity, the data obtained allow a prediction for how 1:1 stoichiometry and

  19. Characterization of the specific interaction between the DNA aptamer sgc8c and protein tyrosine kinase-7 receptors at the surface of T-cells by biosensing AFM.

    Science.gov (United States)

    Leitner, Michael; Poturnayova, Alexandra; Lamprecht, Constanze; Weich, Sabine; Snejdarkova, Maja; Karpisova, Ivana; Hianik, Tibor; Ebner, Andreas

    2017-04-01

    We studied the interaction of the specific DNA aptamer sgc8c immobilized at the AFM tip with its corresponding receptor, the protein tyrosine kinase-7 (PTK7) embedded in the membrane of acute lymphoblastic leukemia (ALL) cells (Jurkat T-cells). Performing single molecule force spectroscopy (SMFS) experiments, we showed that the aptamer sgc8c bound with high probability (38.3 ± 7.48%) and high specificity to PTK7, as demonstrated by receptor blocking experiments and through comparison with the binding behavior of a nonspecific aptamer. The determined kinetic off-rate (koff = 5.16 s-1) indicates low dissociation of the sgc8c-PTK7 complex. In addition to the pulling force experiments, simultaneous topography and recognition imaging (TREC) experiments using AFM tips functionalized with sgc8c aptamers were realized on the outer regions surface of surface-immobilized Jurkat cells for the first time. This allowed determination of the distribution of PTK7 without any labeling and at near physiological conditions. As a result, we could show a homogeneous distribution of PTK7 molecules on the outer regions of ALL cells with a surface density of 325 ± 12 PTK7 receptors (or small receptor clusters) per μm2. Graphical Abstract The specific interaction of the DNA aptamer sgc8c and protein tyrosine kinase-7 (PTK7) on acute lymphoblastic leukemia (ALL) cells was characterized. AFM based single molecule force spectroscopy (SMFS) yielded a kinetic off-rate of 5.16 s-1 of the complex. Simultaneous topography and recognition imaging (TREC) revealed a PTK7 density of 325 ± 12 molecules or clusters per μm2 in the cell membrane.

  20. Novel receptors for bacterial protein toxins.

    Science.gov (United States)

    Schmidt, Gudula; Papatheodorou, Panagiotis; Aktories, Klaus

    2015-02-01

    While bacterial effectors are often directly introduced into eukaryotic target cells by various types of injection machines, toxins enter the cytosol of host cells from endosomal compartments or after retrograde transport via Golgi from the ER. A first crucial step of toxin-host interaction is receptor binding. Using optimized protocols and new methods novel toxin receptors have been identified, including metalloprotease ADAM 10 for Staphylococcus aureus α-toxin, laminin receptor Lu/BCAM for Escherichia coli cytotoxic necrotizing factor CNF1, lipolysis stimulated lipoprotein receptor (LSR) for Clostridium difficile transferase CDT and low-density lipoprotein receptor-related protein (LRP) 1 for Clostridium perfringens TpeL toxin. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. A nuclear envelope-associated kinase phosphorylates arginine-serine motifs and modulates interactions between the lamin B receptor and other nuclear proteins.

    Science.gov (United States)

    Nikolakaki, E; Simos, G; Georgatos, S D; Giannakouros, T

    1996-04-05

    Previous studies have identified a subassembly of nuclear envelope proteins, termed "the LBR complex." This complex includes the lamin B receptor protein (LBR or p58), a kinase which phosphorylates LBR in a constitutive fashion (LBR kinase), the nuclear lamins A and B, an 18-kDa polypeptide (p18), and a 34-kDa protein (p34/p32). The latter polypeptide has been shown to interact with the HIV-1 proteins Rev and Tat and with the splicing factor 2 (SF2). Using recombinant proteins produced in bacteria and synthetic peptides representing different regions of LBR, we now show that the LBR kinase modifies specifically arginine-serine (RS) dipeptide motifs located at the nucleoplasmic, NH2-terminal domain of LBR and in members of the SR family of splicing factors. Furthermore, we show that the NH2-terminal domain of LBR binds to p34/p32, whereas a mutated domain lacking the RS region does not. Phosphorylation of LBR by the RS kinase completely abolishes binding of p34/p32, suggesting that this enzyme regulates interactions among the components of the LBR complex.

  2. Bioinformatic Prediction of WSSV-Host Protein-Protein Interaction

    Directory of Open Access Journals (Sweden)

    Zheng Sun

    2014-01-01

    Full Text Available WSSV is one of the most dangerous pathogens in shrimp aquaculture. However, the molecular mechanism of how WSSV interacts with shrimp is still not very clear. In the present study, bioinformatic approaches were used to predict interactions between proteins from WSSV and shrimp. The genome data of WSSV (NC_003225.1 and the constructed transcriptome data of F. chinensis were used to screen potentially interacting proteins by searching in protein interaction databases, including STRING, Reactome, and DIP. Forty-four pairs of proteins were suggested to have interactions between WSSV and the shrimp. Gene ontology analysis revealed that 6 pairs of these interacting proteins were classified into “extracellular region” or “receptor complex” GO-terms. KEGG pathway analysis showed that they were involved in the “ECM-receptor interaction pathway.” In the 6 pairs of interacting proteins, an envelope protein called “collagen-like protein” (WSSV-CLP encoded by an early virus gene “wsv001” in WSSV interacted with 6 deduced proteins from the shrimp, including three integrin alpha (ITGA, two integrin beta (ITGB, and one syndecan (SDC. Sequence analysis on WSSV-CLP, ITGA, ITGB, and SDC revealed that they possessed the sequence features for protein-protein interactions. This study might provide new insights into the interaction mechanisms between WSSV and shrimp.

  3. C1q-tumour necrosis factor-related protein 8 (CTRP8) is a novel interaction partner of relaxin receptor RXFP1 in human brain cancer cells.

    Science.gov (United States)

    Glogowska, Aleksandra; Kunanuvat, Usakorn; Stetefeld, Jörg; Patel, Trushar R; Thanasupawat, Thatchawan; Krcek, Jerry; Weber, Ekkehard; Wong, G William; Del Bigio, Marc R; Hoang-Vu, Cuong; Hombach-Klonisch, Sabine; Klonisch, Thomas

    2013-12-01

    We report a novel ligand-receptor system composed of the leucine-rich G-protein-coupled relaxin receptor, RXFP1, and the C1q-tumour necrosis factor-related protein 8 (CTRP8) in human primary brain cancer, a tumour entity devoid of the classical RXFP1 ligands, RLN1-3. In structural homology studies and computational docking experiments we delineated the N-terminal region of the globular C1q region of CTRP8 and the leucine-rich repeat units 7 and 8 of RXFP1 to mediate this new ligand-receptor interaction. CTRP8 secreted from HEK293T cells, recombinant human (rh) CTRP8, and short synthetic peptides derived from the C1q globular domain of human CTRP8 caused the activation of RXFP1 as determined by elevated intracellular cAMP levels and the induction of a marked pro-migratory phenotype in established glioblastoma (GB) cell lines and primary cells from GB patients. Employing a small competitor peptide, we were able to disrupt the CTRP8-RXFP1-induced increased GB motility. The CTRP8-RXFP1-mediated migration in GB cells involves the activation of PI3K and specific protein kinase C pathways and the increased production/secretion of the potent lysosomal protease cathepsin B (cathB), a known prognostic marker of GB. Specific inhibition of CTRP8-induced cathB activity effectively blocked the ability of primary GB to invade laminin matrices. Finally, co-immunoprecipitation studies revealed the direct interaction of human CTRP8 with RXFP1. Our results support a therapeutic approach in GB aimed at targeting multiple steps of the CTRP8-RXFP1 signalling pathway by a combined inhibitor and peptide-based strategy to block GB dissemination within the brain. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  4. Receptor-DNA interactions: EMSA and footprinting.

    Science.gov (United States)

    Read, Jason T; Cheng, Helen; Hendy, Stephen C; Nelson, Colleen C; Rennie, Paul S

    2009-01-01

    Defining the precise promoter DNA sequence motifs where nuclear receptors and other transcription factors bind is an essential prerequisite for understanding how these proteins modulate the expression of their specific target genes. The purpose of this chapter is to provide the reader with a detailed guide with respect to the materials and the key methods required to perform this type of DNA-binding analysis. Irrespective of whether starting with purified DNA-binding proteins or somewhat crude cellular extracts, the tried-and-true procedures described here will enable one to accurately access the capacity of specific proteins to bind to DNA as well as to determine the exact sequences and DNA contact nucleotides involved. For illustrative purposes, we primarily have used the interaction of the androgen receptor with the rat probasin proximal promoter as our model system.

  5. CB1 Cannabinoid Receptors and their Associated Proteins

    Science.gov (United States)

    Howlett, Allyn C.; Blume, Lawrence C.; Dalton, George D.

    2011-01-01

    CB1 receptors are G-protein coupled receptors (GPCRs) abundant in neurons, in which they modulate neurotransmission. The CB1 receptor influence on memory and learning is well recognized, and disease states associated with CB1 receptors are observed in addiction disorders, motor dysfunction, schizophrenia, and in bipolar, depression, and anxiety disorders. Beyond the brain, CB1 receptors also function in liver and adipose tissues, vascular as well as cardiac tissue, reproductive tissues and bone. Signal transduction by CB1 receptors occurs through interaction with Gi/o proteins to inhibit adenylyl cyclase, activate mitogen-activated protein kinases (MAPK), inhibit voltage-gated Ca2+ channels, activate K+ currents (Kir), and influence Nitric Oxide (NO) signaling. CB1 receptors are observed in internal organelles as well as plasma membrane. β-Arrestins, adaptor protein AP-3, and G-protein receptor-associated sorting protein 1 (GASP1) modulate cellular trafficking. Cannabinoid Receptor Interacting Protein 1a (CRIP1a) is an accessory protein whose function has not been delineated. Factor Associated with Neutral sphingomyelinase (FAN) regulates ceramide signaling. Such diversity in cellular signaling and modulation by interacting proteins suggests that agonists and allosteric modulators could be developed to specifically regulate unique, cell type-specific responses. PMID:20166926

  6. Semiotic Selection of Mutated or Misfolded Receptor Proteins

    DEFF Research Database (Denmark)

    Giorgi, Franco; Bruni, Luis Emilio; Maggio, Roberto

    2013-01-01

    Receptor oligomerization plays a key role in maintaining genome stability and restricting protein mutagenesis. When properly folded, protein monomers assemble as oligomeric receptors and interact with environmental ligands. In a gene-centered view, the ligand specificity expressed by these recept......Receptor oligomerization plays a key role in maintaining genome stability and restricting protein mutagenesis. When properly folded, protein monomers assemble as oligomeric receptors and interact with environmental ligands. In a gene-centered view, the ligand specificity expressed...... for receptor monomers to assemble along the membrane and to sustain meaningful relationships with environmental ligands. How could a cell lineage deal with these loss-of-function mutations during evolution and restrain gene redundancy accordingly? In this paper, we will be arguing that the easiest way...... focused on the significance and semiotic nature of the interplay between membrane receptors and the epigenetic control of gene expression, as mediated by the control of mismatched repairing and protein folding mechanisms....

  7. CIPP, a novel multivalent PDZ domain protein, selectively interacts with Kir4.0 family members, NMDA receptor subunits, neurexins, and neuroligins.

    Science.gov (United States)

    Kurschner, C; Mermelstein, P G; Holden, W T; Surmeier, D J

    1998-06-01

    We report a novel multivalent PDZ domain protein, CIPP (for channel-interacting PDZ domain protein), which is expressed exclusively in brain and kidney. Within the brain, the highest CIPP mRNA levels were found in neurons of the cerebellum, inferior colliculus, vestibular nucleus, facial nucleus, and thalamus. Furthermore, we identified the inward rectifier K+ (Kir) channel, Kir4.1 (also called "Kir1.2"), as a cellular CIPP ligand. Among several other Kir channels tested, only the closely related Kir4.2 (or "Kir1.3") also interacted with CIPP. In addition, specific PDZ domains within CIPP associated selectively with the C-termini of N-methyl-D-aspartate subtypes of glutamate receptors, as well as neurexins and neuroligins, cell surface molecules enriched in synaptic membranes. Thus, CIPP may serve as a scaffold that brings structurally diverse but functionally connected proteins into close proximity at the synapse. The functional consequences of CIPP expression on Kir4.1 channels were studied using whole-cell voltage clamp techniques in Kir4.1 transfected COS-7 cells. On average, Kir4.1 current densities were doubled by cotransfection with CIPP. Copyright 1998 Academic Press.

  8. Interactions between Two Different G Protein-Coupled Receptors in Reproductive Hormone-Producing Cells: The Role of PACAP and Its Receptor PAC1R

    Directory of Open Access Journals (Sweden)

    Haruhiko Kanasaki

    2016-09-01

    Full Text Available Gonadotropin-releasing hormone (GnRH and gonadotropins are indispensable hormones for maintaining female reproductive functions. In a similar manner to other endocrine hormones, GnRH and gonadotropins are controlled by their principle regulators. Although it has been previously established that GnRH regulates the synthesis and secretion of luteinizing hormone (LH and follicle-stimulating hormone (FSH—both gonadotropins—from pituitary gonadotrophs, it has recently become clear that hypothalamic GnRH is under the control of hypothalamic kisspeptin. Prolactin, which is also known as luteotropic hormone and is released from pituitary lactotrophs, stimulates milk production in mammals. Prolactin is also regulated by hypothalamic factors, and it is thought that prolactin synthesis and release are principally under inhibitory control by dopamine through the dopamine D2 receptor. In addition, although it remains unknown whether it is a physiological regulator, thyrotropin-releasing hormone (TRH is a strong secretagogue for prolactin. Thus, GnRH, LH and FSH, and prolactin are mainly regulated by hypothalamic kisspeptin, GnRH, and TRH, respectively. However, the synthesis and release of these hormones is also modulated by other neuropeptides in the hypothalamus. Pituitary adenylate cyclase-activating polypeptide (PACAP is a hypothalamic peptide that was first isolated from sheep hypothalamic extracts based on its ability to stimulate cAMP production in anterior pituitary cells. PACAP acts on GnRH neurons and pituitary gonadotrophs and lactotrophs, resulting in the modulation of their hormone producing/secreting functions. Furthermore, the presence of the PACAP type 1 receptor (PAC1R has been demonstrated in these cells. We have examined how PACAP and PAC1R affect GnRH- and pituitary hormone-secreting cells and interact with their principle regulators. In this review, we describe our understanding of the role of PACAP and PAC1R in the regulation of Gn

  9. TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins.

    Science.gov (United States)

    Moravcova, Radka; Melkes, Barbora; Novotny, Jiri

    2017-11-14

    Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to Gq/11 proteins. We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP). FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of Gq/11α, Gβ, β-arrestin2 and phospholipase Cβ1, but not of Giα1, β-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of Giα1 and β-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling. These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane.

  10. Interactive protein manipulation

    Energy Technology Data Exchange (ETDEWEB)

    SNCrivelli@lbl.gov

    2003-07-01

    We describe an interactive visualization and modeling program for the creation of protein structures ''from scratch''. The input to our program is an amino acid sequence -decoded from a gene- and a sequence of predicted secondary structure types for each amino acid-provided by external structure prediction programs. Our program can be used in the set-up phase of a protein structure prediction process; the structures created with it serve as input for a subsequent global internal energy minimization, or another method of protein structure prediction. Our program supports basic visualization methods for protein structures, interactive manipulation based on inverse kinematics, and visualization guides to aid a user in creating ''good'' initial structures.

  11. Interaction of 14-3-3 proteins with the Estrogen Receptor Alpha F domain provides a drug target interface

    NARCIS (Netherlands)

    De Vries-van Leeuwen, Ingrid J; da Costa Pereira, Daniel; Flach, Koen D; Piersma, Sander R; Haase, Christian; Bier, David; Yalcin, Zeliha; Michalides, Rob; Feenstra, K Anton; Jiménez, Connie R; de Greef, Tom F A; Brunsveld, Luc; Ottmann, Christian; Zwart, Wilbert; de Boer, Albertus H

    2013-01-01

    Estrogen receptor alpha (ERα) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ERα, either by blocking estrogen production through aromatase inhibitors or

  12. Detecting protein-protein interactions in living cells

    DEFF Research Database (Denmark)

    Gottschalk, Marie; Bach, Anders; Hansen, Jakob Lerche

    2009-01-01

    to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C...

  13. Interaction of 16 alpha-[125I]iodo-estradiol with estrogen receptor and other steroid-binding proteins.

    OpenAIRE

    Hochberg, R B; Rosner, W

    1980-01-01

    This communication describes the synthesis of 16 alpha-[125I]iodo-estradiol (125I-E2) with specific activities greater than 1000 Ci/mmol (1 Ci = 3.7 x 10(10) becquerels). We show that it binds to the same specific estrogen receptor sites as does [3H]estradiol and that it does so with an affinity that is indistinguishable from that for the latter steroid. This is true for receptor obtained from calf uterus and from a pool of human mammary carcinomas. There is no significant binding of 125 I-E2...

  14. Complementary roles of Fas-associated death domain (FADD) and receptor interacting protein kinase-3 (RIPK3) in T-cell homeostasis and antiviral immunity

    Science.gov (United States)

    Lu, Jennifer V.; Weist, Brian M.; van Raam, Bram J.; Marro, Brett S.; Nguyen, Long V.; Srinivas, Prathna; Bell, Bryan D.; Luhrs, Keith A.; Lane, Thomas E.; Salvesen, Guy S.; Walsh, Craig M.

    2011-01-01

    Caspase-8 (casp8) is required for extrinsic apoptosis, and mice deficient in casp8 fail to develop and die in utero while ultimately failing to maintain the proliferation of T cells, B cells, and a host of other cell types. Paradoxically, these failures are not caused by a defect in apoptosis, but by a presumed proliferative function of this protease. Indeed, following mitogenic stimulation, T cells lacking casp8 or its adaptor protein FADD (Fas-associated death domain protein) develop a hyperautophagic morphology, and die a programmed necrosis-like death process termed necroptosis. Recent studies have demonstrated that receptor-interacting protein kinases (RIPKs) RIPK1 and RIPK3 together facilitate TNF-induced necroptosis, but the precise role of RIPKs in the demise of T cells lacking FADD or casp8 activity is unknown. Here we demonstrate that RIPK3 and FADD have opposing and complementary roles in promoting T-cell clonal expansion and homeostasis. We show that the defective proliferation of T cells bearing an interfering form of FADD (FADDdd) is rescued by crossing with RIPK3−/− mice, although such rescue ultimately leads to lymphadenopathy. Enhanced recovery of these double-mutant T cells following stimulation demonstrates that FADD, casp8, and RIPK3 are all essential for clonal expansion, contraction, and antiviral responses. Finally, we demonstrate that caspase-mediated cleavage of RIPK1-containing necrosis inducing complexes (necrosomes) is sufficient to prevent necroptosis in the face of death receptor signaling. These studies highlight the “two-faced” nature of casp8 activity, promoting clonal expansion in some situations and apoptotic demise in others. PMID:21876153

  15. Neuroactive Steroids: Receptor Interactions and Responses

    Directory of Open Access Journals (Sweden)

    Kald Beshir Tuem

    2017-08-01

    Full Text Available Neuroactive steroids (NASs are naturally occurring steroids, which are synthesized centrally as de novo from cholesterol and are classified as pregnane, androstane, and sulfated neurosteroids (NSs. NASs modulate many processes via interacting with gamma-aminobutyric acid (GABA, N-methyl-d-aspartate, serotonin, voltage-gated calcium channels, voltage-dependent anion channels, α-adrenoreceptors, X-receptors of the liver, transient receptor potential channels, microtubule-associated protein 2, neurotrophin nerve growth factor, and σ1 receptors. Among these, NSs (especially allopregnanolone have high potency and extensive GABA-A receptors and hence demonstrate anticonvulsant, anesthetic, central cytoprotectant, and baroreflex inhibitory effects. NSs are also involved in mood and learning via serotonin and anti-nociceptive activity via T-type voltage-gated Ca2+ channels. Moreover, they are modulators of mitochondrial function, synaptic plasticity, or regulators of apoptosis, which have a role in neuroprotective via voltage-dependent anion channels receptors. For proper functioning, NASs need to be in their normal level, whereas excess and deficiency may lead to abnormalities. When they are below the normal, NSs could have a part in development of depression, neuro-inflammation, multiple sclerosis, experimental autoimmune encephalitis, epilepsy, and schizophrenia. On the other hand, stress and attention deficit disorder could occur during excessive level. Overall, NASs are very important molecules with major neuropsychiatric activity.

  16. Pyridoxine improves hippocampal cognitive function via increases of serotonin turnover and tyrosine hydroxylase, and its association with CB1 cannabinoid receptor-interacting protein and the CB1 cannabinoid receptor pathway.

    Science.gov (United States)

    Jung, Hyo Young; Kim, Dae Won; Nam, Sung Min; Kim, Jong Whi; Chung, Jin Young; Won, Moo-Ho; Seong, Je Kyung; Yoon, Yeo Sung; Yoo, Dae Young; Hwang, In Koo

    2017-12-01

    In the present study, we investigated the effects of pyridoxine on hippocampal functions and changes in protein profiles based on the proteomic approach. Eight-week-old mice received intraperitoneal injections of physiological saline (vehicle) or 350mg/kg pyridoxine twice a day for 21days. Phosphoglycerate mutase 1 was up-regulated, while CB1 cannabinoid receptor-interacting protein 1 (CRIP1) was down-regulated, in the pyridoxine-treated group. Additionally, the serotonin and tyrosine hydroxylase was increased in the hippocampus of the pyridoxine-treated group than in that of the vehicle-treated group. Furthermore, discrimination indices based on the novel object recognition test were significantly higher in the pyridoxine-treated group than in the vehicle-treated group. Administration of CRIP1a siRNA significantly increases the discrimination index as well as cell proliferation and neuroblast differentiation in the dentate gyrus. In addition, the administration of rimonabant, a CB1 cannabinoid receptor antagonist, for 3weeks significantly decreased the novel object recognition memory, the tyrosine hydroxylase level, the amount of cell proliferation, and neuroblast differentiation in the dentate gyrus. Treatment with pyridoxine significantly increased novel object recognition memory, but slightly ameliorated rimonabant-induced reduction in serotonin, the tyrosine hydroxylase level, the amount of cell proliferation, and neuroblast differentiation in the dentate gyrus. These results suggest that pyridoxine promotes hippocampal functions by increasing serotonin and tyrosine hydroylase immunoreactivity in the hippocampus. This positive effect may be associated with CRIP1a and CB1 cannabinoid receptor function. Vitamin-B6 enhances hippocampal functions and this is closely associated with CRIP1a and CB1 cannabinoid receptors. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Proteinase-activated receptors (PARs) – focus on receptor-receptor-interactions and their physiological and pathophysiological impact

    Science.gov (United States)

    2013-01-01

    Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR1, PAR2, PAR3 and PAR4, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal “tethered ligand“ domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects. In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease. PMID:24215724

  18. Tat-binding protein-1 (TBP-1), an ATPase of 19S regulatory particles of the 26S proteasome, enhances androgen receptor function in cooperation with TBP-1-interacting protein/Hop2.

    Science.gov (United States)

    Satoh, Tetsurou; Ishizuka, Takahiro; Tomaru, Takuya; Yoshino, Satoshi; Nakajima, Yasuyo; Hashimoto, Koshi; Shibusawa, Nobuyuki; Monden, Tsuyoshi; Yamada, Masanobu; Mori, Masatomo

    2009-07-01

    The 26S proteasome, which degrades ubiquitinated proteins, appears to contribute to the cyclical loading of androgen receptor (AR) to androgen response elements of target gene promoters; however, the mechanism whereby the 26S proteasome modulates AR recruitment remains unknown. Using yeast two-hybrid screening, we previously identified Tat-binding protein-1 (TBP-1), an adenosine triphosphatase of 19S regulatory particles of the 26S proteasome, as a transcriptional coactivator of thyroid hormone receptor. Independently, TBP-1-interacting protein (TBPIP) was also identified as a coactivator of several nuclear receptors, including AR. Here, we investigated whether TBP-1 could interact with and modulate transcriptional activation by AR cooperatively with TBPIP. TBP-1 mRNA was ubiquitously expressed in human tissues, including the testis and prostate, as well as in LNCaP cells. TBP-1 directly bound TBPIP through the amino-terminal domain possessing the leucine zipper structure. AR is physically associated with TBP-1 and TBPIP in vitro and in LNCaP cells. TBP-1 similarly and additively augmented AR-mediated transcription upon coexpression with TBPIP, and the ATPase domain, as well as leucine zipper structure in TBP-1, was essential for transcriptional enhancement. Overexpression of TBP-1 did not alter AR protein and mRNA levels. In the chromatin immunoprecipitation assay, TBP-1 was transiently recruited to the proximal androgen response element of the prostate-specific antigen gene promoter in a ligand-dependent manner in LNCaP cells. These findings suggest that a component of 19S regulatory particles directly binds AR and might participate in AR-mediated transcriptional activation in cooperation with TBPIP.

  19. Interaction between Endothelial Protein C Receptor and Intercellular Adhesion Molecule 1 to Mediate Binding of Plasmodium falciparum-Infected Erythrocytes to Endothelial Cells.

    Science.gov (United States)

    Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell; Brazier, Andrew Jay; Smith, Joseph D

    2016-07-12

    Intercellular adhesion molecule 1 (ICAM-1) and the endothelial protein C receptor (EPCR) are candidate receptors for the deadly complication cerebral malaria. However, it remains unclear if Plasmodium falciparum parasites with dual binding specificity are involved in cytoadhesion or different parasite subpopulations bind in brain microvessels. Here, we investigated this issue by studying different subtypes of ICAM-1-binding parasite lines. We show that two parasite lines expressing domain cassette 13 (DC13) of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family have dual binding specificity for EPCR and ICAM-1 and further mapped ICAM-1 binding to the first DBLβ domain following the PfEMP1 head structure in both proteins. As PfEMP1 head structures have diverged between group A (EPCR binders) and groups B and C (CD36 binders), we also investigated how ICAM-1-binding parasites with different coreceptor binding traits influence P. falciparum-infected erythrocyte binding to endothelial cells. Whereas levels of binding to tumor necrosis factor alpha (TNF-α)-stimulated endothelial cells from the lung and brain by all ICAM-1-binding parasite lines increased, group A (EPCR and ICAM-1) was less dependent than group B (CD36 and ICAM-1) on ICAM-1 upregulation. Furthermore, both group A DC13 parasite lines had higher binding levels to brain endothelial cells (a microvascular niche with limited CD36 expression). This study shows that ICAM-1 is a coreceptor for a subset of EPCR-binding parasites and provides the first evidence of how EPCR and ICAM-1 interact to mediate parasite binding to both resting and TNF-α-activated primary brain and lung endothelial cells. Cerebral malaria is a severe neurological complication of P. falciparum infection associated with infected erythrocyte (IE) binding in cerebral vessels. Yet little is known about the mechanisms by which parasites adhere in the brain or other microvascular sites. Here, we studied parasite lines

  20. The proteins interacting with C-terminal of μ receptor are identified by bacterial two-hybrid system from brain cDNA library in morphine-dependent rats.

    Science.gov (United States)

    Zhou, Peilan; Jiang, Jiebing; Dong, Zhaoqi; Yan, Hui; You, Zhendong; Su, Ruibin; Gong, Zehui

    2015-12-15

    Opioid addiction is associated with long-term adaptive changes in the brain that involve protein expression. The carboxyl-terminal of the μ opioid receptor (MOR-C) is important for receptor signal transduction under opioid treatment. However, the proteins that interact with MOR-C after chronic morphine exposure remain unknown. The brain cDNA library of chronic morphine treatment rats was screened using rat MOR-C to investigate the regulator of opioids dependence in the present study. The brain cDNA library from chronic morphine-dependent rats was constructed using the SMART (Switching Mechanism At 5' end of RNA Transcript) technique. Bacterial two-hybrid system was used to screening the rat MOR-C interacting proteins from the cDNA library. RT-qPCR and immunoblotting were used to determine the variation of MOR-C interacting proteins in rat brain after chronic morphine treatment. Column overlay assays, immunocytochemistry and coimmunoprecipitation were used to demonstrate the interaction of MOR-C and p75NTR-associated cell death executor (NADE). 21 positive proteins, including 19 known proteins were screened to interact with rat MOR-C. Expression of several of these proteins was altered in specific rat brain regions after chronic morphine treatment. Among these proteins, NADE was confirmed to interact with rat MOR-C by in vitro protein-protein binding and coimmunoprecipitation in Chinese hamster ovary (CHO) cells and rat brain with or without chronic morphine treatment. Understanding the rat MOR-C interacting proteins and the proteins variation under chronic morphine treatment may be critical for determining the pathophysiological basis of opioid tolerance and addiction. Copyright © 2015. Published by Elsevier Inc.

  1. Interaction entropy for protein-protein binding

    Science.gov (United States)

    Sun, Zhaoxi; Yan, Yu N.; Yang, Maoyou; Zhang, John Z. H.

    2017-03-01

    Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interaction entropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interaction entropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

  2. A leu-rich repeat receptor-like protein kinase, FaRIPK1, interacts with ABA receptor, FaABAR, to regulate strawberry (Fragaria × ananassa) fruit ripening.

    Science.gov (United States)

    Hou, Bing-Zhu; Xu, Cheng; Shen, Yuan-Yue

    2017-12-21

    Strawberry (Fragaria × ananassa) is a model plant for studying non-climacteric fruit ripening regulated by abscisic acid (ABA), however, its defined molecular mechanisms are yet not fully understood. Here, a strawberry predicted leu-rich repeat (LRR) receptor-like kinase, red-initial protein kinase 1 (FaRIPK1), was screened and shown to interact with a putative ABA receptor, FaABAR, using a yeast two-hybrid assay. This association was confirmed by bimolecular fluorescence complementation and co-immunoprecipitation assays, and shown to occur in the nucleus. Expression analysis by real-time PCR showed that FaRIPK1 is expressed in roots, stems, leaves, flowers and fruits, with a particularly high expression in white fruit at the onset of coloration. Downregulation of FaRIPK1 expression in strawberry fruit, using tobacco rattle virus, inhibited ripening, as evidenced by suppression of ripening-related physiological changes and reduced expression of several genes involved in softening, sugar contents, pigmentation, and ABA biosynthesis and signaling. The yeast-expressed LRR and STK (serine/threonine protein kinase) domains of FaRIPK1 bound ABA and showed kinase activity, respectively. A fruit-disc-incubation test revealed that FaRIPK1 expression was induced by ABA and ethylene. The synergistic action of FaRIPK1 with FaABAR in regulation of strawberry fruit ripening is discussed. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  3. Immunocytochemical study of estrogen receptor activation factor (E-RAF and the proteins that interact with nuclear estrogen receptor II (nER II in epithelial endometrial cells, in the presence and in the absence of estradiol

    Directory of Open Access Journals (Sweden)

    OM Echeverría

    2009-06-01

    Full Text Available The localization and abundance of the estrogen receptor activation factor (E-RAF and a small nuclear ribonucleoprotein (snRNP complex containing three proteins, p32, p55 and p60, which interact with the nuclear estrogen receptor II (nER II, have been studied in rat endometrial epithelial cells by means of immunofluorescence and high resolution quantitative immunocytochemistry. In the cytoplasm E-RAF is associated with the rough endoplasmic reticulum. In the nucleus it is mainly localized at the interchromatin space, and surrounding the clumps of compact or semi-condensed chromatin. Quantitative analyses show that the abundance of E-RAF in the nucleus increases after ovariectomy and decreases 3 minutes after estradiol administration. These results are in agreement with the currently available biochemical data. Double immunolocalizations demonstrate that p32, p55, p60 co-localize with other splicing-related protein. High resolution immunolocalization shows that p32, p55, p60 are associated with perichromatin fibrils (co-transcriptional splicing and with clusters of interchromatin granules (storage of splicing-related molecules. The nuclear abundance of the snRNP complex decreases with ovariectomy, increases within 3 minutes after estradiol administration and remains higher than that in ovariectomized animals for 27 minutes. These results strongly support the previous data on the role of nER-II in the regulation of mRNA transcription and its export from the nucleus to the cytoplasm.

  4. Predicting Protein Interactions by Brownian Dynamics Simulations

    Directory of Open Access Journals (Sweden)

    Xuan-Yu Meng

    2012-01-01

    Full Text Available We present a newly adapted Brownian-Dynamics (BD-based protein docking method for predicting native protein complexes. The approach includes global BD conformational sampling, compact complex selection, and local energy minimization. In order to reduce the computational costs for energy evaluations, a shell-based grid force field was developed to represent the receptor protein and solvation effects. The performance of this BD protein docking approach has been evaluated on a test set of 24 crystal protein complexes. Reproduction of experimental structures in the test set indicates the adequate conformational sampling and accurate scoring of this BD protein docking approach. Furthermore, we have developed an approach to account for the flexibility of proteins, which has been successfully applied to reproduce the experimental complex structure from the structure of two unbounded proteins. These results indicate that this adapted BD protein docking approach can be useful for the prediction of protein-protein interactions.

  5. Interactions of p60, a mediator of progesterone receptor assembly, with heat shock proteins hsp90 and hsp70

    DEFF Research Database (Denmark)

    Chen, S; Prapapanich, V; Rimerman, R A

    1996-01-01

    mature PR complexes. In the present study we observe that a monoclonal antibody specific for p60 can, on the one hand, inhibit formation of mature PR complexes containing heat shock protein 90 (hsp90), p23, and immunophilins and, on the other, enhance recovery of early PR complexes containing hsp70...

  6. The urokinase plasminogen activator receptor-associated protein/endo180 is coexpressed with its interaction partners urokinase plasminogen activator receptor and matrix metalloprotease-13 during osteogenesis

    DEFF Research Database (Denmark)

    Engelholm, L H; Nielsen, B S; Netzel-Arnett, S

    2001-01-01

    ), and collagen V on the cell surface. We have determined the sites of expression of this novel receptor during murine postimplantation development. uPARAP/Endo180 was expressed in all tissues undergoing primary ossification, including the developing bones of the viscerocranium and calvarium that ossify...... intramembranously, and developing long bones undergoing endochondral ossification. uPARAP/Endo180 mRNA was expressed by both immature osteoblasts and by mature osteocalcin-producing osteoblasts-osteocytes, and was coexpressed with MMP-13. Interestingly, osteoblasts also expressed uPAR. Besides bone-forming tissues...

  7. The urokinase plasminogen activator receptor-associated protein/endo180 is coexpressed with its interaction partners urokinase plasminogen activator receptor and matrix metalloprotease-13 during osteogenesis

    DEFF Research Database (Denmark)

    Engelholm, L H; Nielsen, B S; Netzel-Arnett, Sarah

    2001-01-01

    intramembranously, and developing long bones undergoing endochondral ossification. uPARAP/Endo180 mRNA was expressed by both immature osteoblasts and by mature osteocalcin-producing osteoblasts-osteocytes, and was coexpressed with MMP-13. Interestingly, osteoblasts also expressed uPAR. Besides bone-forming tissues......), and collagen V on the cell surface. We have determined the sites of expression of this novel receptor during murine postimplantation development. uPARAP/Endo180 was expressed in all tissues undergoing primary ossification, including the developing bones of the viscerocranium and calvarium that ossify...

  8. Regulation of Nuclear Receptor Interacting Protein 1 (NRIP1) Gene Expression in Response to Weight Loss and Exercise in Humans

    DEFF Research Database (Denmark)

    De Marinis, Yang Z; Sun, Jiangming; Bompada, Pradeep

    2017-01-01

    : NRIP1 expression was measured by microarray and serum NRIP1 by ELISA and Western blotting. Skeletal muscle transcriptomes were analyzed from Gene Expression Omnibus databases. Network-based proximity analysis was performed on the proximity of NRIP1 interacting genes in the human interactome. Results...... expression by 80%, and strength training increased expression by ∼25% compared to baseline. Following rest, NRIP1 expression became sensitive to insulin stimulation. After re-training, NRIP1 expression decreased. Interactome analysis showed significant proximity of NRIP1 interacting partners to the obesity...... network/module. Conclusions: NRIP1 gene expression and serum levels are strongly associated with metabolic states such as obesity, weight loss, different types of exercise, and peripheral tissue insulin resistance, potentially as a mediator of sedentary effects....

  9. Receptor-interacting protein 140 as a co-repressor of Heat Shock Factor 1 regulates neuronal stress response.

    Science.gov (United States)

    Lin, Yu-Lung; Tsai, Hong-Chieh; Liu, Pei-Yao; Benneyworth, Michael; Wei, Li-Na

    2017-12-12

    Heat shock response (HSR) is a highly conserved transcriptional program that protects organisms against various stressful conditions. However, the molecular mechanisms modulating HSR, especially the suppression of HSR, is poorly understood. Here, we found that RIP140, a wide-spectrum cofactor of nuclear hormone receptors, acts as a co-repressor of heat shock factor 1 (HSF1) to suppress HSR in healthy neurons. When neurons are stressed such as by heat shock or sodium arsenite (As), cells engage specific proteosome-mediated degradation to reduce RIP140 level, thereby relieving the suppression and activating HSR. RIP140 degradation requires specific Tyr-phosphorylation by Syk that is activated in stressful conditions. Lowering RIP140 level protects hippocampal neurons from As stress, significantly it increases neuron survival and improves spine density. Reducing hippocampal RIP140 in the mouse rescues chronic As-induced spatial learning deficits. This is the first study elucidating RIP140-mediated suppression of HSF1-activated HSR in neurons and brain. Importantly, degradation of RIP140 in stressed neurons relieves this suppression, allowing neurons to efficiently and timely engage HSR programs and recover. Therefore, stimulating RIP140 degradation to activate anti-stress program provides a potential preventive or therapeutic strategy for neurodegeneration diseases.

  10. Alterations of Dopamine D2 Receptors and Related Receptor-Interacting Proteins in Schizophrenia: The Pivotal Position of Dopamine Supersensitivity Psychosis in Treatment-Resistant Schizophrenia

    Directory of Open Access Journals (Sweden)

    Yasunori Oda

    2015-12-01

    Full Text Available Although the dopamine D2 receptor (DRD2 has been a main target of antipsychotic pharmacotherapy for the treatment of schizophrenia, the standard treatment does not offer sufficient relief of symptoms to 20%–30% of patients suffering from this disorder. Moreover, over 80% of patients experience relapsed psychotic episodes within five years following treatment initiation. These data strongly suggest that the continuous blockade of DRD2 by antipsychotic(s could eventually fail to control the psychosis in some point during long-term treatment, even if such treatment has successfully provided symptomatic improvement for the first-episode psychosis, or stability for the subsequent chronic stage. Dopamine supersensitivity psychosis (DSP is historically known as a by-product of antipsychotic treatment in the manner of tardive dyskinesia or transient rebound psychosis. Numerous data in psychopharmacological studies suggest that the up-regulation of DRD2, caused by antipsychotic(s, is likely the mechanism underlying the development of the dopamine supersensitivity state. However, regardless of evolving notions of dopamine signaling, particularly dopamine release, signal transduction, and receptor recycling, most of this research has been conducted and discussed from the standpoint of disease etiology or action mechanism of the antipsychotic, not of DSP. Hence, the mechanism of the DRD2 up-regulation or mechanism evoking clinical DSP, both of which are caused by pharmacotherapy, remains unknown. Once patients experience a DSP episode, they become increasingly difficult to treat. Light was recently shed on a new aspect of DSP as a treatment-resistant factor. Clarification of the detailed mechanism of DSP is therefore crucial, and a preventive treatment strategy for DSP or treatment-resistant schizophrenia is urgently needed.

  11. Molecular basis of the γ-aminobutyric acid A receptor α3 subunit interaction with the clustering protein gephyrin

    DEFF Research Database (Denmark)

    Tretter, Verena; Kerschner, Bernd; Milenkovic, Ivan

    2011-01-01

    subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABA(A)R α3 M3-M4 domain and the motif SMDKAFITVL at the N terminus...... of the gephyrin E domain. GABA(A)R α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABA(A)Rs in recombinant systems. Interestingly, motifs...

  12. Augmented Growth Hormone Secretion and Stat3 Phosphorylation in an Aryl Hydrocarbon Receptor Interacting Protein (AIP-Disrupted Somatotroph Cell Line.

    Directory of Open Access Journals (Sweden)

    Takashi Fukuda

    Full Text Available Aryl hydrocarbon receptor interacting protein (AIP is thought to be a tumor suppressor gene, as indicated by a mutational analysis of pituitary somatotroph adenomas. However, the physiological significance of AIP inactivation in somatotroph cells remains unclear. Using CRISPR/Cas9, we identified a GH3 cell clone (termed GH3-FTY in which Aip was genetically disrupted, and subsequently investigated its character with respect to growth hormone (Gh synthesis and proliferation. Compared with GH3, GH3-FTY cells showed remarkably increased Gh production and a slight increase in cell proliferation. Gh-induced Stat3 phosphorylation is known to be a mechanism of Gh oversecretion in GH3. Interestingly, phosphorylated-Stat3 expression in GH3-FTY cells was increased more compared with GH3 cells, suggesting a stronger drive for this mechanism in GH3-FTY. The phenotypes of GH3-FTY concerning Gh overproduction, cell proliferation, and increased Stat3 phosphorylation were significantly reversed by the exogenous expression of Aip. GH3-FTY cells were less sensitive to somatostatin than GH3 cells in the suppression of cell proliferation, which might be associated with the reduced expression of somatostatin receptor type 2. GH3-FTY xenografts in BALB/c nude mice (GH3-FTY mice formed more mitotic somatotroph tumors than GH3 xenografts (GH3 mice, as also evidenced by increased Ki67 scores. GH3-FTY mice were also much larger and had significantly higher plasma Gh levels than GH3 mice. Furthermore, GH3-FTY mice showed relative insulin resistance compared with GH3 mice. In conclusion, we established a somatotroph cell line, GH3-FTY, which possessed prominent Gh secretion and mitotic features associated with the disruption of Aip.

  13. Evolution of protein-protein interactions

    Indian Academy of Sciences (India)

    Evolution of protein-protein interactions · Our interests in protein-protein interactions · Slide 3 · Slide 4 · Slide 5 · Slide 6 · Slide 7 · Slide 8 · Slide 9 · Slide 10 · Slide 11 · Slide 12 · Slide 13 · Slide 14 · Slide 15 · Slide 16 · Slide 17 · Slide 18 · Slide 19 · Slide 20.

  14. The urokinase receptor associated protein (uPARAP/endo180)

    DEFF Research Database (Denmark)

    Engelholm, L H; Nielsen, B S; Danø, K

    2001-01-01

    of this proteolytic system. uPARAP is a high molecular weight type-1 membrane protein, belonging to the macrophage mannose receptor protein family. On the surface of certain cells, uPARAP forms a ternary complex with the pro-form of the urokinase-type plasminogen activator (uPA) and its primary receptor (uPAR). While......The urokinase-mediated plasminogen activation system plays a central role in the extracellular proteolytic degradation reactions in cancer invasion. In this review article we discuss a number of recent findings identifying a new cellular receptor protein, uPARAP, that interacts with components...

  15. Host-parasite interaction: selective Pv-fam-a family proteins of Plasmodium vivax bind to a restricted number of human erythrocyte receptors.

    Science.gov (United States)

    Zeeshan, Mohammad; Tyagi, Rupesh Kumar; Tyagi, Kriti; Alam, Mohd Shoeb; Sharma, Yagya Dutta

    2015-04-01

    Plasmodium vivax synthesizes the largest number of 36 tryptophan-rich proteins belonging to the Pv-fam-a family. These parasite proteins need to be characterized for their biological function because tryptophan-rich proteins from other Plasmodium species have been proposed as vaccine candidates. Recombinant P. vivax tryptophan-rich antigens (PvTRAgs) were used to determine their erythrocyte-binding activity by a cell-based enzyme-linked immunosorbent assay, flow cytometry, and a rosetting assay. Only 4 (PvTRAg26.3, PvTRAg34, PvTRAg36, and PvTRAg36.6) of 21 PvTRAgs bind to host erythrocytes. The cross-competition data indicated that PvTRAg36 and PvTRAg34 share their erythrocyte receptors with previously described proteins PvTRAg38 and PvTRAg33.5, respectively. On the other hand, PvTRAg26.3 and PvTRAg36.6 cross-compete with each other and not with any other PvTRAg, indicating that these 2 proteins bind to the same but yet another set of erythrocyte receptor(s). Together, 10 of 36 PvTRAgs possess erythrocyte-binding activity in which each protein recognizes >1 erythrocyte receptor. Further, each erythrocyte receptor is shared by >1 PvTRAg. This redundancy may be useful for the parasite to invade red blood cells and cause disease pathogenesis, and it can be exploited to develop therapeutics against P. vivax malaria. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. Live-cell visualization of intracellular interaction between a nuclear migration protein (hNUDC and the thrombopoietin receptor (Mpl.

    Directory of Open Access Journals (Sweden)

    Yuan-Bin Zheng

    Full Text Available We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER, Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami resulted in increased levels of hNUDC or hNUDC(1-159 secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway.

  17. Studies on the glycoprotein nature of the thyrotropin receptor: interaction with lectins and purification of the bovine protein with the use of Bandeiraea (Griffonia) simplicifolia I affinity chromatography.

    Science.gov (United States)

    Kress, B C; Spiro, R G

    1986-03-01

    The TSH receptor from Triton-solubilized bovine microsomal membranes was found to bind to a substantial extent to columns of the immobilized lectins Bandeiraea (Griffonia) simplicifolia I, Ricinus communis I, wheat germ, and Concanavalin A, whereas it was not retained by Dolichos biflorus. Elution of TSH receptor activity from these lectins could be achieved with the appropriate saccharides in all cases except Concanavalin A. The most extensive adsorption of the receptor occurred on B. simplicifolia I-agarose (84%), and the terminal alpha-D-galactosyl specificity of this interaction was substantiated by its susceptibility to alpha-galactosidase treatment. Whereas TSH itself was not bound to this immobilized lectin, a complex of this hormone with its receptor did interact and could be eluted with methyl-alpha-D-galactoside. Purification (800-fold) of the bovine TSH receptor was achieved by a combination of TSH and B. simplicifolia I affinity chromatographies. Polyacrylamide gel electrophoresis of the purified TSH receptor after radioiodination revealed three major components with apparent mol wt of 316,000, 115,000, and 54,000.

  18. Dynamic fluctuations of protein-carbohydrate interactions promote protein aggregation.

    Directory of Open Access Journals (Sweden)

    Vladimir Voynov

    2009-12-01

    Full Text Available Protein-carbohydrate interactions are important for glycoprotein structure and function. Antibodies of the IgG class, with increasing significance as therapeutics, are glycosylated at a conserved site in the constant Fc region. We hypothesized that disruption of protein-carbohydrate interactions in the glycosylated domain of antibodies leads to the exposure of aggregation-prone motifs. Aggregation is one of the main problems in protein-based therapeutics because of immunogenicity concerns and decreased efficacy. To explore the significance of intramolecular interactions between aromatic amino acids and carbohydrates in the IgG glycosylated domain, we utilized computer simulations, fluorescence analysis, and site-directed mutagenesis. We find that the surface exposure of one aromatic amino acid increases due to dynamic fluctuations. Moreover, protein-carbohydrate interactions decrease upon stress, while protein-protein and carbohydrate-carbohydrate interactions increase. Substitution of the carbohydrate-interacting aromatic amino acids with non-aromatic residues leads to a significantly lower stability than wild type, and to compromised binding to Fc receptors. Our results support a mechanism for antibody aggregation via decreased protein-carbohydrate interactions, leading to the exposure of aggregation-prone regions, and to aggregation.

  19. Functional interaction between Lypd6 and nicotinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Arvaniti, Maria; Jensen, Majbrit M; Soni, Neeraj

    2016-01-01

    Nicotinic acetylcholine receptors (nAChRs) affect multiple physiological functions in the brain and their functions are modulated by regulatory proteins of the Lynx family. Here, we report for the first time a direct interaction of the Lynx protein LY6/PLAUR domain-containing 6 (Lypd6) with n...... findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain, and that Lypd6 is dysregulated by nicotine exposure during early development. Regulatory proteins of the Lynx family modulate the function of nicotinic receptors (nAChRs). We report for the first time that the Lynx...

  20. Familial Isolated Pituitary Adenomas (FIPA) and the Pituitary Adenoma Predisposition due to Mutations in the Aryl Hydrocarbon Receptor Interacting Protein (AIP) Gene

    Science.gov (United States)

    Aaltonen, Lauri A.; Daly, Adrian F.

    2013-01-01

    Pituitary adenomas are one of the most frequent intracranial tumors and occur with a prevalence of approximately 1:1000 in the developed world. Pituitary adenomas have a serious disease burden, and their management involves neurosurgery, biological therapies, and radiotherapy. Early diagnosis of pituitary tumors while they are smaller may help increase cure rates. Few genetic predictors of pituitary adenoma development exist. Recent years have seen two separate, complimentary advances in inherited pituitary tumor research. The clinical condition of familial isolated pituitary adenomas (FIPA) has been described, which encompasses the familial occurrence of isolated pituitary adenomas outside of the setting of syndromic conditions like multiple endocrine neoplasia type 1 and Carney complex. FIPA families comprise approximately 2% of pituitary adenomas and represent a clinical entity with homogeneous or heterogeneous pituitary adenoma types occurring within the same kindred. The aryl hydrocarbon receptor interacting protein (AIP) gene has been identified as causing a pituitary adenoma predisposition of variable penetrance that accounts for 20% of FIPA families. Germline AIP mutations have been shown to associate with the occurrence of large pituitary adenomas that occur at a young age, predominantly in children/adolescents and young adults. AIP mutations are usually associated with somatotropinomas, but prolactinomas, nonfunctioning pituitary adenomas, Cushing disease, and other infrequent clinical adenoma types can also occur. Gigantism is a particular feature of AIP mutations and occurs in more than one third of affected somatotropinoma patients. Study of pituitary adenoma patients with AIP mutations has demonstrated that these cases raise clinical challenges to successful treatment. Extensive research on the biology of AIP and new advances in mouse Aip knockout models demonstrate multiple pathways by which AIP may contribute to tumorigenesis. This review assesses

  1. Phosphorylation of the cytoplasmic tail of the 300-kDa mannose 6-phosphate receptor is required for the interaction with a cytosolic protein

    DEFF Research Database (Denmark)

    Rosorius, O; Issinger, O G; Braulke, T

    1993-01-01

    ) and to 35- and 91-kDa proteins salt-washed from bovine brain membranes. Gel filtration suggested that TIP 35 is part of a higher molecular mass complex of approximately 130-150 kDa. Inhibition studies, using non-phosphorylated and phosphorylated MPR 300-CT and cross-linking, indicate that the interaction...... with a cytosolic protein depending on the phosphorylation by a casein kinase II-like kinase. The cross-linking with salt-washed membrane proteins, however, is inhibited by non-phosphorylated MPR 300-CT, suggesting that different structural determinants in the MPR 300-CT interact with cytosol- and membrane...

  2. A large set of estrogen receptor β-interacting proteins identified by tandem affinity purification in hormone-responsive human breast cancer cell nuclei.

    Science.gov (United States)

    Nassa, Giovanni; Tarallo, Roberta; Ambrosino, Concetta; Bamundo, Angela; Ferraro, Lorenzo; Paris, Ornella; Ravo, Maria; Guzzi, Pietro H; Cannataro, Mario; Baumann, Marc; Nyman, Tuula A; Nola, Ernesto; Weisz, Alessandro

    2011-01-01

    Estrogen receptors α (ER-α) and β (ER-β) play distinct biological roles in onset and progression of hormone-responsive breast cancer, with ER-β exerting a modulatory activity on ER-α-mediated estrogen signaling and stimulation of cell proliferation by mechanisms still not fully understood. We stably expressed human ER-β fused to a tandem affinity purification-tag in estrogen-responsive MCF-7 cells and applied tandem affinity purification and nanoLC-MS/MS to identify the ER-β interactome of this cell type. Functional annotation by bioinformatics analyses of the 303 proteins that co-purify with ER-β from nuclear extracts identify several new molecular partners of this receptor subtype that represents nodal points of a large protein network controlling multiple processes and functions in breast cancer cells. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Regulation of G protein-coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins.

    Science.gov (United States)

    Hendriks-Balk, Mariëlle C; Peters, Stephan L M; Michel, Martin C; Alewijnse, Astrid E

    2008-05-13

    G protein-coupled receptors (GPCRs) are involved in many biological processes. Therefore, GPCR function is tightly controlled both at receptor level and at the level of signalling components. Well-known mechanisms by which GPCR function can be regulated comprise desensitization/resensitization processes and GPCR up- and downregulation. GPCR function can also be regulated by several proteins that directly interact with the receptor and thereby modulate receptor activity. An additional mechanism by which receptor signalling is regulated involves an emerging class of proteins, the so-called regulators of G protein signalling (RGS). In this review we will describe some of these control mechanisms in more detail with some specific examples in the cardiovascular system. In addition, we will provide an overview on RGS proteins and the involvement of RGS proteins in cardiovascular function.

  4. Genetic interactions between neurofibromin and endothelin receptor B in mice.

    Directory of Open Access Journals (Sweden)

    Mugdha Deo

    Full Text Available When mutations in two different genes produce the same mutant phenotype, it suggests that the encoded proteins either interact with each other, or act in parallel to fulfill a similar purpose. Haploinsufficiency of Neurofibromin and over-expression of Endothelin 3 both cause increased numbers of melanocytes to populate the dermis during mouse development, and thus we are interested in how these two signaling pathways might intersect. Neurofibromin is mutated in the human genetic disease, neurofibromatosis type 1, which is characterized by the development of Schwann cell based tumors and skin hyper-pigmentation. Neurofibromin is a GTPase activating protein, while the Endothelin 3 ligand activates Endothelin receptor B, a G protein coupled receptor. In order to study the genetic interactions between endothelin and neurofibromin, we defined the deletion breakpoints of the classical Ednrb piebald lethal allele (Ednrb(s-l and crossed these mice to mice with a loss-of-function mutation in neurofibromin, Dark skin 9 (Dsk9. We found that Neurofibromin haploinsufficiency requires Endothelin receptor B to darken the tail dermis. In contrast, Neurofibromin haploinsufficiency increases the area of the coat that is pigmented in Endothelin receptor B null mice. We also found an oncogenic mutation in the G protein alpha subunit, GNAQ, which couples to Endothelin receptor B, in a uveal melanoma from a patient with neurofibromatosis type 1. Thus, this data suggests that there is a complex relationship between Neurofibromin and Endothelin receptor B.

  5. Interaction network of ABA receptors in grey poplar.

    Science.gov (United States)

    Papacek, Michael; Christmann, Alexander; Grill, Erwin

    2017-10-01

    The plant hormone abscisic acid (ABA) is a key player in responses to abiotic stress. ABA regulates a plant's water status and mediates drought tolerance by controlling stomatal gas exchange, water conductance and differential gene expression. ABA is recognized and bound by the Regulatory Component of ABA Receptors (RCARs)/PYR1/PYL (Pyrabactin Resistance 1/PYR1-like). Ligand binding stabilizes the interaction of RCARs with type 2C protein phosphatases (PP2C), which are ABA co-receptors. While the core pathway of ABA signalling has been elucidated, the large number of different ABA receptors and co-receptors within a plant species generates a complexity of heteromeric receptor complexes that has not functionally been resolved in any plant species to date. In this study, we characterized ABA receptors and co-receptors of grey poplar (Populus x canescens [Ait.] Sm.) and their capacity to regulate ABA responses. We observed a high number of regulatory combinations of holo-receptor complexes, but also some preferential and selective RCAR-PP2C interactions. Poplar and Arabidopsis ABA receptor components revealed a strong structural and functional conservation. Heterologous receptor complexes of poplar and Arabidopsis components showed functionality in vitro and regulated ABA-responsive gene expression in cells of both species. ABA-responsive promoters of Arabidopsis were also active in poplar, which was explored to generate poplar reporter lines expressing green fluorescent protein in response to ABA. The study presents a detailed analysis of receptor complexes of a tree species and shows high conservation of ABA receptor components between an annual and a perennial plant. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  6. Analysis of leukocyte membrane protein interactions using protein microarrays

    Directory of Open Access Journals (Sweden)

    Foster-Cuevas Mildred

    2005-03-01

    Full Text Available Abstract Background Protein microarrays represent an emerging class of proteomic tools to investigate multiple protein-protein interactions in parallel. A sufficient proportion of immobilized proteins must maintain an active conformation and an orientation that allows for the sensitive and specific detection of antibody and ligand binding. In order to establish protein array technology for the characterization of the weak interactions between leukocyte membrane proteins, we selected the human leukocyte membrane protein CD200 (OX2 and its cell surface receptor (hCD200R as a model system. As antibody-antigen reactions are generally of higher affinity than receptor-ligand binding, we first analyzed the reactivity of monoclonal antibodies (mAb to normal and mutant forms of immobilized CD200R. Results Fluorescently labelled mAb DX147, DX136 and OX108 were specifically reactive with immobilized recombinant hCD200R extracellular region, over a range of 0.1–40 μg ml-1 corresponding to a limit of sensitivity of 0.01–0.05 femtomol per spot. Orientating hCD200R using capture antibodies, showed that DX147 reacts with an epitope spatially distinct from the more closely related DX136 and OX108 epitopes. A panel of soluble recombinant proteins with mutations in hCD200R domain 1 produced by transiently transfected cells, was arrayed directly without purification and screened for binding to the three mAb. Several showed decreased binding to the blocking mAb DX136 and OX108, suggesting close proximity of these epitopes to the CD200 binding site. Binding of hCD200 to directly immobilized rat, mouse, and hCD200R was achieved with multimeric ligands, in the form of biotinylated-hCD200 coupled to FITC-labelled avidin coated beads. Conclusion We have achieved sensitive, specific and reproducible detection of immobilized CD200R with different antibodies and mapped antigenic epitopes for two mAb in the vicinity of the ligand binding site using protein microarrays

  7. Heterotypic interactions between transferrin receptor and transferrin receptor 2

    OpenAIRE

    Vogt, TM; Blackwell, AD; Giannetti, AM; Bjorkman, PJ; Enns, CA

    2003-01-01

    Cellular iron uptake in most tissues occurs via endocytosis of diferric transferrin (Tf) bound to the transferrin receptor (TfR). Recently, a second transferrin receptor, transferrin receptor 2 (TfR2), has been identified and shown to play a critical role in iron metabolism. TfR2 is capable of Tf-mediated iron uptake and mutations in this gene result in a rare form of hereditary hemochromatosis unrelated to the hereditary hemochromatosis protein, HFE. Unlike TfR, TfR2 expression is not contro...

  8. Furosemide interactions with brain GABAA receptors

    OpenAIRE

    Korpi, Esa R; Lüddens, Hartmut

    1997-01-01

    The loop diuretic furosemide is known to antagonize the function of γ-aminobutyric acid type A (GABAA) receptors. The purpose of the present study was to examine the direct interaction of furosemide with the GABAA receptors by autoradiography and ligand binding studies with native rat and human receptors and with recombinant receptors composed of rat subunits.Autoradiography with [35S]-t-butylbicyclophosphorothionate ([35S]-TBPS) as a ligand indicated that furosemide (0.1–1 mM) reversed the 5...

  9. Interactions of the bovine brain A1-adenosine receptor with recombinant G protein alpha-subunits. Selectivity for rGi alpha-3.

    Science.gov (United States)

    Freissmuth, M; Schütz, W; Linder, M E

    1991-09-25

    The ability of the bovine brain A1-adenosine receptor to discriminate between different G protein subtypes was tested using G protein alpha-subunits synthesized in Escherichia coli (rG alpha-subunits). When combined with a 3-fold molar excess of beta gamma-subunit purified from bovine brain and used at high concentrations, all three subtypes of rGi alpha (rGi alpha-1, rGi alpha-2, and rGi alpha-3) and rGo alpha were capable of reconstituting guanine nucleotide-sensitive high-affinity binding of the agonist radioligand (-)-N6-3-[125I] (iodo-4-hydroxyphenylisopropyl) adenosine ([125I]HPIA) to the purified A1-adenosine receptor (Kd approximately 1.2 nM). Titration of the A1-adenosine receptor with increasing amounts of rG alpha revealed a approximately 10-fold higher affinity for rGi alpha-3 compared with rGi alpha-1, rGi alpha-2, and rGo alpha. This selectivity was also observed in the absence of beta gamma. Other alpha-subunits (rGs alpha-s, rGs alpha-L, rGs alpha PT, and rGz alpha) did not promote [125I]HPIA binding to the purified receptor. In N-ethylmaleimide-treated bovine brain membranes, rGi alpha-3 was the only rG alpha-subunit capable of reconstituting high-affinity agonist binding. Similarly, rGi alpha-3 competed potently with rGo alpha for activation by the agonist-liganded A1-adenosine receptor, whereas a approximately 50-fold molar excess of rGo alpha was required to quench the receptor-mediated release of [alpha-32P]GDP from rGi alpha-3. Hence, in spite of the extensive homology between alpha-subunits belonging to the Gi/Go group, the A1-adenosine receptor appears to discriminate between the subtypes. This specificity is likely to govern transmembrane signaling pathways in vivo.

  10. Opioid receptor trafficking and interaction in nociceptors

    Science.gov (United States)

    Zhang, X; Bao, L; Li, S

    2015-01-01

    Opiate analgesics such as morphine are often used for pain therapy. However, antinociceptive tolerance and dependence may develop with long-term use of these drugs. It was found that μ-opioid receptors can interact with δ-opioid receptors, and morphine antinociceptive tolerance can be reduced by blocking δ-opioid receptors. Recent studies have shown that μ- and δ-opioid receptors are co-expressed in a considerable number of small neurons in the dorsal root ganglion. The interaction of μ-opioid receptors with δ-opioid receptors in the nociceptive afferents is facilitated by the stimulus-induced cell-surface expression of δ-opioid receptors, and contributes to morphine tolerance. Further analysis of the molecular, cellular and neural circuit mechanisms that regulate the trafficking and interaction of opioid receptors and related signalling molecules in the pain pathway would help to elucidate the mechanism of opiate analgesia and improve pain therapy. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24611685

  11. The effect of a high-protein, high-sodium diet on calcium and bone metabolism in postmenopausal women and its interaction with vitamin D receptor genotype

    DEFF Research Database (Denmark)

    Harrington, M.; Bennett, T.; Jakobsen, Jette

    2004-01-01

    The influence of a high-Na, high-protein (calciuric) diet on Ca and bone metabolism was investigated in postmenopausal women (aged 5067 years) who were stratified by vitamin D receptor (VDR) genotype. In a crossover trial, twenty-four women were randomly assigned to a diet high in protein (90 g...... no differences in serum markers or urinary minerals between the basal and calciuric diet in either VDR genotype groups. While the calciuric diet significantly increased urinary NTx (by 25.6%, PVDR group (n 10; carrying one or more (f) Fok I alleles), it had no effect in the f - VDR group (n 14......; not carrying any Fok I alleles). It is concluded that the Na- and protein-induced urinary Ca loss is compensated for by increased bone resorption and that this response may be influenced by VDR genotype....

  12. The urokinase receptor associated protein (uPARAP/endo180)

    DEFF Research Database (Denmark)

    Engelholm, L H; Nielsen, B S; Danø, K

    2001-01-01

    The urokinase-mediated plasminogen activation system plays a central role in the extracellular proteolytic degradation reactions in cancer invasion. In this review article we discuss a number of recent findings identifying a new cellular receptor protein, uPARAP, that interacts with components...... of this proteolytic system. uPARAP is a high molecular weight type-1 membrane protein, belonging to the macrophage mannose receptor protein family. On the surface of certain cells, uPARAP forms a ternary complex with the pro-form of the urokinase-type plasminogen activator (uPA) and its primary receptor (uPAR). While...... the biological consequences of this reaction have not yet been verified experimentally, a likely event is ligand internalization because uPARAP is a constitutively recycling internalization receptor. uPARAP also binds at least one component, collagen type V, in the extracellular matrix meshwork, pointing...

  13. Analysis of odorant receptor protein function in the yellow fever mosquito, aedes aegypti

    Science.gov (United States)

    Odorant receptors (ORs) in insects are ligand-gated ion channels comprised of two subunits: a variable receptor and an obligatory co-receptor (Orco). This protein receptor complex of unknown stoichiometry interacts with an odor molecule leading to changes in permeability of the sensory dendrite, th...

  14. Interaction proteomics reveals brain region-specific AMPA receptor complexes

    NARCIS (Netherlands)

    Chen, N.; Pandya, N.J.; Koopmans, F.T.W.; Castelo-Szekelv, V.; van der Schors, R.C.; Smit, A.B.; Li, K.W.

    2014-01-01

    Fast excitatory synaptic transmission in the brain is mediated by glutamate acting on postsynaptic AMPA receptors. Recent studies have revealed a substantial number of AMPA receptor auxiliary proteins, which potentially contribute to the regulation of AMPA receptor trafficking, subcellular receptor

  15. Discovering functional interaction patterns in protein-protein interaction networks

    Directory of Open Access Journals (Sweden)

    Can Tolga

    2008-06-01

    Full Text Available Abstract Background In recent years, a considerable amount of research effort has been directed to the analysis of biological networks with the availability of genome-scale networks of genes and/or proteins of an increasing number of organisms. A protein-protein interaction (PPI network is a particular biological network which represents physical interactions between pairs of proteins of an organism. Major research on PPI networks has focused on understanding the topological organization of PPI networks, evolution of PPI networks and identification of conserved subnetworks across different species, discovery of modules of interaction, use of PPI networks for functional annotation of uncharacterized proteins, and improvement of the accuracy of currently available networks. Results In this article, we map known functional annotations of proteins onto a PPI network in order to identify frequently occurring interaction patterns in the functional space. We propose a new frequent pattern identification technique, PPISpan, adapted specifically for PPI networks from a well-known frequent subgraph identification method, gSpan. Existing module discovery techniques either look for specific clique-like highly interacting protein clusters or linear paths of interaction. However, our goal is different; instead of single clusters or pathways, we look for recurring functional interaction patterns in arbitrary topologies. We have applied PPISpan on PPI networks of Saccharomyces cerevisiae and identified a number of frequently occurring functional interaction patterns. Conclusion With the help of PPISpan, recurring functional interaction patterns in an organism's PPI network can be identified. Such an analysis offers a new perspective on the modular organization of PPI networks. The complete list of identified functional interaction patterns is available at http://bioserver.ceng.metu.edu.tr/PPISpan/.

  16. GPCR-interacting proteins (GIPs): nature and functions.

    Science.gov (United States)

    Bockaert, J; Roussignol, G; Bécamel, C; Gavarini, S; Joubert, L; Dumuis, A; Fagni, L; Marin, P

    2004-11-01

    The simplistic idea that seven transmembrane receptors are single monomeric proteins that interact with heterotrimeric G-proteins after agonist binding is definitively out of date. Indeed, GPCRs (G-protein-coupled receptors) are part of multiprotein networks organized around scaffolding proteins. These GIPs (GPCR-interacting proteins) are either transmembrane or cytosolic proteins. Proteomic approaches can be used to get global pictures of these 'receptosomes'. This approach allowed us to identify direct but also indirect binding partners of serotonin receptors. GIPs are involved in a wide range of functions including control of the targeting, trafficking and signalling of GPCRs. One of them, Shank, which is a secondary and tertiary partner of metabotropic and ionotropic glutamate receptors, respectively, can induce the formation of a whole functional glutamate 'receptosome' and the structure to which it is associated, the dendritic spine.

  17. Channel-interacting PDZ protein “CIPP” interacts with proteins involved in cytoskeletal dynamics

    OpenAIRE

    Alpi, Emanuele; Landi, Elena; Barilari, Manuela; Serresi, Michela; Salvadori, Piero; Bachi, Angela; Dente, Luciana

    2009-01-01

    Abstract Neuronal CIPP is a multivalent PDZ protein that interacts with specific channels and receptors, highly expressed in the brain. It is composed of four PDZ domains that behave as a scaffold to clusterize functionally connected proteins. In this study, we selected a set of potential CIPP interactors that are directly or indirectly involved in mechanisms of cytoskeletal remodeling and membrane protrusions formation. For some of these, we first proved the direct binding to spec...

  18. Detecting mutually exclusive interactions in protein-protein interaction maps.

    Directory of Open Access Journals (Sweden)

    Carmen Sánchez Claros

    Full Text Available Comprehensive protein interaction maps can complement genetic and biochemical experiments and allow the formulation of new hypotheses to be tested in the system of interest. The computational analysis of the maps may help to focus on interesting cases and thereby to appropriately prioritize the validation experiments. We show here that, by automatically comparing and analyzing structurally similar regions of proteins of known structure interacting with a common partner, it is possible to identify mutually exclusive interactions present in the maps with a sensitivity of 70% and a specificity higher than 85% and that, in about three fourth of the correctly identified complexes, we also correctly recognize at least one residue (five on average belonging to the interaction interface. Given the present and continuously increasing number of proteins of known structure, the requirement of the knowledge of the structure of the interacting proteins does not substantially impact on the coverage of our strategy that can be estimated to be around 25%. We also introduce here the Estrella server that embodies this strategy, is designed for users interested in validating specific hypotheses about the functional role of a protein-protein interaction and it also allows access to pre-computed data for seven organisms.

  19. Detecting mutually exclusive interactions in protein-protein interaction maps.

    KAUST Repository

    Sánchez Claros, Carmen

    2012-06-08

    Comprehensive protein interaction maps can complement genetic and biochemical experiments and allow the formulation of new hypotheses to be tested in the system of interest. The computational analysis of the maps may help to focus on interesting cases and thereby to appropriately prioritize the validation experiments. We show here that, by automatically comparing and analyzing structurally similar regions of proteins of known structure interacting with a common partner, it is possible to identify mutually exclusive interactions present in the maps with a sensitivity of 70% and a specificity higher than 85% and that, in about three fourth of the correctly identified complexes, we also correctly recognize at least one residue (five on average) belonging to the interaction interface. Given the present and continuously increasing number of proteins of known structure, the requirement of the knowledge of the structure of the interacting proteins does not substantially impact on the coverage of our strategy that can be estimated to be around 25%. We also introduce here the Estrella server that embodies this strategy, is designed for users interested in validating specific hypotheses about the functional role of a protein-protein interaction and it also allows access to pre-computed data for seven organisms.

  20. Modeling disordered protein interactions from biophysical principles.

    Directory of Open Access Journals (Sweden)

    Lenna X Peterson

    2017-04-01

    Full Text Available Disordered protein-protein interactions (PPIs, those involving a folded protein and an intrinsically disordered protein (IDP, are prevalent in the cell, including important signaling and regulatory pathways. IDPs do not adopt a single dominant structure in isolation but often become ordered upon binding. To aid understanding of the molecular mechanisms of disordered PPIs, it is crucial to obtain the tertiary structure of the PPIs. However, experimental methods have difficulty in solving disordered PPIs and existing protein-protein and protein-peptide docking methods are not able to model them. Here we present a novel computational method, IDP-LZerD, which models the conformation of a disordered PPI by considering the biophysical binding mechanism of an IDP to a structured protein, whereby a local segment of the IDP initiates the interaction and subsequently the remaining IDP regions explore and coalesce around the initial binding site. On a dataset of 22 disordered PPIs with IDPs up to 69 amino acids, successful predictions were made for 21 bound and 18 unbound receptors. The successful modeling provides additional support for biophysical principles. Moreover, the new technique significantly expands the capability of protein structure modeling and provides crucial insights into the molecular mechanisms of disordered PPIs.

  1. Sigma 1 receptor modulation of G-protein-coupled receptor signaling: potentiation of opioid transduction independent from receptor binding.

    Science.gov (United States)

    Kim, Felix J; Kovalyshyn, Ivanka; Burgman, Maxim; Neilan, Claire; Chien, Chih-Cheng; Pasternak, Gavril W

    2010-04-01

    sigma Ligands modulate opioid actions in vivo, with agonists diminishing morphine analgesia and antagonists enhancing the response. Using human BE(2)-C neuroblastoma cells that natively express opioid receptors and human embryonic kidney (HEK) cells transfected with a cloned mu opioid receptor, we now demonstrate a similar modulation of opioid function, as assessed by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding, by sigma(1) receptors. sigma Ligands do not compete opioid receptor binding. Administered alone, neither sigma agonists nor antagonists significantly stimulated [(35)S]GTP gamma S binding. Yet sigma receptor selective antagonists, but not agonists, shifted the EC(50) of opioid-induced stimulation of [(35)S]GTP gamma S binding by 3- to 10-fold to the left. This enhanced potency was seen without a change in the efficacy of the opioid, as assessed by the maximal stimulation of [(35)S]GTP gamma S binding. sigma(1) Receptors physically associate with mu opioid receptors, as shown by coimmunoprecipitation studies in transfected HEK cells, implying a direct interaction between the proteins. Thus, sigma receptors modulate opioid transduction without influencing opioid receptor binding. RNA interference knockdown of sigma(1) in BE(2)-C cells also potentiated mu opioid-induced stimulation of [(35)S]GTP gamma S binding. These modulatory actions are not limited to mu and delta opioid receptors. In mouse brain membrane preparations, sigma(1)-selective antagonists also potentiated both opioid receptor and muscarinic acetylcholine receptor-mediated stimulation of [(35)S]GTP gamma S binding, suggesting a broader role for sigma receptors in modulating G-protein-coupled receptor signaling.

  2. Channel-interacting PDZ protein, 'CIPP', interacts with proteins involved in cytoskeletal dynamics.

    Science.gov (United States)

    Alpi, Emanuele; Landi, Elena; Barilari, Manuela; Serresi, Michela; Salvadori, Piero; Bachi, Angela; Dente, Luciana

    2009-04-15

    Neuronal CIPP (channel-interacting PDZ protein) is a multivalent PDZ protein that interacts with specific channels and receptors highly expressed in the brain. It is composed of four PDZ domains that behave as a scaffold to clusterize functionally connected proteins. In the present study, we selected a set of potential CIPP interactors that are involved directly or indirectly in mechanisms of cytoskeletal remodelling and membrane protrusion formation. For some of these, we first proved the direct binding to specific CIPP PDZ domains considered as autonomous elements, and then confirmed the interaction with the whole protein. In particular, the small G-protein effector IRSp53 (insulin receptor tyrosine kinase substrate protein p53) specifically interacts with the second PDZ domain of CIPP and, when co-transfected in cultured mammalian cells with a tagged full-length CIPP, it induces a marked reorganization of CIPP cytoplasmic localization. Large punctate structures are generated as a consequence of CIPP binding to the IRSp53 C-terminus. Analysis of the puncta nature, using various endocytic markers, revealed that they are not related to cytoplasmic vesicles, but rather represent multi-protein assemblies, where CIPP can tether other potential interactors.

  3. Database of Interacting Proteins (DIP)

    Data.gov (United States)

    U.S. Department of Health & Human Services — The DIP database catalogs experimentally determined interactions between proteins. It combines information from a variety of sources to create a single, consistent...

  4. The melanocortin receptors and their accessory proteins

    Directory of Open Access Journals (Sweden)

    Shwetha eRamachandrappa

    2013-02-01

    Full Text Available The five melanocortin receptors named MC1R-MC5R have diverse physiological roles encompassing pigmentation, steroidogenesis, energy homeostasis and feeding behaviour as well as exocrine function. Since their identification almost 20 years ago much has been learnt about these receptors. As well as interacting with their endogenous ligands the melanocortin peptides, there is now a growing list of important peptides that can modulate the way these receptors signal, acting as agonists, antagonists and inverse agonists. The discovery of MRAPs as a novel accessory factor to the melanocortin receptors provides further insight into the regulation of these important GPCRs.

  5. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Shoko, E-mail: satosho@rs.tus.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Shirakawa, Hitoshi, E-mail: shirakah@m.tohoku.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Tomita, Shuhei, E-mail: tomita@med.tottori-u.ac.jp [Division of Molecular Pharmacology, Department of Pathophysiological and Therapeutic Science, Yonago 683-8503 (Japan); Tohkin, Masahiro, E-mail: tohkin@phar.nagoya-cu.ac.jp [Department of Medical Safety Science, Graduate School of Pharmaceutical Science, Nagoya City University, Nagoya 267-8603 (Japan); Gonzalez, Frank J., E-mail: gonzalef@mail.nih.gov [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Komai, Michio, E-mail: mkomai@m.tohoku.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan)

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.

  6. A casein kinase II phosphorylation site in the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor determines the high affinity interaction of the AP-1 Golgi assembly proteins with membranes.

    Science.gov (United States)

    Mauxion, F; Le Borgne, R; Munier-Lehmann, H; Hoflack, B

    1996-01-26

    The transport of proteins from the secretory to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi assembly proteins and clathrin. The mannose 6-phosphate receptors (MPHs) are two major transmembrane proteins segregated into these transport vesicles. Together with the GTPase ARF-1, these cargo proteins are essential components for the efficient translocation of the cytosolic AP-1 onto membranes of the trans-Golgi network, the first step of clathrin coat assembly, MPR-negative fibroblasts have a low capacity of recruiting AP-1 which can be restored by re-expressing the MPRs in these cells. This property was used to identify the protein motif of the cation-dependent mannose 6-phosphate receptor (CD-MPR) cytoplasmic domain that is essential for these interactions. Thus, the affinity of AP-1 for membranes and in vivo transport of cathepsin D were measured for MPR-negative cells re-expressing various CD-MPR mutants. The results indicate that the targeting of lysosomal enzymes requires the CD-PDR cytoplasmic domain that are different from tyrosine-based endocytosis motifs. The first is a casein kinase II phosphorylation site (ESEER) that is essential for high affinity binding of AP-1 and therefore probably acts as a dominant determinant controlling CD-MPR sorting in the trans-Golgi network. The second is the adjacent di-leucine motif (HLLPM), which, by itself, is not critical for AP-1 binding, but is absolutely required for a downstream sorting event.

  7. A Toll/interleukin (IL)-1 receptor domain protein from Yersinia pestis interacts with mammalian IL-1/Toll-like receptor pathways but does not play a central role in the virulence of Y. pestis in a mouse model of bubonic plague.

    Science.gov (United States)

    Spear, Abigail M; Rana, Rohini R; Jenner, Dominic C; Flick-Smith, Helen C; Oyston, Petra C F; Simpson, Peter; Matthews, Stephen J; Byrne, Bernadette; Atkins, Helen S

    2012-06-01

    The Toll/interleukin (IL)-1 receptor (TIR) domain is an essential component of eukaryotic innate immune signalling pathways. Interaction between TIR domains present in Toll-like receptors and associated adaptors initiates and propagates an immune signalling cascade. Proteins containing TIR domains have also been discovered in bacteria. Studies have subsequently shown that these proteins are able to modulate mammalian immune signalling pathways dependent on TIR interactions and that this may represent an evasion strategy for bacterial pathogens. Here, we investigate a TIR domain protein from the highly virulent bacterium Yersinia pestis, the causative agent of plague. When overexpressed in vitro this protein is able to downregulate IL-1β- and LPS-dependent signalling to NFκB and to interact with the TIR adaptor protein MyD88. This interaction is dependent on a single proline residue. However, a Y. pestis knockout mutant lacking the TIR domain protein was not attenuated in virulence in a mouse model of bubonic plague. Minor alterations in the host cytokine response to the mutant were indicated, suggesting a potential subtle role in pathogenesis. The Y. pestis mutant also showed increased auto-aggregation and reduced survival in high-salinity conditions, phenotypes which may contribute to pathogenesis or survival.

  8. Regulation of Insulin Receptor Trafficking by Bardet Biedl Syndrome Proteins.

    Directory of Open Access Journals (Sweden)

    Rachel D Starks

    2015-06-01

    Full Text Available Insulin and its receptor are critical for the regulation of metabolic functions, but the mechanisms underlying insulin receptor (IR trafficking to the plasma membrane are not well understood. Here, we show that Bardet Biedl Syndrome (BBS proteins are necessary for IR localization to the cell surface. We demonstrate that the IR interacts physically with BBS proteins, and reducing the expression of BBS proteins perturbs IR expression in the cell surface. We show the consequence of disrupting BBS proteins for whole body insulin action and glucose metabolism using mice lacking different BBS genes. These findings demonstrate the importance of BBS proteins in underlying IR cell surface expression. Our data identify defects in trafficking and localization of the IR as a novel mechanism accounting for the insulin resistance commonly associated with human BBS. This is supported by the reduced surface expression of the IR in fibroblasts derived from patients bearing the M390R mutation in the BBS1 gene.

  9. Toward fluorescent probes for G-protein-coupled receptors (GPCRs).

    Science.gov (United States)

    Ma, Zhao; Du, Lupei; Li, Minyong

    2014-10-23

    G-protein-coupled receptors (GPCRs), a superfamily of cell-surface receptors that are the targets of about 40% of prescription drugs on the market, can sense numerous critical extracellular signals. Recent breakthroughs in structural biology, especially in holo-form X-ray crystal structures, have contributed to our understanding of GPCR signaling. However, actions of GPCRs at the cellular and molecular level, interactions between GPCRs, and the role of protein dynamics in receptor activities still remain controversial. To overcome these dilemmas, fluorescent probes of GPCRs have been employed, which have advantages of in vivo safety and real-time monitoring. Various probes that depend on specific mechanisms and/or technologies have been used to study GPCRs. The present review focuses on surveying the design and applications of fluorescent probes for GPCRs that are derived from small molecules or using protein-labeling techniques, as well as discussing some design strategies for new probes.

  10. Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Martini, Lene; Schwartz, Thue W

    2005-01-01

    that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization...... for the fusion constructs was observed. We conclude that the glucagon-like peptide 1 fusion construct mimics the natural interaction of the receptor with (beta)arr2 with respect to binding peptide ligands, G protein-mediated signaling and internalization, and that this distinct molecular phenotype is reminiscent......To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting...

  11. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling

    DEFF Research Database (Denmark)

    Blagoev, B.; Kratchmarova, I.; Ong, S.E.

    2003-01-01

    Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we em...

  12. Biased Gs Versus Gq Proteins and β-Arrestin Signaling in the NK1 Receptor Determined by Interactions in the Water Hydrogen Bond Network

    DEFF Research Database (Denmark)

    Valentin-Hansen, Louise; Frimurer, Thomas M; Mokrosinski, Jacek

    2015-01-01

    of the highly conserved AspII:10 (2.50). Here, we find that this GluII:10 occupies the space of a putative allosteric modulating Na(+) ion and makes direct inter-helical interactions in particular with SerIII:15 (3.39) and AsnVII:16 (7.49) of the NPXXY motif. Mutational changes in the interface between GluII:10......X-ray structures, molecular dynamics simulations, and mutational analysis have previously indicated that an extended water hydrogen bond network between trans-membranes I-III, VI, and VII constitutes an allosteric interface essential for stabilizing different active and inactive helical....... It is concluded that the interface between position II:10 (2.50), III:15 (3.39), and VII:16 (7.49) in the center of the water hydrogen bond network constitutes a focal point for fine-tuning seven trans-membrane receptor conformations activating different signal transduction pathways....

  13. Interaction of lipids with the neurotensin receptor 1.

    Science.gov (United States)

    Bolivar, Juan H; Muñoz-García, Juan C; Castro-Dopico, Tomas; Dijkman, Patricia M; Stansfeld, Phillip J; Watts, Anthony

    2016-06-01

    Information about lipid-protein interactions for G protein-coupled receptors (GPCRs) is scarce. Here, we use electron spin resonance (ESR) and spin-labelled lipids to study lipid interactions with the rat neurotensin receptor 1 (NTS1). A fusion protein containing rat NTS1 fully able to bind its ligand neurotensin was reconstituted into phosphatidylcholine (PC) bilayers at specific lipid:protein molar ratios. The fraction of motionally restricted lipids in the range of 40:1 to 80:1 lipids per receptor suggested an oligomeric state of the protein, and the result was unaffected by increasing the hydrophobic thickness of the lipid bilayer from C-18 to C-20 or C-22 chain length PC membranes. Comparison of the ESR spectra of different spin-labelled lipids allowed direct measurement of lipid binding constants relative to PC (Kr), with spin-labelled phosphatidylethanolamine (PESL), phosphatidylserine (PSSL), stearic acid (SASL), and a spin labelled cholesterol analogue (CSL) Kr values of 1.05±0.05, 1.92±0.08, 5.20±0.51 and 0.91±0.19, respectively. The results contrast with those from rhodopsin, the only other GPCR studied this way, which has no selectivity for the lipids analysed here. Molecular dynamics simulations of NTS1 in bilayers are in agreement with the ESR data, and point to sites in the receptor where PS could interact with higher affinity. Lipid selectivity could be necessary for regulation of ligand binding, oligomerisation and/or G protein activation processes. Our results provide insight into the potential modulatory mechanisms that lipids can exert on GPCRs. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Modeling multivalent ligand-receptor interactions with steric constraints on configurations of cell surface receptor aggregates

    Energy Technology Data Exchange (ETDEWEB)

    Monine, Michael [Los Alamos National Laboratory; Posner, Richard [TRANSLATION GENOMICS RESAEARCH INSTITUTE; Savage, Paul [BYU; Faeder, James [UNIV OF PITTSBURGH; Hlavacek, William S [UNM

    2008-01-01

    Signal transduction generally involves multivalent protein-protein interactions, which can produce various protein complexes and post-translational modifications. The reaction networks that characterize these interactions tend to be so large as to challenge conventional simulation procedures. To address this challenge, a kinetic Monte Carlo (KMC) method has been developed that can take advantage of a model specification in terms of reaction rules for molecular interactions. A set of rules implicitly defines the reactions that can occur as a result of the interactions represented by the rules. With the rule-based KMC method, explicit generation of the underlying chemical reaction network implied by rules is avoided. Here, we apply and extend this method to characterize the interactions of a trivalent ligand with a bivalent cell-surface receptor. This system is also studied experimentally. We consider the following kinetic models: an equivalent-site model, an extension of this model, which takes into account steric constraints on the configurations of receptor aggregates, and finally, a model that accounts for cyclic receptor aggregates. Simulation results for the equivalent-site model are consistent with an equilibrium continuum model. Using these models, we investigate the effects of steric constraints and the formation of cyclic aggregates on the kinetics and equilibria of small and large aggregate formation and the percolation phase transition that occurs in this system.

  15. Leptin and the OB-receptor as anti-obesity target: recent in silico advances in the comprehension of the protein-protein interaction and rational drug design of anti- obesity lead compounds.

    Science.gov (United States)

    Tutone, Marco; Lauria, Antonino; Almerico, Anna Maria

    2014-01-01

    The OB-receptor or leptin receptor (LR) is crucial for energy homeostasis and regulation of food uptake. Leptin is a 16 kDa hormone that is mainly secreted by fat cells into the bloodstream. Under normal circumstances, circulating leptin levels are proportionate to the fat body mass. Sensing of elevated leptin levels by the hypothalamic neuro-circuitry activates a negative feedback loop resulting in reduced food intake and increased energy expenditure. Decreased leptin concentrations lead to opposite effects. Therefore, rational design of leptin agonists/antagonists could be an appealing challenge in the battle against obesity. The Leptin/LR interactions have been studied in several works by means of different molecular modelling approaches, spreading from homology modelling to manual docking. No small molecules have ever been proposed as agonists of the Ob receptor but researchers' efforts focused only on leptin-related synthetic peptides as receptor antagonists and on peptidomimetics. In this review we try to track a timeline of obtained in silico information to clarify the mechanism of interaction between leptin and its receptor, together to summarize the more recent efforts to propose new drugs usable in anti-obesity therapy. Final considerations could be useful starting points for the rational drug design of new lead compounds.

  16. Key region of laminin receptor 1 for interaction with human period 1

    African Journals Online (AJOL)

    GREGORY

    2010-09-20

    Sep 20, 2010 ... The 67 kDa laminin receptor 1 (Lamr1) is a novel protein that interacts with human circadian clock protein period 1 (hPer1). We confirmed the interaction between hPer1 and complete Lamr1 (295 amino acids) through yeast two-hybrid system in the present study. And we identified the interaction between.

  17. Multifactorial Regulation of G Protein-Coupled Receptor Endocytosis

    Science.gov (United States)

    Zhang, Xiaohan; Kim, Kyeong-Man

    2017-01-01

    Endocytosis is a process by which cells absorb extracellular materials via the inward budding of vesicles formed from the plasma membrane. Receptor-mediated endocytosis is a highly selective process where receptors with specific binding sites for extracellular molecules internalize via vesicles. G protein-coupled receptors (GPCRs) are the largest single family of plasma-membrane receptors with more than 1000 family members. But the molecular mechanisms involved in the regulation of GPCRs are believed to be highly conserved. For example, receptor phosphorylation in collaboration with β-arrestins plays major roles in desensitization and endocytosis of most GPCRs. Nevertheless, a number of subsequent studies showed that GPCR regulation, such as that by endocytosis, occurs through various pathways with a multitude of cellular components and processes. This review focused on i) functional interactions between homologous and heterologous pathways, ii) methodologies applied for determining receptor endocytosis, iii) experimental tools to determine specific endocytic routes, iv) roles of small guanosine triphosphate-binding proteins in GPCR endocytosis, and v) role of post-translational modification of the receptors in endocytosis. PMID:28035080

  18. The interaction of Clostridium perfringens enterotoxin with receptor claudins

    Science.gov (United States)

    Shrestha, Archana; Uzal, Francisco A.; McClane, Bruce A.

    2016-01-01

    Clostridium perfringens enterotoxin (CPE) has significant medical importance due to its involvement in several common human gastrointestinal diseases. This 35 kDa single polypeptide toxin consists of two domains: a C-terminal domain involved in receptor binding and an N-terminal domain involved in oligomerization, membrane insertion and pore formation. The action of CPE starts with its binding to receptors, which include certain members of the claudin tight junction protein family; bound CPE then forms a series of complexes, one of which is a pore that causes the calcium influx responsible for host cell death. Recent studies have revealed that CPE binding to claudin receptors involves interactions between the C-terminal CPE domain and both the 1st and 2nd extracellular loops (ECL-1 and ECL-2) of claudin receptors. Of particular importance for this binding is the docking of ECL-2 into a pocket present in the C-terminal domain of the toxin. This increased understanding of CPE interactions with claudin receptors is now fostering the development of receptor decoy therapeutics for CPE-mediated gastrointestinal disease, reagents for cancer therapy/diagnoses and enhancers of drug delivery. PMID:27090847

  19. Yeast Interacting Proteins Database: YJL199C, YJL199C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available d in closely related Saccharomyces species; protein detected in large-scale protein-protein interaction studies...cies; protein detected in large-scale protein-protein interaction studies Rows with this prey as prey (4) Ro...n; not conserved in closely related Saccharomyces species; protein detected in large-scale protein-protein interaction studies... species; protein detected in large-scale protein-protein interaction studies Rows with this prey as prey Ro

  20. Self-organized criticality in proteins: Hydropathic roughening profiles of G-protein-coupled receptors

    Science.gov (United States)

    Phillips, J. C.

    2013-03-01

    Proteins appear to be the most dramatic natural example of self-organized criticality (SOC), a concept that explains many otherwise apparently unlikely phenomena. Protein conformational functionality is often dominated by long-range hydrophobic or hydrophilic interactions which both drive protein compaction and mediate protein-protein interactions. Superfamily transmembrane G-protein-coupled receptors (GPCRs) are the largest family of proteins in the human genome; their amino acid sequences form the largest database for protein-membrane interactions. While there are now structural data on the heptad transmembrane structures of representatives of several heptad families, here we show how fresh insights into global and some local chemical trends in GPCR properties can be obtained accurately from sequences alone, especially by algebraically separating the extracellular and cytoplasmic loops from transmembrane segments. The global mediation of long-range water-protein interactions occurs in conjunction with modulation of these interactions by roughened interfaces. Hydropathic roughening profiles are defined here solely in terms of amino acid sequences, and knowledge of protein coordinates is not required. Roughening profiles both for GPCR and some simpler protein families display accurate and transparent connections to protein functionality, and identify natural length scales for protein functionality.

  1. Direct interaction of Gβγ with a C-terminal Gβγ-binding domain of the Ca2+ channel α1 subunit is responsible for channel inhibition by G protein-coupled receptors

    Science.gov (United States)

    Qin, Ning; Platano, Daniela; Olcese, Riccardo; Stefani, Enrico; Birnbaumer, Lutz

    1997-01-01

    Several classes of voltage-gated Ca2+ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca2+ channels the executing arm of the activated G protein is its βγ dimer (Gβγ). We report below the existence of two Gβγ-binding sites on the A-, B-, and E-type α1 subunits that form non-L-type Ca2+ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both Gβγ-binding regions also bind the Ca2+ channel β subunit (CCβ), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in α1E of loop 1 with that of the G protein-insensitive and Gβγ-binding-negative loop 1 of α1C did not abolish inhibition by G proteins, but the exchange of the α1E C terminus with that of α1C did. This and properties of α1E C-terminal truncations indicated that the Gβγ-binding site mediating the inhibition of Ca2+ channel activity is the one in the C terminus. Binding of Gβγ to this site was inhibited by an α1-binding domain of CCβ, thus providing an explanation for the functional antagonism existing between CCβ and G protein inhibition. The data do not support proposals that Gβγ inhibits α1 function by interacting with the site located in the loop I–II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission. PMID:9238069

  2. Direct interaction of gbetagamma with a C-terminal gbetagamma-binding domain of the Ca2+ channel alpha1 subunit is responsible for channel inhibition by G protein-coupled receptors.

    Science.gov (United States)

    Qin, N; Platano, D; Olcese, R; Stefani, E; Birnbaumer, L

    1997-08-05

    Several classes of voltage-gated Ca2+ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca2+ channels the executing arm of the activated G protein is its betagamma dimer (Gbetagamma). We report below the existence of two Gbetagamma-binding sites on the A-, B-, and E-type alpha1 subunits that form non-L-type Ca2+ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both Gbetagamma-binding regions also bind the Ca2+ channel beta subunit (CCbeta), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in alpha1E of loop 1 with that of the G protein-insensitive and Gbetagamma-binding-negative loop 1 of alpha1C did not abolish inhibition by G proteins, but the exchange of the alpha1E C terminus with that of alpha1C did. This and properties of alpha1E C-terminal truncations indicated that the Gbetagamma-binding site mediating the inhibition of Ca2+ channel activity is the one in the C terminus. Binding of Gbetagamma to this site was inhibited by an alpha1-binding domain of CCbeta, thus providing an explanation for the functional antagonism existing between CCbeta and G protein inhibition. The data do not support proposals that Gbetagamma inhibits alpha1 function by interacting with the site located in the loop I-II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.

  3. Structural basis of ligand interaction with atypical chemokine receptor 3

    Science.gov (United States)

    Gustavsson, Martin; Wang, Liwen; van Gils, Noortje; Stephens, Bryan S.; Zhang, Penglie; Schall, Thomas J.; Yang, Sichun; Abagyan, Ruben; Chance, Mark R.; Kufareva, Irina; Handel, Tracy M.

    2017-01-01

    Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally driven models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor.

  4. Structural basis of ligand interaction with atypical chemokine receptor 3

    Energy Technology Data Exchange (ETDEWEB)

    Gustavsson, Martin; Wang, Liwen; van Gils, Noortje; Stephens, Bryan S.; Zhang, Penglie; Schall, Thomas J.; Yang, Sichun; Abagyan, Ruben; Chance, Mark R.; Kufareva, Irina; Handel, Tracy M.

    2017-01-18

    Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally driven models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor.

  5. Single methyl groups can act as toggle switches to specify transmembrane protein-protein interactions

    DEFF Research Database (Denmark)

    He, Li; Steinocher, Helena; Shelar, Ashish

    2017-01-01

    Transmembrane domains (TMDs) engage in protein-protein interactions that regulate many cellular processes, but the rules governing the specificity of these interactions are poorly understood. To discover these principles, we analyzed 26-residue model transmembrane proteins consisting exclusively...... productively with the TMD of the human EPOR, the mouse EPOR, or both receptors. Association of the traptamers with the EPOR induced EPOR oligomerization in an orientation that stimulated receptor activity. These results highlight the high intrinsic specificity of TMD interactions, demonstrate that a single...

  6. Membrane cholesterol access into a G-protein-coupled receptor

    Science.gov (United States)

    Guixà-González, Ramon; Albasanz, José L.; Rodriguez-Espigares, Ismael; Pastor, Manuel; Sanz, Ferran; Martí-Solano, Maria; Manna, Moutusi; Martinez-Seara, Hector; Hildebrand, Peter W.; Martín, Mairena; Selent, Jana

    2017-02-01

    Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.

  7. Protein mixtures: interactions and gelation

    NARCIS (Netherlands)

    Ersch, C.

    2015-01-01

    Gelation is a ubiquitous process in the preparation of foods. As most foods are multi constituent mixtures, understanding gelation in mixtures is an important goal in food science. Here we presented a systematic investigation on the influence of molecular interactions on the gelation in protein

  8. Role of Phosphodiesterases on the Function of Aryl Hydrocarbon Receptor-Interacting Protein (AIP) in the Pituitary Gland and on the Evaluation of AIP Gene Variants.

    Science.gov (United States)

    Hernández-Ramírez, Laura C; Trivellin, Giampaolo; Stratakis, Constantine A

    2017-04-01

    Familial isolated pituitary adenoma (FIPA) is caused in about 20% of cases by loss-of-function germline mutations in the AIP gene. Patients harboring AIP mutations usually present with somatotropinomas resulting either in gigantism or young-onset acromegaly. AIP encodes for a co-chaperone protein endowed with tumor suppressor properties in somatotroph cells. Among other mechanisms proposed to explain this function, a regulatory effect over the 3',5'-cyclic adenosine monophosphate (cAMP) signaling pathway seems to play a prominent role. In this setting, the well-known interaction between AIP and 2 different isoforms of phosphodiesterases (PDEs), PDE2A3 and PDE4A5, is of particular interest. While the interaction with over-expressed AIP does not seem to affect PDE2A3 function, the reported effect on PDE4A5 is, in contrast, reduced enzymatic activity. In this review, we explore the possible implications of these molecular interactions for the function of somatotroph cells. In particular, we discuss how both PDEs and AIP could act as negative regulators of the cAMP pathway in the pituitary, probably both by shared and independent mechanisms. Moreover, we describe how the evaluation of the AIP-PDE4A5 interaction has proven to be a useful tool for testing AIP mutations, complementing other in silico, in vitro, and in vivo analyses. Improved assessment of the pathogenicity of AIP mutations is indeed paramount to provide adequate guidance for genetic counseling and clinical screening in AIP mutation carriers, which can lead to prospective diagnosis of pituitary adenomas. © Georg Thieme Verlag KG Stuttgart · New York.

  9. Protein- protein interaction detection system using fluorescent protein microdomains

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  10. Structural organization of G-protein-coupled receptors

    Science.gov (United States)

    Lomize, Andrei L.; Pogozheva, Irina D.; Mosberg, Henry I.

    1999-07-01

    Atomic-resolution structures of the transmembrane 7-α-helical domains of 26 G-protein-coupled receptors (GPCRs) (including opsins, cationic amine, melatonin, purine, chemokine, opioid, and glycoprotein hormone receptors and two related proteins, retinochrome and Duffy erythrocyte antigen) were calculated by distance geometry using interhelical hydrogen bonds formed by various proteins from the family and collectively applied as distance constraints, as described previously [Pogozheva et al., Biophys. J., 70 (1997) 1963]. The main structural features of the calculated GPCR models are described and illustrated by examples. Some of the features reflect physical interactions that are responsible for the structural stability of the transmembrane α-bundle: the formation of extensive networks of interhelical H-bonds and sulfur-aromatic clusters that are spatially organized as 'polarity gradients' the close packing of side-chains throughout the transmembrane domain; and the formation of interhelical disulfide bonds in some receptors and a plausible Zn2+ binding center in retinochrome. Other features of the models are related to biological function and evolution of GPCRs: the formation of a common 'minicore' of 43 evolutionarily conserved residues; a multitude of correlated replacements throughout the transmembrane domain; an Na+-binding site in some receptors, and excellent complementarity of receptor binding pockets to many structurally dissimilar, conformationally constrained ligands, such as retinal, cyclic opioid peptides, and cationic amine ligands. The calculated models are in good agreement with numerous experimental data.

  11. Furosemide interactions with brain GABAA receptors

    Science.gov (United States)

    Korpi, Esa R; Lüddens, Hartmut

    1997-01-01

    The loop diuretic furosemide is known to antagonize the function of γ-aminobutyric acid type A (GABAA) receptors. The purpose of the present study was to examine the direct interaction of furosemide with the GABAA receptors by autoradiography and ligand binding studies with native rat and human receptors and with recombinant receptors composed of rat subunits.Autoradiography with [35S]-t-butylbicyclophosphorothionate ([35S]-TBPS) as a ligand indicated that furosemide (0.1–1 mM) reversed the 5 μM GABA-induced inhibition of binding only in the cerebellar granule cell layer of rat brain sections. In all other regions studied, notably also in the hippocampal and thalamic areas, furosemide failed to antagonize GABA. Furosemide 1 mM decreased [35S]-TBPS binding only in a limited number of brain regions, but facilitation of the GABA-inhibition of the binding was much more widespread.In well-washed rat cerebellar, but not cerebrocortical, membranes, furosemide enhanced the [35S]-TBPS binding over basal level in the absence of added GABA. The GABAA antagonist, SR 95531, and the convulsant, Ro 5-4864, blocked this furosemide-induced increase. Both interactions with the furosemide enhancement are likely to be allosteric, since furosemide affected the binding of [3H]-SR 95531 and [3H]-Ro 5-4864 identically in the cerebellar and cerebrocortical membranes. Maximal GABA-antagonism induced by furosemide in cerebellar membranes was further increased by SR 95531 but not by Ro 5-4864, indicating additive antagonism only for SR 95531. In human cerebellar receptors, only GABA antagonism by furosemide, but not the enhancement without added GABA, was observed.In recombinant GABAA receptors, furosemide antagonism of GABA-inhibition of [35S]-TBPS binding depended only on the presence of α6 and β2/3 subunits, irrespective of the presence or absence of γ2 or δ subunits.In α6β3γ2 receptors, clozapine reversed the enhancement of [35S]-TBPS binding by furosemide in the absence

  12. Analysis of protein interactions at native chloroplast membranes by ellipsometry.

    Directory of Open Access Journals (Sweden)

    Verena Kriechbaumer

    Full Text Available Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE. We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins.

  13. Sphingolipids in the function of G protein-coupled receptors.

    Science.gov (United States)

    Jafurulla, Mohammad; Chattopadhyay, Amitabha

    2015-09-15

    G protein-coupled receptors (GPCRs) constitute the largest and most diverse protein family in mammals and are involved in information transfer across cellular membranes. GPCRs are known to regulate multiple physiological functions and therefore represent major drug targets in all clinical areas. The fact that GPCRs are integral membrane proteins raises the possibility of their interaction with functionally important membrane lipids such as sphingolipids. Sphingolipids are essential membrane components and are recognized as diverse and dynamic regulators of a multitude of cellular processes. Interaction with sphingolipids could lead to modulation of GPCR structure and function. In this review, we highlight the role of sphingolipids in the function of GPCRs with specific examples. A comprehensive understanding of molecular events involved in GPCR-lipid interaction would provide better insight into GPCR function in health and disease. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Human Dopamine Receptors Interaction Network (DRIN): a systems biology perspective on topology, stability and functionality of the network.

    Science.gov (United States)

    Podder, Avijit; Jatana, Nidhi; Latha, N

    2014-09-21

    Dopamine receptors (DR) are one of the major neurotransmitter receptors present in human brain. Malfunctioning of these receptors is well established to trigger many neurological and psychiatric disorders. Taking into consideration that proteins function collectively in a network for most of the biological processes, the present study is aimed to depict the interactions between all dopamine receptors following a systems biology approach. To capture comprehensive interactions of candidate proteins associated with human dopamine receptors, we performed a protein-protein interaction network (PPIN) analysis of all five receptors and their protein partners by mapping them into human interactome and constructed a human Dopamine Receptors Interaction Network (DRIN). We explored the topology of dopamine receptors as molecular network, revealing their characteristics and the role of central network elements. More to the point, a sub-network analysis was done to determine major functional clusters in human DRIN that govern key neurological pathways. Besides, interacting proteins in a pathway were characterized and prioritized based on their affinity for utmost drug molecules. The vulnerability of different networks to the dysfunction of diverse combination of components was estimated under random and direct attack scenarios. To the best of our knowledge, the current study is unique to put all five dopamine receptors together in a common interaction network and to understand the functionality of interacting proteins collectively. Our study pinpointed distinctive topological and functional properties of human dopamine receptors that have helped in identifying potential therapeutic drug targets in the dopamine interaction network. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Portraying G Protein-Coupled Receptors with Fluorescent Ligands

    Science.gov (United States)

    2015-01-01

    The thermodynamics of ligand–receptor interactions at the surface of living cells represents a fundamental aspect of G protein-coupled receptor (GPCR) biology; thus, its detailed elucidation constitutes a challenge for modern pharmacology. Interestingly, fluorescent ligands have been developed for a variety of GPCRs in order to monitor ligand–receptor binding in living cells. Accordingly, new methodological strategies derived from noninvasive fluorescence-based approaches, especially fluorescence resonance energy transfer (FRET), have been successfully developed to characterize ligand–receptor interactions. Importantly, these technologies are supplanting more hazardous and expensive radioactive binding assays. In addition, FRET-based tools have also become extremely powerful approaches for visualizing receptor–receptor interactions (i.e., GPCR oligomerization) in living cells. Thus, by means of the synthesis of compatible fluorescent ligands these novel techniques can be implemented to demonstrate the existence of GPCR oligomerization not only in heterologous systems but also in native tissues. Finally, there is no doubt that these methodologies would also be relevant in drug discovery in order to develop new high-throughput screening approaches or to identify new therapeutic targets. Overall, herein, we provide a thorough assessment of all technical and biological aspects, including strengths and weaknesses, of these fluorescence-based methodologies when applied to the study of GPCR biology at the plasma membrane of living cells. PMID:25010291

  16. ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

    Energy Technology Data Exchange (ETDEWEB)

    HOWITT,J.; ANDERSON,C.W.; FREIMUTH,P.

    2001-08-01

    The mechanism of adenovirus attachment to the host cell plasma membrane has been revealed in detail by research over the past 10 years. It has long been known that receptor binding activity is associated with the viral fibers, trimeric spike proteins that protrude radially from the vertices of the icosahedral capsid (Philipson et al. 1968). In some adenovirus serotypes, fiber and other virus structural proteins are synthesized in excess and accumulate in the cell nucleus during late stages of infection. Fiber protein can be readily purified from lysates of cells infected with subgroup C viruses, for example Ad2 and Ad5 (Boulanger and Puvion 1973). Addition of purified fiber protein to virus suspensions during adsorption strongly inhibits infection, indicating that fiber and intact virus particles compete for binding sites on host cells (Philipson et al. 1968; Hautala et al. 1998). Cell binding studies using purified radiolabeled fiber demonstrated that fiber binds specifically and with high affinity to the cell plasma membrane, and that cell lines typically used for laboratory propagation of adenovirus have approximately 10{sup 4} high-affinity receptor sites per cell (Persson et al. 1985; Freimuth 1996). Similar numbers of high-affinity binding sites for radiolabeled intact virus particles also were observed (Seth et al. 1994).

  17. Allosteric modulation of G-protein coupled receptors

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Spalding, Tracy A

    2004-01-01

    The superfamily of G-protein coupled receptors (GPCRs) has more than 1000 members and is the largest family of proteins in the body. GPCRs mediate signalling of stimuli as diverse as light, ions, small molecules, peptides and proteins and are the targets for many pharmaceuticals. Most GPCR ligands....... In recent years, combinatorial chemistry and high throughput screening have helped identify several allosteric GPCR modulators with novel structures, several of which already have become valuable pharmacological tools and may be candidates for clinical testing in the near future. This mini review outlines...... the current status and perspectives of allosteric modulation of GPCR function with emphasis on the pharmacology of endogenous and synthesised modulators, their receptor interactions and the therapeutic prospects of allosteric ligands compared to orthosteric ligands....

  18. Association of dopamine D(3) receptors with actin-binding protein 280 (ABP-280).

    Science.gov (United States)

    Li, Ming; Li, Chuanyu; Weingarten, Paul; Bunzow, James R; Grandy, David K; Zhou, Qun Yong

    2002-03-01

    Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280.

  19. REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity

    OpenAIRE

    Susann Björk; Hurt, Carl M.; Ho, Vincent K.; Timothy Angelotti

    2013-01-01

    Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi t...

  20. Receptor kinase signaling pathways in plant-microbe interactions.

    Science.gov (United States)

    Antolín-Llovera, Meritxell; Ried, Martina K; Binder, Andreas; Parniske, Martin

    2012-01-01

    Plant receptor-like kinases (RLKs) function in diverse signaling pathways, including the responses to microbial signals in symbiosis and defense. This versatility is achieved with a common overall structure: an extracytoplasmic domain (ectodomain) and an intracellular protein kinase domain involved in downstream signal transduction. Various surfaces of the leucine-rich repeat (LRR) ectodomain superstructure are utilized for interaction with the cognate ligand in both plant and animal receptors. RLKs with lysin-motif (LysM) ectodomains confer recognitional specificity toward N-acetylglucosamine-containing signaling molecules, such as chitin, peptidoglycan (PGN), and rhizobial nodulation factor (NF), that induce immune or symbiotic responses. Signaling downstream of RLKs does not follow a single pattern; instead, the detailed analysis of brassinosteroid (BR) signaling, innate immunity, and symbiosis revealed at least three largely nonoverlapping pathways. In this review, we focus on RLKs involved in plant-microbe interactions and contrast the signaling pathways leading to symbiosis and defense.

  1. Insights into cellular signalling by G protein coupled receptor transactivation of cell surface protein kinase receptors.

    Science.gov (United States)

    Chaplin, Rebecca; Thach, Lyna; Hollenberg, Morley D; Cao, Yingnan; Little, Peter J; Kamato, Danielle

    2017-06-01

    G protein coupled receptor (GPCR) signalling is mediated by transactivation independent and transactivation dependent pathways. GPCRs transactivate protein tyrosine kinase receptors (PTKRs) and protein serine/threonine kinase receptors (PS/TKR). Since the initial observations of transactivation dependent signalling, there has been an effort to understand the mechanisms behind this phenomena. GPCR signalling has evolved to include biased signalling. Biased signalling, whereby selected ligands can activate the same GPCR that can generate multiple signals, but drive only a unique response. To date, there has been no focus on the ability of biased agonists to activate the PTKR and PS/TKR transactivation pathways differentially. As such, this represents a novel direction for future research. This review will discuss the main mechanisms of GPCR mediated receptor transactivation and the pathways involved in intracellular responses.

  2. G Protein-coupled Receptor Biased Agonism.

    Science.gov (United States)

    Hodavance, Sima Y; Gareri, Clarice; Torok, Rachel D; Rockman, Howard A

    2016-03-01

    G protein-coupled receptors are the largest family of targets for current therapeutics. The classic model of their activation was binary, where agonist binding induced an active conformation and subsequent downstream signaling. Subsequently, the revised concept of biased agonism emerged, where different ligands at the same G protein-coupled receptor selectively activate one downstream pathway versus another. Advances in understanding the mechanism of biased agonism have led to the development of novel ligands, which have the potential for improved therapeutic and safety profiles. In this review, we summarize the theory and most recent breakthroughs in understanding biased signaling, examine recent laboratory investigations concerning biased ligands across different organ systems, and discuss the promising clinical applications of biased agonism.

  3. Characterization of hyaluronan-binding proteins on guinea pig polymorphonuclear leukocytes: possible involvement of complement receptor type 3 (CR3, CD11b/CD18) in the hyaluronan-leukocyte interaction.

    Science.gov (United States)

    Nochi, Hiromi; Shinomiya, Takahisa; Tamoto, Koichi

    2006-01-01

    Hyaluronan (HA), a high-molecular-weight glycosaminoglycan ubiquitously present in the extracellular matrices (ECMs) of animals, plays important roles in ECM organization and cell behavior through binding to hyaluronan-binding proteins (HABPs). We previously reported that HA has anti-inflammatory effects on guinea pig phagocytes, although the nature of guinea pig HABPs was unknown. In this study, we characterized guinea pig HABPs on peritoneal polymorphonuclear leukocytes (PMNs) and blood neutrophils by flow cytometry and affinity chromatography. It was found that PMNs express diverse HABPs with different molecular weights. These HABPs maximally bound with HA over a wide pH range (6-8), and recognized HAs as small as the pentadisaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine. Furthermore, they could be divided into Mg(2+)-dependent and Ca(2+)/Mg(2+)-independent groups. Interestingly, two proteins in the Mg(2+)-dependent group were found to be the two subunits of complement receptor type 3 (CR3, CD11b/CD18). Unlike PMNs, blood neutrophils expressed several functionally inactive HABPs. Among these inactive HABPs, Mg(2+)-dependent proteins including CR3 but not Ca(2+)/Mg(2+)-independent proteins were activated on phorbol ester-stimulation. These results show the existence of diverse HABPs on guinea pig neutrophils and the cell activation-dependent activation of HABPs. It is also suggested that the CR3-HA interaction is possibly involved in the regulation of neutrophil function.

  4. Quantitative receptor radioautography in the study of receptor-receptor interactions in the nucleus tractus solitarii

    Directory of Open Access Journals (Sweden)

    Fior-Chadi D.R.

    1998-01-01

    Full Text Available The nucleus tractus solitarii (NTS in the dorsomedial medulla comprises a wide range of neuropeptides and biogenic amines. Several of them are related to mechanisms of central blood pressure control. Angiotensin II (Ang II, neuropeptide Y (NPY and noradrenaline (NA are found in the NTS cells, as well as their receptors. Based on this observation we have evaluated the modulatory effect of these peptide receptors on a2-adrenoceptors in the NTS. Using quantitative receptor radioautography, we observed that NPY and Ang II receptors decreased the affinity of a2-adrenoceptors for their agonists in the NTS of the rat. Cardiovascular experiments agreed with the in vitro data. Coinjection of a threshold dose of Ang II or of the NPY agonists together with an ED50 dose of adrenergic agonists such as NA, adrenaline and clonidine counteracted the depressor effect produced by the a2-agonist in the NTS. The results provide evidence for the existence of an antagonistic interaction between Ang II at1 receptors and NPY receptor subtypes with the a2-adrenoceptors in the NTS. This receptor interaction may reduce the transduction over the a2-adrenoceptors which can be important in central cardiovascular regulation and in the development of hypertension

  5. Salivary agglutinin and lung scavenger receptor cysteine-rich glycoprotein 340 have broad anti-influenza activities and interactions with surfactant protein D that vary according to donor source and sialylation

    DEFF Research Database (Denmark)

    Hartshorn, Kevan L.; Ligtenberg, Antoon; White, Mitchell R.

    2006-01-01

    has co-operative interactions with SP-D in viral neutralization and aggregation assays. We now report that salivary gp-340 can, in some cases, strongly antagonize certain antiviral activities of SP-D. This effect was associated with greater binding of salivary gp-340 to the carbohydrate recognition......We previously found that scavenger receptor cysteine-rich gp-340 (glycoprotein-340), isolated from lung or saliva, directly inhibits human IAVs (influenza A viruses). We now show that salivary gp-340 has broad antiviral activity against human, equine and porcine IAV strains. Although lung...... and salivary gp-340 are identical in protein sequence, salivary gp-340 from one donor had significantly greater antiviral activity against avian-like IAV strains which preferentially bind sialic acids in alpha(2,3) linkage. A greater density of alpha(2,3)-linked sialic acids was present on the salivary gp-340...

  6. The Arabidopsis kinase-associated protein phosphatase controls internalization of the somatic embryogenesis receptor kinase 1

    NARCIS (Netherlands)

    Shah, K.; Russinova, E.; Gadella, T.W.J.; Willemse, J.; Vries, de S.C.

    2002-01-01

    The AtSERK1 protein is a plasma membrane-located LRR receptor-like serine threonine kinase that is transiently expressed during plant embryogenesis. Our results show that AtSERK1 interacts with the kinase-associated protein phosphatase (KAPP) in vitro. The kinase interaction (KI) domain of KAPP does

  7. A receptor for infectious and cellular prion protein

    Directory of Open Access Journals (Sweden)

    V.R. Martins

    1999-07-01

    Full Text Available Prions are an unconventional form of infectious agents composed only of protein and involved in transmissible spongiform encephalopathies in humans and animals. The infectious particle is composed by PrPsc which is an isoform of a normal cellular glycosyl-phosphatidylinositol (GPI anchored protein, PrPc, of unknown function. The two proteins differ only in conformation, PrPc is composed of 40% a helix while PrPsc has 60% ß-sheet and 20% a helix structure. The infection mechanism is trigged by interaction of PrPsc with cellular prion protein causing conversion of the latter's conformation. Therefore, the infection spreads because new PrPsc molecules are generated exponentially from the normal PrPc. The accumulation of insoluble PrPsc is probably one of the events that lead to neuronal death. Conflicting data in the literature showed that PrPc internalization is mediated either by clathrin-coated pits or by caveolae-like membranous domains. However, both pathways seem to require a third protein (a receptor or a prion-binding protein either to make the connection between the GPI-anchored molecule to clathrin or to convert PrPc into PrPsc. We have recently characterized a 66-kDa membrane receptor which binds PrPc in vitro and in vivo and mediates the neurotoxicity of a human prion peptide. Therefore, the receptor should have a role in the pathogenesis of prion-related diseases and in the normal cellular process. Further work is necessary to clarify the events triggered by the association of PrPc/PrPsc with the receptor.

  8. G Protein-Coupled Receptors: Extranuclear Mediators for the Non-Genomic Actions of Steroids

    Directory of Open Access Journals (Sweden)

    Chen Wang

    2014-09-01

    Full Text Available Steroids hormones possess two distinct actions, a delayed genomic effect and a rapid non-genomic effect. Rapid steroid-triggered signaling is mediated by specific receptors localized most often to the plasma membrane. The nature of these receptors is of great interest and accumulated data suggest that G protein-coupled receptors (GPCRs are appealing candidates. Increasing evidence regarding the interaction between steroids and specific membrane proteins, as well as the involvement of G protein and corresponding downstream signaling, have led to identification of physiologically relevant GPCRs as steroid extranuclear receptors. Examples include G protein-coupled receptor 30 (GPR30 for estrogen, membrane progestin receptor for progesterone, G protein-coupled receptor family C group 6 member A (GPRC6A and zinc transporter member 9 (ZIP9 for androgen, and trace amine associated receptor 1 (TAAR1 for thyroid hormone. These receptor-mediated biological effects have been extended to reproductive development, cardiovascular function, neuroendocrinology and cancer pathophysiology. However, although great progress have been achieved, there are still important questions that need to be answered, including the identities of GPCRs responsible for the remaining steroids (e.g., glucocorticoid, the structural basis of steroids and GPCRs’ interaction and the integration of extranuclear and nuclear signaling to the final physiological function. Here, we reviewed the several significant developments in this field and highlighted a hypothesis that attempts to explain the general interaction between steroids and GPCRs.

  9. Genetic-epigenetic interaction modulates μ-opioid receptor regulation.

    Science.gov (United States)

    Oertel, Bruno G; Doehring, Alexandra; Roskam, Bianca; Kettner, Mattias; Hackmann, Nadja; Ferreirós, Nerea; Schmidt, Peter H; Lötsch, Jörn

    2012-11-01

    Genetic and epigenetic mechanisms play important roles in protein expression, although at different levels. Genetic variations can alter CpG sites and thus influence the epigenetic regulation of mRNA expression, providing an increasingly recognized mechanism of functional consequences of genetic polymorphisms. One of those genetic effects is the association of reduced μ-opioid receptor expression with the functional genetic variant N40D (OPRM1 118A>G, rs1799971) that causes an amino acid exchange in the extracellular terminal of the μ-opioid receptor. We report that the nucleotide exchange at gene position +118 introduces a new CpG-methylation site into the OPRM1 DNA at position +117. This leads to an enhanced methylation of the OPRM1 DNA at this site and downstream. This epigenetic mechanism impedes μ-opioid receptor upregulation in brain tissue of Caucasian chronic opiate addicts, assessed postmortem. While in wild-type subjects, a reduced signalling efficiency associated with chronic heroin exposure was compensated by an increased receptor density, this upregulation was absent in carriers of the 118G receptor variant due to a diminished OPRM1 mRNA transcription. Thus, the OPRM1 118A>G SNP variant not only reduces µ-opioid receptor signalling efficiency, but, by a genetic-epigenetic interaction, reduces opioid receptor expression and therefore, depletes the opioid system of a compensatory reaction to chronic exposure. This demonstrates that a change in the genotype can cause a change in the epigenotype with major functional consequences.

  10. Minireview: Role of Intracellular Scaffolding Proteins in the Regulation of Endocrine G Protein-Coupled Receptor Signaling

    Science.gov (United States)

    Walther, Cornelia

    2015-01-01

    The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies. PMID:25942107

  11. Identification of a preassembled TRH receptor-G(q/11) protein complex in HEK293 cells.

    Science.gov (United States)

    Drastichova, Zdenka; Novotny, Jiri

    2012-01-01

    Protein-protein interactions define specificity in signal transduction and these interactions are central to transmembrane signaling by G-protein-coupled receptors (GPCRs). It is not quite clear, however, whether GPCRs and the regulatory trimeric G-proteins behave as freely and independently diffusible molecules in the plasma membrane or whether they form some preassociated complexes. Here we used clear-native polyacrylamide gel electrophoresis (CN-PAGE) to investigate the presumed coupling between thyrotropin-releasing hormone (TRH) receptor and its cognate G(q/11) protein in HEK293 cells expressing high levels of these proteins. Under different solubilization conditions, the TRH receptor (TRH-R) was identified to form a putative pentameric complex composed of TRH-R homodimer and G(q/11) protein. The presumed association of TRH-R with G(q/11)α or Gβ proteins in plasma membranes was verified by RNAi experiments. After 10- or 30-min hormone treatment, TRH-R signaling complexes gradually dissociated with a concomitant release of receptor homodimers. These observations support the model in which GPCRs can be coupled to trimeric G-proteins in preassembled signaling complexes, which might be dynamically regulated upon receptor activation. The precoupling of receptors with their cognate G-proteins can contribute to faster G-protein activation and subsequent signal transfer into the cell interior.

  12. Protein Interactions Investigated by the Raman Spectroscopy for Biosensor Applications

    Directory of Open Access Journals (Sweden)

    R. P. Kengne-Momo

    2012-01-01

    Full Text Available Interaction and surface binding characteristics of staphylococcal protein A (SpA and an anti-Escherichia coli immunoglobulin G (IgG were studied using the Raman spectroscopy. The tyrosine amino acid residues present in the α-helix structure of SpA were found to be involved in interaction with IgG. In bulk interaction condition the native structure of proteins was almost preserved where interaction-related changes were observed in the overall secondary structure (α-helix of SpA. In the adsorbed state, the protein structure was largely modified, which allowed the identification of tyrosine amino acids involved in SpA and IgG interaction. This study constitutes a direct Raman spectroscopic investigation of SpA and IgG (receptor-antibody interaction mechanism in the goal of a future biosensor application for detection of pathogenic microorganisms.

  13. Pichia pastoris Pex 14p, a phosphorylated peroxisomal membrane protein, is part of a PTS-receptor docking complex and interacts with many peroxins

    NARCIS (Netherlands)

    Johnson, Monique A.; Snyder, William B.; Cereghino, Joan Lin; Veenhuis, Marten; Subramani, Suresh; Cregg, James M.

    2001-01-01

    The peroxisomal protein import machinery plays a central role in the assembly of this organelle in all eukaryotes. Genes encoding components of this machinery, termed peroxins or Pex proteins, have been isolated and characterized in several yeast species and in mammals, including humans. Here we

  14. Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom.

    Science.gov (United States)

    Xu, Yanjie; Xia, Jixiang; Liu, Suxuan; Stein, Sam; Ramon, Cueto; Xi, Hang; Wang, Luqiao; Xiong, Xinyu; Zhang, Lixiao; He, Dingwen; Yang, William; Zhao, Xianxian; Cheng, Xiaoshu; Yang, Xiaofeng; Wang, Hong

    2017-03-01

    Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via its binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complexes and their intracellular trafficking based on protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair.

  15. Molecular interactions of agonist and inverse agonist ligands at serotonin 5-HT2C G protein-coupled receptors: computational ligand docking and molecular dynamics studies validated by experimental mutagenesis results

    Science.gov (United States)

    Córdova-Sintjago, Tania C.; Liu, Yue; Booth, Raymond G.

    2015-02-01

    To understand molecular determinants for ligand activation of the serotonin 5-HT2C G protein-coupled receptor (GPCR), a drug target for obesity and neuropsychiatric disorders, a 5-HT2C homology model was built according to an adrenergic β2 GPCR (β2AR) structure and validated using a 5-HT2B GPCR crystal structure. The models were equilibrated in a simulated phosphatidyl choline membrane for ligand docking and molecular dynamics studies. Ligands included (2S, 4R)-(-)-trans-4-(3'-bromo- and trifluoro-phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalene-2-amine (3'-Br-PAT and 3'-CF3-PAT), a 5-HT2C agonist and inverse agonist, respectively. Distinct interactions of 3'-Br-PAT and 3'-CF3-PAT at the wild-type (WT) 5-HT2C receptor model were observed and experimental 5-HT2C receptor mutagenesis studies were undertaken to validate the modelling results. For example, the inverse agonist 3'-CF3-PAT docked deeper in the WT 5-HT2C binding pocket and altered the orientation of transmembrane helices (TM) 6 in comparison to the agonist 3'-Br-PAT, suggesting that changes in TM orientation that result from ligand binding impact function. For both PATs, mutation of 5-HT2C residues S3.36, T3.37, and F5.47 to alanine resulted in significantly decreased affinity, as predicted from modelling results. It was concluded that upon PAT binding, 5-HT2C residues T3.37 and F5.47 in TMs 3 and 5, respectively, engage in inter-helical interactions with TMs 4 and 6, respectively. The movement of TMs 5 and 6 upon agonist and inverse agonist ligand binding observed in the 5-HT2C receptor modelling studies was similar to movements reported for the activation and deactivation of the β2AR, suggesting common mechanisms among aminergic neurotransmitter GPCRs.

  16. Mimicking Intermolecular Interactions of Tight Protein-Protein Complexes for Small-Molecule Antagonists.

    Science.gov (United States)

    Xu, David; Bum-Erdene, Khuchtumur; Si, Yubing; Zhou, Donghui; Ghozayel, Mona K; Meroueh, Samy O

    2017-11-08

    Tight protein-protein interactions (Kd 1000 Å2 ) are highly challenging to disrupt with small molecules. Historically, the design of small molecules to inhibit protein-protein interactions has focused on mimicking the position of interface protein ligand side chains. Here, we explore mimicry of the pairwise intermolecular interactions of the native protein ligand with residues of the protein receptor to enrich commercial libraries for small-molecule inhibitors of tight protein-protein interactions. We use the high-affinity interaction (Kd =1 nm) between the urokinase receptor (uPAR) and its ligand urokinase (uPA) to test our methods. We introduce three methods for rank-ordering small molecules docked to uPAR: 1) a new fingerprint approach that represents uPA's pairwise interaction energies with uPAR residues; 2) a pharmacophore approach to identify small molecules that mimic the position of uPA interface residues; and 3) a combined fingerprint and pharmacophore approach. Our work led to small molecules with novel chemotypes that inhibited a tight uPAR⋅uPA protein-protein interaction with single-digit micromolar IC50 values. We also report the extensive work that identified several of the hits as either lacking stability, thiol reactive, or redox active. This work suggests that mimicking the binding profile of the native ligand and the position of interface residues can be an effective strategy to enrich commercial libraries for small-molecule inhibitors of tight protein-protein interactions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Yeast Interacting Proteins Database: YEL043W, YOR164C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available on quantitative analysis of protein-protein interaction maps; may interact with ribosomes, based on co-purification studies...ing based on quantitative analysis of protein-protein interaction maps; may interact with ribosomes, based on co-purification studies

  18. Coarse-grain modelling of protein-protein interactions

    NARCIS (Netherlands)

    Baaden, Marc; Marrink, Siewert J.

    2013-01-01

    Here, we review recent advances towards the modelling of protein-protein interactions (PPI) at the coarse-grained (CG) level, a technique that is now widely used to understand protein affinity, aggregation and self-assembly behaviour. PPI models of soluble proteins and membrane proteins are

  19. An RBCC protein implicated in maintenance of steady-state neuregulin receptor levels.

    Science.gov (United States)

    Diamonti, A John; Guy, Pamela M; Ivanof, Caryn; Wong, Karen; Sweeney, Colleen; Carraway, Kermit L

    2002-03-05

    Despite numerous recent advances in our understanding of the molecular mechanisms underlying receptor tyrosine kinase down-regulation and degradation in response to growth factor binding, relatively little is known about ligand-independent receptor tyrosine kinase degradation mechanisms. In a screen for proteins that might regulate the trafficking or localization of the ErbB3 receptor, we have identified a tripartite or RBCC (RING, B-box, coiled-coil) protein that interacts with the cytoplasmic tail of the receptor in an activation-independent manner. We have named this protein Nrdp1 for neuregulin receptor degradation protein-1. Northern blotting reveals ubiquitous distribution of Nrdp1 in human adult tissues, but message is particularly prominent in heart, brain, and skeletal muscle. Nrdp1 interacts specifically with the neuregulin receptors ErbB3 and ErbB4 and not with epidermal growth factor receptor or ErbB2. When coexpressed in COS7 cells, Nrdp1 mediates the redistribution of ErbB3 from the cell surface to intracellular compartments and induces the suppression of ErbB3 and ErbB4 receptor levels but not epidermal growth factor receptor or ErbB2 levels. A putative dominant-negative form of Nrdp1 potentiates neuregulin-stimulated Erk1/2 activity in transfected MCF7 breast tumor cells. Our observations suggest that Nrdp1 may act to regulate steady-state cell surface neuregulin receptor levels, thereby influencing the efficiency of neuregulin signaling.

  20. Immunoprecipitation-based analysis of protein-protein interactions.

    Science.gov (United States)

    Speth, Corinna; Toledo-Filho, Luis A A; Laubinger, Sascha

    2014-01-01

    Several techniques allow the detection of protein-protein interactions. In vivo co-immunoprecipitation (Co-IP) studies are an important complement to other commonly used techniques such as yeast two-hybrid or fluorescence complementation, as they reveal interactions between functional proteins at physiological relevant concentrations. Here, we describe an in vivo Co-IP approach using either GFP affinity matrix or specific antibodies to purify proteins of interests and their interacting partners.

  1. Analysis of Protein-Membrane Interactions

    DEFF Research Database (Denmark)

    Kemmer, Gerdi Christine

    are implemented by soluble proteins reversibly binding to, as well as by integral membrane proteins embedded in, cellular membranes. The activity and interaction of these proteins is furthermore modulated by the lipids of the membrane. Here, liposomes were used as model membrane systems to investigate...... interactions between proteins and lipids. First, interactions of soluble proteins with membranes and specific lipids were studied, using two proteins: Annexin V and Tma1. The protein was first subjected to a lipid/protein overlay assay to identify candidate interaction partners in a fast and efficient way....... Discovered interactions were then probed on the level of the membrane using liposome-based assays. In the second part, a transmembrane protein was investigated. Assays to probe activity of the plasma membrane ATPase (Arabidopsis thaliana H+ -ATPase isoform 2 (AHA2)) in single liposomes using both giant...

  2. Pichia pastoris Pex 14p, a phosphorylated peroxisomal membrane protein, is part of a PTS-receptor docking complex and interacts with many peroxins

    OpenAIRE

    Johnson, Monique A.; Snyder, William B.; Cereghino, Joan Lin; Veenhuis, Marten; Subramani, Suresh; Cregg, James M.

    2001-01-01

    The peroxisomal protein import machinery plays a central role in the assembly of this organelle in all eukaryotes. Genes encoding components of this machinery, termed peroxins or Pex proteins, have been isolated and characterized in several yeast species and in mammals, including humans. Here we report on one of these components, Pex14p, from the methylotrophic yeast Pichia pastoris. Work in other organisms has shown that Pex14p is located on the cytoplasmic surface of the peroxisomal membran...

  3. Alternative splicing determines the interaction of SMRT isoforms with nuclear receptor-DNA complexes.

    Science.gov (United States)

    Faist, Flavie; Short, Stephen; Kneale, G Geoff; Sharpe, Colin R

    2009-06-01

    Signalling by small molecules, such as retinoic acid, is mediated by heterodimers comprising a class II nuclear receptor and an RXR (retinoid X receptor) subunit. The receptors bind to DNA response elements and act as ligand-dependent transcription factors, but, in the absence of signal, the receptors bind the co-repressors SMRT [silencing mediator for RAR (retinoic acid receptor) and TR (thyroid hormone receptor)] and NCoR (nuclear receptor co-repressor) and repress gene expression. Alternative splicing of the SMRT transcript in mammals generates six isoforms containing 1, 2 or 3 CoRNR (co-repressor for nuclear receptor) box motifs which are responsible for the interactions with nuclear receptors. We show that human cell lines express all six SMRT isoforms and then determine the binding affinity of mouse SMRT isoforms for RAR/RXR and three additional class II nuclear receptor-DNA complexes. This approach demonstrates the importance of the full complement of CoRNR boxes within each SMRT protein, rather than the identity of individual CoRNR boxes, in directing the interaction of SMRT with nuclear receptors. Each class of SMRT isoform displays a distinct feature, as the 1-box isoform discriminates between DNA response elements, the 2-box isoforms promote high-affinity binding to TR complexes and the 3-box isoforms show differential binding to nuclear receptors. Consequently, the differential deployment of SMRT isoforms observed in vivo could significantly expand the regulatory capacity of nuclear receptor signalling.

  4. Yeast Interacting Proteins Database: YGL161C, YDR084C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YGL161C YIP5 Protein that interacts with Rab GTPases, localized to late Golgi vesicles; computational...GTPases, localized to late Golgi vesicles; computational analysis of large-scale protein-protein interaction

  5. Yeast Interacting Proteins Database: YPL095C, YGL198W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available d to late Golgi vesicles; computational analysis of large-scale protein-protein interaction data suggests a ...gene name YIP4 Prey description Protein that interacts with Rab GTPases, localized to late Golgi vesicles; computational

  6. Ric-8A, a Gα protein guanine nucleotide exchange factor potentiates taste receptor signaling

    Directory of Open Access Journals (Sweden)

    Claire J Fenech

    2009-10-01

    Full Text Available Taste receptors for sweet, bitter and umami tastants are G-protein coupled receptors (GPCRs. While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS, RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Gα subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Gα-gustducin and Gαi2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

  7. Building blocks for protein interaction devices

    OpenAIRE

    Gr?nberg, Raik; Ferrar, Tony S.; van der Sloot, Almer M.; Constante, Marco; Serrano, Luis

    2010-01-01

    Here, we propose a framework for the design of synthetic protein networks from modular protein?protein or protein?peptide interactions and provide a starter toolkit of protein building blocks. Our proof of concept experiments outline a general work flow for part?based protein systems engineering. We streamlined the iterative BioBrick cloning protocol and assembled 25 synthetic multidomain proteins each from seven standardized DNA fragments. A systematic screen revealed two main factors contro...

  8. The G protein-coupled receptor, class C, group 6, subtype A (GPRC6A) receptor

    DEFF Research Database (Denmark)

    Clemmensen, C; Smajilovic, S; Wellendorph, P

    2014-01-01

    GPRC6A (G protein-coupled receptor, class C, group 6, subtype A) is a class C G protein-coupled receptor, that has been cloned from human, mouse and rat. Several groups have shown that the receptor is activated by a range of basic and small aliphatic L-α-amino acids of which L-arginine, L-lysine...

  9. PSAIA – Protein Structure and Interaction Analyzer

    Directory of Open Access Journals (Sweden)

    Vlahoviček Kristian

    2008-04-01

    Full Text Available Abstract Background PSAIA (Protein Structure and Interaction Analyzer was developed to compute geometric parameters for large sets of protein structures in order to predict and investigate protein-protein interaction sites. Results In addition to most relevant established algorithms, PSAIA offers a new method PIADA (Protein Interaction Atom Distance Algorithm for the determination of residue interaction pairs. We found that PIADA produced more satisfactory results than comparable algorithms implemented in PSAIA. Particular advantages of PSAIA include its capacity to combine different methods to detect the locations and types of interactions between residues and its ability, without any further automation steps, to handle large numbers of protein structures and complexes. Generally, the integration of a variety of methods enables PSAIA to offer easier automation of analysis and greater reliability of results. PSAIA can be used either via a graphical user interface or from the command-line. Results are generated in either tabular or XML format. Conclusion In a straightforward fashion and for large sets of protein structures, PSAIA enables the calculation of protein geometric parameters and the determination of location and type for protein-protein interaction sites. XML formatted output enables easy conversion of results to various formats suitable for statistic analysis. Results from smaller data sets demonstrated the influence of geometry on protein interaction sites. Comprehensive analysis of properties of large data sets lead to new information useful in the prediction of protein-protein interaction sites.

  10. NPIDB: nucleic acid?protein interaction database

    OpenAIRE

    Kirsanov, Dmitry D.; Zanegina, Olga N.; Aksianov, Evgeniy A.; Spirin, Sergei A.; Karyagina, Anna S.; Alexeevski, Andrei V

    2012-01-01

    The Nucleic acid?Protein Interaction DataBase (http://npidb.belozersky.msu.ru/) contains information derived from structures of DNA?protein and RNA?protein complexes extracted from the Protein Data Bank (3846 complexes in October 2012). It provides a web interface and a set of tools for extracting biologically meaningful characteristics of nucleoprotein complexes. The content of the database is updated weekly. The current version of the Nucleic acid?Protein Interaction DataBase is an upgrade ...

  11. BIOLUMINISCENCE RESONANCE ENERGY TRANSFER (BRET) METHODS TO STUDY G PROTEIN-COUPLED RECEPTOR - RECEPTOR TYROSINE KINASE HETERORECEPTOR COMPLEXES

    OpenAIRE

    Borroto-Escuela, Dasiel O.; Flajolet, Marc; Agnati, Luigi F.; Greengard, Paul; Fuxe, Kjell

    2013-01-01

    A large body of evidence indicates that G protein-coupled receptors (GPCRs) and Receptor tyrosine kinases (RTKs) can form heteroreceptor complexes. In these complexes, the signalling from each interacting protomer is modulated to produce an integrated and therefore novel response upon agonist(s) activation. In the GPCR-RTK heteroreceptor complexes, GPCRs can activate RTK in the absence of added growth factor through the use of RTK signalling molecules. This integrative phenomenon is reciproca...

  12. Yeast Interacting Proteins Database: YNL189W, YJL199C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available tein; not conserved in closely related Saccharomyces species; protein detected in large-scale protein-protein interaction studies...myces species; protein detected in large-scale protein-protein interaction studies Rows with this prey as pr

  13. Chapter 4: Protein interactions and disease.

    Directory of Open Access Journals (Sweden)

    Mileidy W Gonzalez

    Full Text Available Proteins do not function in isolation; it is their interactions with one another and also with other molecules (e.g. DNA, RNA that mediate metabolic and signaling pathways, cellular processes, and organismal systems. Due to their central role in biological function, protein interactions also control the mechanisms leading to healthy and diseased states in organisms. Diseases are often caused by mutations affecting the binding interface or leading to biochemically dysfunctional allosteric changes in proteins. Therefore, protein interaction networks can elucidate the molecular basis of disease, which in turn can inform methods for prevention, diagnosis, and treatment. In this chapter, we will describe the computational approaches to predict and map networks of protein interactions and briefly review the experimental methods to detect protein interactions. We will describe the application of protein interaction networks as a translational approach to the study of human disease and evaluate the challenges faced by these approaches.

  14. Inferring interaction partners from protein sequences

    CERN Document Server

    Bitbol, Anne-Florence; Colwell, Lucy J; Wingreen, Ned S

    2016-01-01

    Specific protein-protein interactions are crucial in the cell, both to ensure the formation and stability of multi-protein complexes, and to enable signal transduction in various pathways. Functional interactions between proteins result in coevolution between the interaction partners. Hence, the sequences of interacting partners are correlated. Here we exploit these correlations to accurately identify which proteins are specific interaction partners from sequence data alone. Our general approach, which employs a pairwise maximum entropy model to infer direct couplings between residues, has been successfully used to predict the three-dimensional structures of proteins from sequences. Building on this approach, we introduce an iterative algorithm to predict specific interaction partners from among the members of two protein families. We assess the algorithm's performance on histidine kinases and response regulators from bacterial two-component signaling systems. The algorithm proves successful without any a pri...

  15. The receptor-binding site of the measles virus hemagglutinin protein itself constitutes a conserved neutralizing epitope.

    Science.gov (United States)

    Tahara, Maino; Ohno, Shinji; Sakai, Kouji; Ito, Yuri; Fukuhara, Hideo; Komase, Katsuhiro; Brindley, Melinda A; Rota, Paul A; Plemper, Richard K; Maenaka, Katsumi; Takeda, Makoto

    2013-03-01

    Here, we provide direct evidence that the receptor-binding site of measles virus (MV) hemagglutinin protein itself forms an effective conserved neutralizing epitope (CNE). Several receptor-interacting residues constitute the CNE. Thus, viral escape from neutralization has to be associated with loss of receptor-binding activity. Since interactions with both the signaling lymphocyte activation molecule (SLAM) and nectin4 are critical for MV pathogenesis, its escape, which results from loss of receptor-binding activity, should not occur in nature.

  16. Yeast Interacting Proteins Database: YNL189W, YOR284W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ait as prey (0) YOR284W HUA2 Cytoplasmic protein of unknown function; computational...protein of unknown function; computational analysis of large-scale protein-protein interaction data suggests

  17. The protein interaction map of bacteriophage lambda

    Directory of Open Access Journals (Sweden)

    Uetz Peter

    2011-09-01

    Full Text Available Abstract Background Bacteriophage lambda is a model phage for most other dsDNA phages and has been studied for over 60 years. Although it is probably the best-characterized phage there are still about 20 poorly understood open reading frames in its 48-kb genome. For a complete understanding we need to know all interactions among its proteins. We have manually curated the lambda literature and compiled a total of 33 interactions that have been found among lambda proteins. We set out to find out how many protein-protein interactions remain to be found in this phage. Results In order to map lambda's interactions, we have cloned 68 out of 73 lambda open reading frames (the "ORFeome" into Gateway vectors and systematically tested all proteins for interactions using exhaustive array-based yeast two-hybrid screens. These screens identified 97 interactions. We found 16 out of 30 previously published interactions (53%. We have also found at least 18 new plausible interactions among functionally related proteins. All previously found and new interactions are combined into structural and network models of phage lambda. Conclusions Phage lambda serves as a benchmark for future studies of protein interactions among phage, viruses in general, or large protein assemblies. We conclude that we could not find all the known interactions because they require chaperones, post-translational modifications, or multiple proteins for their interactions. The lambda protein network connects 12 proteins of unknown function with well characterized proteins, which should shed light on the functional associations of these uncharacterized proteins.

  18. Understanding Protein-Protein Interactions Using Local Structural Features

    DEFF Research Database (Denmark)

    Planas-Iglesias, Joan; Bonet, Jaume; García-García, Javier

    2013-01-01

    Protein-protein interactions (PPIs) play a relevant role among the different functions of a cell. Identifying the PPI network of a given organism (interactome) is useful to shed light on the key molecular mechanisms within a biological system. In this work, we show the role of structural features...... (loops and domains) to comprehend the molecular mechanisms of PPIs. A paradox in protein-protein binding is to explain how the unbound proteins of a binary complex recognize each other among a large population within a cell and how they find their best docking interface in a short timescale. We use...... interacting and non-interacting protein pairs to classify the structural features that sustain the binding (or non-binding) behavior. Our study indicates that not only the interacting region but also the rest of the protein surface are important for the interaction fate. The interpretation...

  19. Complex formation and functional interaction between adenosine A1 receptor and type-1 metabotropic glutamate receptor

    Directory of Open Access Journals (Sweden)

    Yuji Kamikubo

    2015-07-01

    Full Text Available The adenosine A1 receptor (A1R is a G protein-coupled receptor (GPCR for adenosine, a ubiquitous neuromodulator, and thus regulates neuronal excitability, as well as arousal and sensitivity to pain. In addition, we have previously described a new mode of action for A1R: in cerebellar Purkinje cells, its activation attenuates neuronal responses to glutamate, as mediated by the type-1 metabotropic glutamate receptor (mGluR1. mGluR1 is also a GPCR, and elicits such responses as long-term depression of the postsynaptic response to glutamate, a cellular basis for cerebellar motor learning. Here, we explore in greater detail the interaction between A1R and mGluR1 using non-neuronal cells. Co-immunoprecipitation and Förster resonance energy transfer (FRET analysis reveal that A1R and mGluR1 form a complex. Furthermore, we found that mGluR1 activation inhibits A1R signaling, as measured by changes in intracellular cAMP. These findings demonstrate that A1R and mGluR1 have the intrinsic ability to form a heteromeric complex and mutually modulate signaling. This interaction may represent a new form of intriguing GPCR-mediated cellular responses.

  20. Biophysical approaches to G protein-coupled receptors: Structure, function and dynamics

    Science.gov (United States)

    Chollet, André; Turcatti, Gerardo

    1999-05-01

    G protein-coupled receptors (GPCR) represent a large family of drug targets for which there is no high-resolution structural information. In order to understand the mechanisms of ligand recognition and receptor activation, there is a strong need for novel biophysical methods. In this Perspective we provide an overview of recent experimental approaches used to explore the molecular architecture and dynamics of GPCR and their interactions with ligands and G proteins using biophysical, non-crystallographic, methods.

  1. A conserved mammalian protein interaction network.

    Directory of Open Access Journals (Sweden)

    Åsa Pérez-Bercoff

    Full Text Available Physical interactions between proteins mediate a variety of biological functions, including signal transduction, physical structuring of the cell and regulation. While extensive catalogs of such interactions are known from model organisms, their evolutionary histories are difficult to study given the lack of interaction data from phylogenetic outgroups. Using phylogenomic approaches, we infer a upper bound on the time of origin for a large set of human protein-protein interactions, showing that most such interactions appear relatively ancient, dating no later than the radiation of placental mammals. By analyzing paired alignments of orthologous and putatively interacting protein-coding genes from eight mammals, we find evidence for weak but significant co-evolution, as measured by relative selective constraint, between pairs of genes with interacting proteins. However, we find no strong evidence for shared instances of directional selection within an interacting pair. Finally, we use a network approach to show that the distribution of selective constraint across the protein interaction network is non-random, with a clear tendency for interacting proteins to share similar selective constraints. Collectively, the results suggest that, on the whole, protein interactions in mammals are under selective constraint, presumably due to their functional roles.

  2. Yeast Interacting Proteins Database: YDR425W, YGL161C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available with this bait as prey (0) YGL161C YIP5 Protein that interacts with Rab GTPases, localized to late Golgi vesicles; computational...IP5 Prey description Protein that interacts with Rab GTPases, localized to late Golgi vesicles; computatio...nal analysis of large-scale protein-protein interaction data suggests a possible ro

  3. Yeast Interacting Proteins Database: YDR425W, YGL198W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available with this bait as prey (0) YGL198W YIP4 Protein that interacts with Rab GTPases, localized to late Golgi vesicles; computational...IP4 Prey description Protein that interacts with Rab GTPases, localized to late Golgi vesicles; computatio...nal analysis of large-scale protein-protein interaction data suggests a possible ro

  4. The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses.

    Science.gov (United States)

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

    2015-04-01

    The pepper receptor-like cytoplasmic protein kinase, CaPIK1, which mediates signalling of plant cell death and defence responses was previously identified. Here, the identification of a class IV chitinase, CaChitIV, from pepper plants (Capsicum annuum), which interacts with CaPIK1 and promotes CaPIK1-triggered cell death and defence responses, is reported. CaChitIV contains a signal peptide, chitin-binding domain, and glycol hydrolase domain. CaChitIV expression was up-regulated by Xanthomonas campestris pv. vesicatoria (Xcv) infection. Notably, avirulent Xcv infection rapidly induced CaChitIV expression in pepper leaves. Bimolecular fluorescence complementation and co-immunoprecipitation revealed that CaPIK1 interacts with CaChitIV in planta, and that the CaPIK1-CaChitIV complex is localized mainly in the cytoplasm and plasma membrane. CaChitIV is also localized in the endoplasmic reticulum. Transient co-expression of CaChitIV with CaPIK1 enhanced CaPIK1-triggered cell death response and reactive oxygen species (ROS) and nitric oxide (NO) bursts. Co-silencing of both CaChitIV and CaPIK1 in pepper plants conferred enhanced susceptibility to Xcv infection, which was accompanied by a reduced induction of cell death response, ROS and NO bursts, and defence response genes. Ectopic expression of CaPIK1 in Arabidopsis enhanced basal resistance to Hyaloperonospora arabidopsidis infection. Together, the results suggest that CaChitIV positively regulates CaPIK1-triggered cell death and defence responses through its interaction with CaPIK1. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  5. MTUS1, a gene encoding angiotensin-II type 2 (AT2) receptor-interacting proteins, in health and disease, with special emphasis on its role in carcinogenesis.

    Science.gov (United States)

    Bozgeyik, Ibrahim; Yumrutas, Onder; Bozgeyik, Esra

    2017-08-30

    Loss of tumor suppressor activity is a frequent event in the formation and progression of tumors and has been listed as an important hallmark of cancers. Microtubule-Associated Scaffold Protein 1 (MTUS1) is a candidate tumor suppressor gene which is reported to be frequently down-regulated in a variety of human cancers including pancreas, colon, bladder, head-and-neck, ovarian, breast cancers, gastric, lung cancers. It is also reported to be implicated in several types of pathologies such as cardiac hypertrophy, atherosclerosis, and SLE-like lymphoproliferative diseases. Moreover, MTUS1-encoded proteins are shown to be involved in the regulation of vital cellular processes such as proliferation, differentiation, DNA repair, inflammation, vascular remodeling and senescence. However, the current knowledge is very limited about the role of this gene in human cancers as well as other type diseases. Besides, there is no literature report which summarizes and criticizes the importance of MTUS1 in the cellular processes, especially in the processes of carcinogenesis. Accordingly, in this comprehensive review, we tried to shed light on the role of tumor suppressor MTUS1/ATIP in health and disease, putting special emphasis on its role in the development and progression of human cancers as well as associated molecular mechanisms and the reasons behind MTUS1/ATIP deficiency, which have been not well documented previously. Copyright © 2017. Published by Elsevier B.V.

  6. The repertoire of olfactory C family G protein-coupled receptors in zebrafish: candidate chemosensory receptors for amino acids

    Directory of Open Access Journals (Sweden)

    Ngai John

    2006-12-01

    Full Text Available Abstract Background Vertebrate odorant receptors comprise at least three types of G protein-coupled receptors (GPCRs: the OR, V1R, and V2R/V2R-like receptors, the latter group belonging to the C family of GPCRs. These receptor families are thought to receive chemosensory information from a wide spectrum of odorant and pheromonal cues that influence critical animal behaviors such as feeding, reproduction and other social interactions. Results Using genome database mining and other informatics approaches, we identified and characterized the repertoire of 54 intact "V2R-like" olfactory C family GPCRs in the zebrafish. Phylogenetic analysis – which also included a set of 34 C family GPCRs from fugu – places the fish olfactory receptors in three major groups, which are related to but clearly distinct from other C family GPCRs, including the calcium sensing receptor, metabotropic glutamate receptors, GABA-B receptor, T1R taste receptors, and the major group of V2R vomeronasal receptor families. Interestingly, an analysis of sequence conservation and selective pressure in the zebrafish receptors revealed the retention of a conserved sequence motif previously shown to be required for ligand binding in other amino acid receptors. Conclusion Based on our findings, we propose that the repertoire of zebrafish olfactory C family GPCRs has evolved to allow the detection and discrimination of a spectrum of amino acid and/or amino acid-based compounds, which are potent olfactory cues in fish. Furthermore, as the major groups of fish receptors and mammalian V2R receptors appear to have diverged significantly from a common ancestral gene(s, these receptors likely mediate chemosensation of different classes of chemical structures by their respective organisms.

  7. Mining protein networks for synthetic genetic interactions

    Directory of Open Access Journals (Sweden)

    Zhao Shan

    2008-10-01

    Full Text Available Abstract Background The local connectivity and global position of a protein in a protein interaction network are known to correlate with some of its functional properties, including its essentiality or dispensability. It is therefore of interest to extend this observation and examine whether network properties of two proteins considered simultaneously can determine their joint dispensability, i.e., their propensity for synthetic sick/lethal interaction. Accordingly, we examine the predictive power of protein interaction networks for synthetic genetic interaction in Saccharomyces cerevisiae, an organism in which high confidence protein interaction networks are available and synthetic sick/lethal gene pairs have been extensively identified. Results We design a support vector machine system that uses graph-theoretic properties of two proteins in a protein interaction network as input features for prediction of synthetic sick/lethal interactions. The system is trained on interacting and non-interacting gene pairs culled from large scale genetic screens as well as literature-curated data. We find that the method is capable of predicting synthetic genetic interactions with sensitivity and specificity both exceeding 85%. We further find that the prediction performance is reasonably robust with respect to errors in the protein interaction network and with respect to changes in the features of test datasets. Using the prediction system, we carried out novel predictions of synthetic sick/lethal gene pairs at a genome-wide scale. These pairs appear to have functional properties that are similar to those that characterize the known synthetic lethal gene pairs. Conclusion Our analysis shows that protein interaction networks can be used to predict synthetic lethal interactions with accuracies on par with or exceeding that of other computational methods that use a variety of input features, including functional annotations. This indicates that protein

  8. Does protein binding modulate the effect of angiotensin II receptor antagonists?

    Directory of Open Access Journals (Sweden)

    Marc P Maillard

    2001-03-01

    Full Text Available IntroductionAngiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%. With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists.MethodsA radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin.ResultsThe in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists.ConclusionAlthough the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors.

  9. Yeast Interacting Proteins Database: YML064C, YJL199C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available y related Saccharomyces species; protein detected in large-scale protein-protein interaction studies Rows wi...in-protein interaction studies Rows with this prey as prey (4) Rows with this prey as bait (1) 28 6 3 4 0 0 ...d in closely related Saccharomyces species; protein detected in large-scale prote

  10. Yeast Interacting Proteins Database: YLR291C, YJL199C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ved in closely related Saccharomyces species; protein detected in large-scale protein-protein interaction studies...in large-scale protein-protein interaction studies Rows with this prey as prey Rows with this prey as prey (

  11. Engineering High Affinity Protein-Protein Interactions Using a High-Throughput Microcapillary Array Platform.

    Science.gov (United States)

    Lim, Sungwon; Chen, Bob; Kariolis, Mihalis S; Dimov, Ivan K; Baer, Thomas M; Cochran, Jennifer R

    2017-02-17

    Affinity maturation of protein-protein interactions requires iterative rounds of protein library generation and high-throughput screening to identify variants that bind with increased affinity to a target of interest. We recently developed a multipurpose protein engineering platform, termed μSCALE (Microcapillary Single Cell Analysis and Laser Extraction). This technology enables high-throughput screening of libraries of millions of cell-expressing protein variants based on their binding properties or functional activity. Here, we demonstrate the first use of the μSCALE platform for affinity maturation of a protein-protein binding interaction. In this proof-of-concept study, we engineered an extracellular domain of the Axl receptor tyrosine kinase to bind tighter to its ligand Gas6. Within 2 weeks, two iterative rounds of library generation and screening resulted in engineered Axl variants with a 50-fold decrease in kinetic dissociation rate, highlighting the use of μSCALE as a new tool for directed evolution.

  12. Modulation of dopamine D(2) receptor signaling by actin-binding protein (ABP-280).

    Science.gov (United States)

    Li, M; Bermak, J C; Wang, Z W; Zhou, Q Y

    2000-03-01

    Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling. In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding. The functional significance of the D(2) receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280. D(2) receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells. Maximal inhibitory responses of D(2) receptor activation were also reduced. Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D(2) receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358). Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation. This mutant (D(2)S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase. PKC activation also generated D(2) receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D(2)-ABP association. A mechanism for these results may be derived from a role of ABP-280 in the clustering of D(2) receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells. Our results suggest a new molecular mechanism of modulating D(2) receptor signaling by cytoskeletal protein interaction.

  13. Electrostatic similarities between protein and small molecule ligands facilitate the design of protein-protein interaction inhibitors.

    Directory of Open Access Journals (Sweden)

    Arnout Voet

    Full Text Available One of the underlying principles in drug discovery is that a biologically active compound is complimentary in shape and molecular recognition features to its receptor. This principle infers that molecules binding to the same receptor may share some common features. Here, we have investigated whether the electrostatic similarity can be used for the discovery of small molecule protein-protein interaction inhibitors (SMPPIIs. We have developed a method that can be used to evaluate the similarity of electrostatic potentials between small molecules and known protein ligands. This method was implemented in a software called EleKit. Analyses of all available (at the time of research SMPPII structures indicate that SMPPIIs bear some similarities of electrostatic potential with the ligand proteins of the same receptor. This is especially true for the more polar SMPPIIs. Retrospective analysis of several successful SMPPIIs has shown the applicability of EleKit in the design of new SMPPIIs.

  14. Steered Molecular Dynamics for Investigating the Interactions Between Insulin Receptor Tyrosine Kinase (IRK) and Variants of Protein Tyrosine Phosphatase 1B (PTP1B).

    Science.gov (United States)

    Nguyen, Hung; Do, Nhat; Phan, Tuyn; Pham, Tri

    2018-02-01

    The aim of this study is to use steered molecular dynamics to investigate the dissociation process between IRK and PTP1Bs for wild type and five mutants (consisting of p.D181E, p.D181A, p.Q262A, p.D181A-Y46F, and p.D181A-Q262A). The gained results are observed not only the unbinding mechanism of IRK-PTP1B complexes came from pulling force profile, number of hydrogen bonds, and interaction energy between IRK and PTP1Bs but also described PTP1B's point mutations could variably change its binding affinity towards IRK. Additionally, the binding free energy calculated by Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) is also revealed that electrostatic energy and polar solvation energy mainly made up the binding free energy of PTP1B-IRK complexes.

  15. Regulation of transferrin receptor 2 protein levels by transferrin.

    Science.gov (United States)

    Robb, Aeisha; Wessling-Resnick, Marianne

    2004-12-15

    Transferrin receptor 2 (TfR2) plays a critical role in iron homeostasis because patients carrying disabling mutations in the TFR2 gene suffer from hemochromatosis. In this study, iron-responsive regulation of TfR2 at the protein level was examined in vitro and in vivo. HepG2 cell TfR2 protein levels were up-regulated after exposure to holotransferrin (holoTf) in a time- and dose-responsive manner. ApoTf or high-iron treatment with non-Tf-bound iron failed to elicit similar effects, suggesting that TfR2 regulation reflects interactions of the iron-bound ligand. Hepatic TfR2 protein levels also reflected an adaptive response to changing iron status in vivo. Liver TfR2 protein levels were down- and up-regulated in rats fed an iron-deficient and a high-iron diet, respectively. TfR2 was also up-regulated in Hfe(-/-) mice, an animal model that displays liver iron loading. In contrast, TfR2 levels were reduced in hypotransferrinemic mice despite liver iron overload, supporting the idea that regulation of the receptor is dependent on Tf. This idea is confirmed by up-regulation of TfR2 in beta-thalassemic mice, which, like hypotransferrinemic mice, are anemic and incur iron loading, but have functional Tf. Based on these combined results, we hypothesize that TfR2 acts as a sensor of iron status such that receptor levels reflect Tf saturation.

  16. Extracellular Protein Interactions Mediated by the Neural Cell Adhesion Molecule, NCAM: Heterophilic Interactions Between NCAM and Cell Adhesion Molecules, Extracellular Matrix Proteins, and Viruses

    DEFF Research Database (Denmark)

    Nielsen, Janne; Kulahin, Nikolaj; Walmod, Peter

    2008-01-01

    interactions, thereby modulating a range of biological processes. This review summarizes interactions between NCAM and other CAMs and ECM proteins. Additionally, the role of NCAM as a receptor for rabies virus, and its implications in rabies infections is briefly described. Interactions between NCAM and its...

  17. Mass spectrometric analysis of protein interactions

    DEFF Research Database (Denmark)

    Borch, Jonas; Jørgensen, Thomas J. D.; Roepstorff, Peter

    2005-01-01

    Mass spectrometry is a powerful tool for identification of interaction partners and structural characterization of protein interactions because of its high sensitivity, mass accuracy and tolerance towards sample heterogeneity. Several tools that allow studies of protein interaction are now...... available and recent developments that increase the confidence of studies of protein interaction by mass spectrometry include quantification of affinity-purified proteins by stable isotope labeling and reagents for surface topology studies that can be identified by mass-contributing reporters (e.g. isotope...... labels, cleavable cross-linkers or fragment ions. The use of mass spectrometers to study protein interactions using deuterium exchange and for analysis of intact protein complexes recently has progressed considerably....

  18. Role for protein-protein interaction databases in human genetics.

    Science.gov (United States)

    Pattin, Kristine A; Moore, Jason H

    2009-12-01

    Proteomics and the study of protein-protein interactions are becoming increasingly important in our effort to understand human diseases on a system-wide level. Thanks to the development and curation of protein-interaction databases, up-to-date information on these interaction networks is accessible and publicly available to the scientific community. As our knowledge of protein-protein interactions increases, it is important to give thought to the different ways that these resources can impact biomedical research. In this article, we highlight the importance of protein-protein interactions in human genetics and genetic epidemiology. Since protein-protein interactions demonstrate one of the strongest functional relationships between genes, combining genomic data with available proteomic data may provide us with a more in-depth understanding of common human diseases. In this review, we will discuss some of the fundamentals of protein interactions, the databases that are publicly available and how information from these databases can be used to facilitate genome-wide genetic studies.

  19. Molecular basis for activation of G protein-coupled receptor kinases

    Energy Technology Data Exchange (ETDEWEB)

    Boguth, Cassandra A.; Singh, Puja; Huang, Chih-chin; Tesmer, John J.G. (Michigan)

    2012-03-16

    G protein-coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N-terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N-terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.

  20. Protein-protein interactions and cancer: targeting the central dogma.

    Science.gov (United States)

    Garner, Amanda L; Janda, Kim D

    2011-01-01

    Between 40,000 and 200,000 protein-protein interactions have been predicted to exist within the human interactome. As these interactions are of a critical nature in many important cellular functions and their dysregulation is causal of disease, the modulation of these binding events has emerged as a leading, yet difficult therapeutic arena. In particular, the targeting of protein-protein interactions relevant to cancer is of fundamental importance as the tumor-promoting function of several aberrantly expressed proteins in the cancerous state is directly resultant of its ability to interact with a protein-binding partner. Of significance, these protein complexes play a crucial role in each of the steps of the central dogma of molecular biology, the fundamental processes of genetic transmission. With the many important discoveries being made regarding the mechanisms of these genetic process, the identification of new chemical probes are needed to better understand and validate the druggability of protein-protein interactions related to the central dogma. In this review, we provide an overview of current small molecule-based protein-protein interaction inhibitors for each stage of the central dogma: transcription, mRNA splicing and translation. Importantly, through our analysis we have uncovered a lack of necessary probes targeting mRNA splicing and translation, thus, opening up the possibility for expansion of these fields.

  1. The lactate receptor, G-protein-coupled receptor 81/hydroxycarboxylic acid receptor 1

    DEFF Research Database (Denmark)

    Morland, Cecilie; Lauritzen, Knut Huso; Puchades, Maja

    2015-01-01

    , and schizophrenia and in the deposition of phosphorylated tau protein in Alzheimer's disease. HCAR1 could serve to ameliorate these conditions and might also act through downstream mechanisms other than cAMP. Lactate exits cells through monocarboxylate transporters in an equilibrating manner and through astrocyte......We have proposed that lactate is a “volume transmitter” in the brain and underpinned this by showing that the lactate receptor, G-protein-coupled receptor 81 (GPR81, also known as HCA1 or HCAR1), which promotes lipid storage in adipocytes, is also active in the mammalian brain. This includes...... the cerebral neocortex and the hippocampus, where it can be stimulated by physiological concentrations of lactate and by the HCAR1 agonist 3,5-dihydroxybenzoate to reduce cAMP levels. Cerebral HCAR1 is concentrated on the postsynaptic membranes of excitatory synapses and also is enriched at the blood...

  2. Regulation of neuronal communication by G protein-coupled receptors.

    Science.gov (United States)

    Huang, Yunhong; Thathiah, Amantha

    2015-06-22

    Neuronal communication plays an essential role in the propagation of information in the brain and requires a precisely orchestrated connectivity between neurons. Synaptic transmission is the mechanism through which neurons communicate with each other. It is a strictly regulated process which involves membrane depolarization, the cellular exocytosis machinery, neurotransmitter release from synaptic vesicles into the synaptic cleft, and the interaction between ion channels, G protein-coupled receptors (GPCRs), and downstream effector molecules. The focus of this review is to explore the role of GPCRs and G protein-signaling in neurotransmission, to highlight the function of GPCRs, which are localized in both presynaptic and postsynaptic membrane terminals, in regulation of intrasynaptic and intersynaptic communication, and to discuss the involvement of astrocytic GPCRs in the regulation of neuronal communication. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support.

    Directory of Open Access Journals (Sweden)

    Natalie Di Bartolo

    Full Text Available The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs. Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40-70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β1-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β1-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes.

  4. Rap-Interacting Proteins are Key Players in the Rap Symphony Orchestra

    Directory of Open Access Journals (Sweden)

    Xiao-Xi Guo

    2016-06-01

    Full Text Available Rap, a member of the Ras-like small G-protein family, is a key node among G-protein coupled receptors (GPCR, receptor tyrosine kinases (RTKs, ion channels and many other downstream pathways. Rap plays a unique role in cell morphogenesis, adhesion, migration, exocytosis, proliferation, apoptosis and carcinogenesis. The complexity and diversity of Rap functions are tightly regulated by Rap-interacting proteins such as GEFs, GAPs, Rap effectors and scaffold proteins. These interacting proteins decide the subcellular localization of Rap, the interaction modes with downstream Rap effectors and tune Rap as an atypical molecular conductor, coupling extra- and intracellular signals to various pathways. In this review, we summarize four groups of Rap-interacting proteins, highlight their distinctions in Rap-binding properties and interactive modes and discuss their contribution to the spatiotemporal regulation of Rap as well as the implications of targeting Rap-interacting proteins in human cancer therapy.

  5. Agouti-related protein is posttranslationally cleaved by proprotein convertase 1 to generate agouti-related protein (AGRP)83-132: interaction between AGRP83-132 and melanocortin receptors cannot be influenced by syndecan-3.

    Science.gov (United States)

    Creemers, John W M; Pritchard, Lynn E; Gyte, Amy; Le Rouzic, Philippe; Meulemans, Sandra; Wardlaw, Sharon L; Zhu, Xiaorong; Steiner, Donald F; Davies, Nicola; Armstrong, Duncan; Lawrence, Catherine B; Luckman, Simon M; Schmitz, Catherine A; Davies, Rick A; Brennand, John C; White, Anne

    2006-04-01

    Agouti-related protein (AGRP) plays a key role in energy homeostasis. The carboxyl-terminal domain of AGRP acts as an endogenous antagonist of the melanocortin-4 receptor (MC4-R). It has been suggested that the amino-terminal domain of AGRP binds to syndecan-3, thereby modulating the effects of carboxyl-terminal AGRP at the MC4-R. This model assumes that AGRP is secreted as a full-length peptide. In this study we found that AGRP is processed intracellularly after Arg(79)-Glu(80)-Pro(81)-Arg(82). The processing site suggests cleavage by proprotein convertases (PCs). RNA interference and overexpression experiments showed that PC1/3 is primarily responsible for cleavage in vitro, although both PC2 and PC5/6A can also process AGRP. Dual in situ hybridization demonstrated that PC1/3 is expressed in AGRP neurons in the rat hypothalamus. Moreover, hypothalamic extracts from PC1-null mice contained 3.3-fold more unprocessed full-length AGRP, compared with wild-type mice, based on combined HPLC and RIA analysis, demonstrating that PC1/3 plays a role in AGRP cleavage in vivo. We also found that AGRP(83-132) is more potent an antagonist than full-length AGRP, based on cAMP reporter assays, suggesting that posttranslational cleavage is required to potentiate the effect of AGRP at the MC4-R. Because AGRP is cleaved into distinct amino-terminal and carboxyl-terminal peptides, we tested whether amino-terminal peptides modulate food intake. However, intracerebroventricular injection of rat AGRP(25-47) and AGRP(50-80) had no effect on body weight, food intake, or core body temperature. Because AGRP is cleaved before secretion, syndecan-3 must influence food intake independently of the MC4-R.

  6. GPCRDB: an information system for G protein-coupled receptors

    NARCIS (Netherlands)

    Isberg, V.; Vroling, B.; Kant, R.; Li, K.; Vriend, G.; Gloriam, D.

    2014-01-01

    For the past 20 years, the GPCRDB (G protein-coupled receptors database; http://www.gpcr.org/7tm/) has been a 'one-stop shop' for G protein-coupled receptor (GPCR)-related data. The GPCRDB contains experimental data on sequences, ligand-binding constants, mutations and oligomers, as well as many

  7. Physical and functional interaction between CB1 cannabinoid receptors and β2-adrenoceptors

    Science.gov (United States)

    Hudson, Brian D; Hébert, Terence E; Kelly, Melanie EM

    2010-01-01

    Background and purpose: The CB1 cannabinoid receptor and the β2-adrenoceptor are G protein-coupled receptors (GPCRs) co-expressed in many tissues. The present study examined physical and functional interactions between these receptors in a heterologous expression system and in primary human ocular cells. Experimental approach: Physical interactions between CB1 receptors and β2-adrenoceptors were assessed using bioluminescence resonance energy transfer (BRET). Functional interactions between these receptors were evaluated by examining receptor trafficking, as well as extracellular signal-regulated kinase (ERK) and cyclic AMP response element binding protein (CREB) signalling. Key results: Physical interactions between CB1 receptors and β2-adrenoceptors were demonstrated using BRET. In human embryonic kidney (HEK) 293H cells, co-expression of β2-adrenoceptors tempered the constitutive activity and increased cell surface expression of CB1 receptors. Co-expression altered the signalling properties of CB1receptors, resulting in increased Gαi-dependent ERK phosphorylation, but decreased non-Gαi-mediated CREB phosphorylation. The CB1 receptor inverse agonist AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) attenuated β2-adrenoceptor-pERK signalling in cells expressing both receptors, while the CB1 receptor neutral antagonist O-2050 ((6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran) did not. The actions of AM251 and O-2050 were further examined in primary human trabecular meshwork (HTM) cells, which are ocular cells endogenously co-expressing CB1 receptors and β2-adrenoceptors. In HTM cells, as in HEK 293H cells, AM251 but not O-2050, altered the β2-adrenoceptor–pERK response. Conclusion and implications: A complex interaction was demonstrated between CB1 receptors and β2-adrenoceptors in HEK 293H cells. As similar functional interactions were also

  8. Human cancer protein-protein interaction network: a structural perspective.

    Directory of Open Access Journals (Sweden)

    Gozde Kar

    2009-12-01

    Full Text Available Protein-protein interaction networks provide a global picture of cellular function and biological processes. Some proteins act as hub proteins, highly connected to others, whereas some others have few interactions. The dysfunction of some interactions causes many diseases, including cancer. Proteins interact through their interfaces. Therefore, studying the interface properties of cancer-related proteins will help explain their role in the interaction networks. Similar or overlapping binding sites should be used repeatedly in single interface hub proteins, making them promiscuous. Alternatively, multi-interface hub proteins make use of several distinct binding sites to bind to different partners. We propose a methodology to integrate protein interfaces into cancer interaction networks (ciSPIN, cancer structural protein interface network. The interactions in the human protein interaction network are replaced by interfaces, coming from either known or predicted complexes. We provide a detailed analysis of cancer related human protein-protein interfaces and the topological properties of the cancer network. The results reveal that cancer-related proteins have smaller, more planar, more charged and less hydrophobic binding sites than non-cancer proteins, which may indicate low affinity and high specificity of the cancer-related interactions. We also classified the genes in ciSPIN according to phenotypes. Within phenotypes, for breast cancer, colorectal cancer and leukemia, interface properties were found to be discriminating from non-cancer interfaces with an accuracy of 71%, 67%, 61%, respectively. In addition, cancer-related proteins tend to interact with their partners through distinct interfaces, corresponding mostly to multi-interface hubs, which comprise 56% of cancer-related proteins, and constituting the nodes with higher essentiality in the network (76%. We illustrate the interface related affinity properties of two cancer-related hub

  9. Nkrp1 Family, from Lectins to Protein Interacting Molecules

    Directory of Open Access Journals (Sweden)

    Daniel Rozbeský

    2015-02-01

    Full Text Available The C-type lectin-like receptors include the Nkrp1 protein family that regulates the activity of natural killer (NK cells. Rat Nkrp1a was reported to bind monosaccharide moieties in a Ca2+-dependent manner in preference order of GalNac > GlcNAc >> Fuc >> Gal > Man. These findings established for rat Nkrp1a have been extrapolated to all additional Nkrp1 receptors and have been supported by numerous studies over the past two decades. However, since 1996 there has been controversy and another article showed lack of interactions with saccharides in 1999. Nevertheless, several high affinity saccharide ligands were synthesized in order to utilize their potential in antitumor therapy. Subsequently, protein ligands were introduced as specific binders for Nkrp1 proteins and three dimensional models of receptor/protein ligand interaction were derived from crystallographic data. Finally, for at least some members of the NK cell C-type lectin-like proteins, the “sweet story” was impaired by two reports in recent years. It has been shown that the rat Nkrp1a and CD69 do not bind saccharide ligands such as GlcNAc, GalNAc, chitotetraose and saccharide derivatives (GlcNAc-PAMAM do not directly and specifically influence cytotoxic activity of NK cells as it was previously described.

  10. Novel protein-protein interactions inferred from literature context.

    Directory of Open Access Journals (Sweden)

    Herman H H B M van Haagen

    Full Text Available We have developed a method that predicts Protein-Protein Interactions (PPIs based on the similarity of the context in which proteins appear in literature. This method outperforms previously developed PPI prediction algorithms that rely on the conjunction of two protein names in MEDLINE abstracts. We show significant increases in coverage (76% versus 32% and sensitivity (66% versus 41% at a specificity of 95% for the prediction of PPIs currently archived in 6 PPI databases. A retrospective analysis shows that PPIs can efficiently be predicted before they enter PPI databases and before their interaction is explicitly described in the literature. The practical value of the method for discovery of novel PPIs is illustrated by the experimental confirmation of the inferred physical interaction between CAPN3 and PARVB, which was based on frequent co-occurrence of both proteins with concepts like Z-disc, dysferlin, and alpha-actinin. The relationships between proteins predicted by our method are broader than PPIs, and include proteins in the same complex or pathway. Dependent on the type of relationships deemed useful, the precision of our method can be as high as 90%. The full set of predicted interactions is available in a downloadable matrix and through the webtool Nermal, which lists the most likely interaction partners for a given protein. Our framework can be used for prioritizing potential interaction partners, hitherto undiscovered, for follow-up studies and to aid the generation of accurate protein interaction maps.

  11. Helix 3-Helix 5 interactions in Steroid Hormone Receptor Function

    Science.gov (United States)

    Zhang, Junhui; Geller, David S.

    2009-01-01

    Steroid hormones working through their receptors regulate a wide variety of physiologic processes necessary for normal homeostasis. Recent years have witnessed great advances in our understanding of how these hormones interact with their receptors, and have brought us closer to the era of directed drug design. We previously described a novel intramolecular interaction between helix 3 and helix 5 which is responsible for a Mendelian form of human hypertension. Further studies revealed that this interaction is highly conserved throughout the steroid hormone receptor family and functions as a key regulator of steroid hormone receptor sensitivity and specificity. Here, we review the contribution of helix 3-helix 5 interaction to steroid hormone receptor activity, with an eye towards how this knowledge may aid in the creation of novel therapeutic agonists and antagonists. PMID:18502379

  12. Yeast Interacting Proteins Database: YIL007C, YOR117W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YIL007C NAS2 Proteasome-interacting protein involved in the assembly of the base su...tion Proteasome-interacting protein involved in the assembly of the base subcomplex of the 19S proteasomal r

  13. Yeast Interacting Proteins Database: YDL226C, YGL198W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available s bait as prey (0) YGL198W YIP4 Protein that interacts with Rab GTPases, localized to late Golgi vesicles; computational...iption Protein that interacts with Rab GTPases, localized to late Golgi vesicles; computational

  14. Yeast Interacting Proteins Database: YOR158W, YLR424W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YOR158W PET123 Mitochondrial ribosomal protein of the small subunit; PET123 exhibits genetic interactions...al ribosomal protein of the small subunit; PET123 exhibits genetic interactions with PET122, which encodes a

  15. Yeast Interacting Proteins Database: YPR103W, YOR047C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available tein involved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sensors...gulated gene expression; interacts with protein kinase Snf1p, glucose sensors Snf

  16. Yeast Interacting Proteins Database: YNL258C, YKR022C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available membrane protein required for Golgi-to-ER retrograde traffic; component of the ER target site that interacts...membrane protein required for Golgi-to-ER retrograde traffic; component of the ER target site that interacts

  17. Yeast Interacting Proteins Database: YGL145W, YNL258C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available membrane protein required for Golgi-to-ER retrograde traffic; component of the ER target site that interacts...membrane protein required for Golgi-to-ER retrograde traffic; component of the ER target site that interacts

  18. Yeast Interacting Proteins Database: YNL258C, YLR440C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available membrane protein required for Golgi-to-ER retrograde traffic; component of the ER target site that interacts...membrane protein required for Golgi-to-ER retrograde traffic; component of the ER target site that interacts

  19. Yeast Interacting Proteins Database: YNL078W, YKR048C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available Protein localized in the bud neck at G2/M phase; physically interacts with septins; possibly involved in...Protein localized in the bud neck at G2/M phase; physically interacts with septins; possibly involved in

  20. Yeast Interacting Proteins Database: YPR040W, YDL188C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YPR040W TIP41 Protein that interacts physically and genetically with Tap42p, which ...ait ORF YPR040W Bait gene name TIP41 Bait description Protein that interacts physically and genetically

  1. Yeast Interacting Proteins Database: YPR040W, YDL134C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YPR040W TIP41 Protein that interacts physically and genetically with Tap42p, which ...Bait ORF YPR040W Bait gene name TIP41 Bait description Protein that interacts physically and genetically

  2. Modeling disordered protein interactions from biophysical principles

    National Research Council Canada - National Science Library

    Peterson, Lenna X; Roy, Amitava; Christoffer, Charles; Terashi, Genki; Kihara, Daisuke

    2017-01-01

    ...-protein interactions (PPIs) are formed with IDPs [3]. A well-known example is the p53 tumor suppressor, which contains disordered regions that interact with dozens of partner proteins [4]. Due to the abundance and characteristic features of IDPs in PPI networks, including many critical signaling pathways, fully understanding the molecular mechanisms of PPI networ...

  3. Yeast Interacting Proteins Database: YGR268C, YER125W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available larity to that of Type I J-proteins; computational analysis of large-scale protein-protein interaction data ...equence similarity to that of Type I J-proteins; computational analysis of large-scale protein-protein inter

  4. Inferring Domain-Domain Interactions from Protein-Protein Interactions with Formal Concept Analysis

    OpenAIRE

    Susan Khor

    2014-01-01

    Identifying reliable domain-domain interactions will increase our ability to predict novel protein-protein interactions, to unravel interactions in protein complexes, and thus gain more information about the function and behavior of genes. One of the challenges of identifying reliable domain-domain interactions is domain promiscuity. Promiscuous domains are domains that can occur in many domain architectures and are therefore found in many proteins. This becomes a problem for a method where t...

  5. New partner proteins containing novel internal recognition motif for human glutaminase interacting protein (hGIP).

    Science.gov (United States)

    Zencir, Sevil; Banerjee, Monimoy; Dobson, Melanie J; Ayaydin, Ferhan; Fodor, Elfrieda Ayaydin; Topcu, Zeki; Mohanty, Smita

    2013-03-01

    Regulation of gene expression in cells is mediated by protein-protein, DNA-protein and receptor-ligand interactions. PDZ (PSD-95/Discs-large/ZO-1) domains are protein-protein interaction modules. PDZ-containing proteins function in the organization of multi-protein complexes controlling spatial and temporal fidelity of intracellular signaling pathways. In general, PDZ proteins possess multiple domains facilitating distinct interactions. The human glutaminase interacting protein (hGIP) is an unusual PDZ protein comprising entirely of a single PDZ domain and plays pivotal roles in many cellular processes through its interaction with the C-terminus of partner proteins. Here, we report the identification by yeast two-hybrid screening of two new hGIP-interacting partners, DTX1 and STAU1. Both proteins lack the typical C-terminal PDZ recognition motif but contain a novel internal hGIP recognition motif recently identified in a phage display library screen. Fluorescence resonance energy transfer and confocal microscopy analysis confirmed the in vivo association of hGIP with DTX1 and STAU1 in mammalian cells validating the previous discovery of S/T-X-V/L-D as a consensus internal motif for hGIP recognition. Similar to hGIP, DTX1 and STAU1 have been implicated in neuronal function. Identification of these new interacting partners furthers our understanding of GIP-regulated signaling cascades and these interactions may represent potential new drug targets in humans. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Arc Interacts with the Integral Endoplasmic Reticulum Protein, Calnexin

    Directory of Open Access Journals (Sweden)

    Craig Myrum

    2017-09-01

    Full Text Available Activity-regulated cytoskeleton-associated protein, Arc, is a major regulator of long-term synaptic plasticity and memory formation. Here we reveal a novel interaction partner of Arc, a resident endoplasmic reticulum transmembrane protein, calnexin. We show an interaction between recombinantly-expressed GST-tagged Arc and endogenous calnexin in HEK293, SH-SY5Y neuroblastoma and PC12 cells. The interaction was dependent on the central linker region of the Arc protein that is also required for endocytosis of AMPA-type glutamate receptors. High-resolution proximity-ligation assays (PLAs demonstrate molecular proximity of endogenous Arc with the cytosolic C-terminus, but not the lumenal N-terminus of calnexin. In hippocampal neuronal cultures treated with brain-derived neurotrophic factor (BDNF, Arc interacted with calnexin in the perinuclear cytoplasm and dendritic shaft. Arc also interacted with C-terminal calnexin in the adult rat dentate gyrus (DG. After induction of long-term potentiation (LTP in the perforant path projection to the DG of adult anesthetized rats, enhanced interaction between Arc and calnexin was obtained in the dentate granule cell layer (GCL. Although Arc and calnexin are both implicated in the regulation of receptor endocytosis, no modulation of endocytosis was detected in transferrin uptake assays. Previous work showed that Arc interacts with multiple protein partners to regulate synaptic transmission and nuclear signaling. The identification of calnexin as a binding partner further supports the role of Arc as a hub protein and extends the range of Arc function to the endoplasmic reticulum, though the function of the Arc/calnexin interaction remains to be defined.

  7. Building blocks for protein interaction devices.

    Science.gov (United States)

    Grünberg, Raik; Ferrar, Tony S; van der Sloot, Almer M; Constante, Marco; Serrano, Luis

    2010-05-01

    Here, we propose a framework for the design of synthetic protein networks from modular protein-protein or protein-peptide interactions and provide a starter toolkit of protein building blocks. Our proof of concept experiments outline a general work flow for part-based protein systems engineering. We streamlined the iterative BioBrick cloning protocol and assembled 25 synthetic multidomain proteins each from seven standardized DNA fragments. A systematic screen revealed two main factors controlling protein expression in Escherichia coli: obstruction of translation initiation by mRNA secondary structure or toxicity of individual domains. Eventually, 13 proteins were purified for further characterization. Starting from well-established biotechnological tools, two general-purpose interaction input and two readout devices were built and characterized in vitro. Constitutive interaction input was achieved with a pair of synthetic leucine zippers. The second interaction was drug-controlled utilizing the rapamycin-induced binding of FRB(T2098L) to FKBP12. The interaction kinetics of both devices were analyzed by surface plasmon resonance. Readout was based on Förster resonance energy transfer between fluorescent proteins and was quantified for various combinations of input and output devices. Our results demonstrate the feasibility of parts-based protein synthetic biology. Additionally, we identify future challenges and limitations of modular design along with approaches to address them.

  8. A membrane protein / signaling protein interaction network for Arabidopsis version AMPv2

    Directory of Open Access Journals (Sweden)

    Sylvie Lalonde

    2010-09-01

    Full Text Available Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway compatible vector. The mating-based split-ubiquitin system was used to screen for potential protein-protein interactions (pPPIs among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases, 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 387 pPPIs between 179 proteins, yielding a scale-free network (r2=0.863. Eighty of 142 transmembrane receptor-like kinases (RLK tested positive, identifying three homomers, 63 heteromers and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

  9. Structures and Interactions of Proteins in the Brain

    DEFF Research Database (Denmark)

    Nielsen, Lau Dalby

    coding for Arc protein has been domesticated from the same branch of genes that has given rise to retroviruses. We show that even despite the large evolutional distance between Arc and retroviruses. Despite large evolutionary distance Arc still self-assemble into higher order structures that resembles......The protein low density lipoprotein receptor related protein 1 (LRP1) plays multiple roles in the biology of amyloid β peptide (Aβ) and Alzheimer’s disease. LRP1 is very important for clearance of Aβ both in the brain and by facilitating Aβ export over the blood brain barrier. In spite...... the primary nucleation is increased. The data furthermore indicates that there is an interaction with Aβ oligomer state and possible also the fibrils. Another brain protein is the neuronal protein Activity-regulated cytoskeletonassociated protein (Arc) which is important for learning and memory. The gene...

  10. Cannabinoid Receptor 1 Gene by Cannabis Use Interaction on CB1 Receptor Density.

    Science.gov (United States)

    Ketcherside, Ariel; Noble, Lindsey J; McIntyre, Christa K; Filbey, Francesca M

    2017-01-01

    Background: Because delta-9-tetrahydrocannabinol (THC), the primary psychoactive ingredient in cannabis, binds to cannabinoid 1 (CB1) receptors, levels of CB1 protein could serve as a potential biomarker for response to THC. To date, available techniques to characterize CB1 expression and function in vivo are limited. In this study, we developed an assay to quantify CB1 in lymphocytes to determine how it relates to cannabis use in 58 daily cannabis users compared with 47 nonusers. Furthermore, we tested whether CB1 levels are associated with mutations in a single nucleotide polymorphism known to regulate CB1 functioning (i.e., rs2023239). Methods: Total protein concentration was analyzed through the Pierce BCA Protein assay kit. CB1 protein was quantified through CNR1 enzyme-linked immunosorbent assay (ELISA) kit from MyBioSource. CB1 concentration and total protein concentration were quantified and used to calculate a ratio of CB1 to total protein. Results: Inherent levels of peripheral lymphocyte CB1 were sufficient for quantification through ELISA without protein amplification. We found a group×genotype interaction such that users with the G allele had greater CB1 concentration than users with the A/A genotype, and a trend-level difference between genotypes in nonusers. Conclusions: This study demonstrates a minimally invasive technique of CB1 quantification that holds promise for the use of CB1 protein concentration, along with rs2023239 genotype, as a potential biomarker for susceptibility to cannabis use. These results suggest a gene (rs2023239 G)×environment (cannabis use) effect on CB1 density.

  11. Molecular simulations of lipid-mediated protein-protein interactions

    NARCIS (Netherlands)

    de Meyer, F.J.M.; Venturoli, M.; Smit, B.

    2008-01-01

    Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the

  12. A Review of Stapled Peptides and Small Molecules to Inhibit Protein-Protein Interactions in Cancer.

    Science.gov (United States)

    Iyer, Vidhya V

    2016-01-01

    Disruption of binding of two or more molecules to a protein surface is a common basis of inhibition of many biological activities. Smallmolecule inhibitors, antibodies, proteins, and peptidomimetics have been examined as ways to antagonize receptor activity. The peptide α-helix plays a crucial role in the function of many proteins. Hence, much effort has been invested in mimicking α-helices at the binding interface of two proteins to competitively inhibit their interactions. Peptide stapling involves choosing two amino acids on the same face of a native peptide sequence for substitution with non-native amino acids whose side chains can be "stapled" together. The focus of this review is to survey the prevalence in literature of stapled peptides and small-molecule antagonists of interactions of selected mammalian cancer targets, such as β-catenin, BH3-only members of the Bcl-2 family of proteins, eIF4E/G, estrogen receptor complexes, EZH2, Mdm2, Notch, p110α, and survivin. The increasing interest in protein targets currently considered to be "undruggable" with greater selectivity for existing targets, with the goal of overcoming the omnipresent problem of resistance, could be served well by utilizing information about protein-protein interactions to develop both small-molecule and stapled peptide inhibitors.

  13. An Interactive Introduction to Protein Structure

    Science.gov (United States)

    Lee, W. Theodore

    2004-01-01

    To improve student understanding of protein structure and the significance of noncovalent interactions in protein structure and function, students are assigned a project to write a paper complemented with computer-generated images. The assignment provides an opportunity for students to select a protein structure that is of interest and detail…

  14. Differential palmitoylation directs the AMPA receptor-binding protein ABP to spines or to intracellular clusters.

    Science.gov (United States)

    DeSouza, Sunita; Fu, Jie; States, Bradley A; Ziff, Edward B

    2002-05-01

    Long-term changes in excitatory synapse strength are thought to reflect changes in synaptic abundance of AMPA receptors mediated by receptor trafficking. AMPA receptor-binding protein (ABP) and glutamate receptor-interacting protein (GRIP) are two similar PDZ (postsynaptic density 95/Discs large/zona occludens 1) proteins that interact with glutamate receptors 2 and 3 (GluR2 and GluR3) subunits. Both proteins have proposed roles during long-term potentiation and long-term depression in the delivery and anchorage of AMPA receptors at synapses. Here we report a variant of ABP-L (seven PDZ form of ABP) called pABP-L that is palmitoylated at a cysteine residue at position 11 within a novel 18 amino acid N-terminal leader sequence encoded through differential splicing. In cultured hippocampal neurons, nonpalmitoylated ABP-L localizes with internal GluR2 pools expressed from a Sindbis virus vector, whereas pABP-L is membrane targeted and associates with surface-localized GluR2 receptors at the plasma membrane in spines. Mutation of Cys-11 to alanine blocks the palmitoylation of pABP-L and targets the protein to intracellular clusters, confirming that targeting the protein to spines is dependent on palmitoylation. Non-palmitoylated GRIP is primarily intracellular, but a chimera with the pABP-L N-terminal palmitoylation sequence linked to the body of the GRIP protein is targeted to spines. We suggest that pABP-L and ABP-L provide, respectively, synaptic and intracellular sites for the anchorage of AMPA receptors during receptor trafficking to and from the synapse.

  15. Yeast Interacting Proteins Database: YGL198W, YDR084C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YGL198W YIP4 Protein that interacts with Rab GTPases, localized to late Golgi vesicles; computational... GTPases, localized to late Golgi vesicles; computational analysis of large-scale protein-protein interactio

  16. Noninvasive imaging of protein-protein interactions in living animals

    Science.gov (United States)

    Luker, Gary D.; Sharma, Vijay; Pica, Christina M.; Dahlheimer, Julie L.; Li, Wei; Ochesky, Joseph; Ryan, Christine E.; Piwnica-Worms, Helen; Piwnica-Worms, David

    2002-05-01

    Protein-protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein-protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein-protein interactions in living animals.

  17. Yeast Interacting Proteins Database: YLR447C, YOR047C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available xpression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Sp...; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; act

  18. Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor

    Science.gov (United States)

    Pillay, Sirika; Zou, Wei; Cheng, Fang; Puschnik, Andreas S.; Meyer, Nancy L.; Ganaie, Safder S.; Deng, Xuefeng; Wosen, Jonathan E.; Davulcu, Omar; Yan, Ziying; Engelhardt, John F.; Brown, Kevin E.; Chapman, Michael S.

    2017-01-01

    ABSTRACT Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor. IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life

  19. Nuclear Receptors in Drug Metabolism, Drug Response and Drug Interactions

    OpenAIRE

    Prakash, Chandra; Zuniga, Baltazar; Song, Chung Seog; Jiang, Shoulei; Cropper, Jodie; Park, Sulgi; Chatterjee, Bandana

    2015-01-01

    Orally delivered small-molecule therapeutics are metabolized in the liver and intestine by phase I and phase II drug-metabolizing enzymes (DMEs), and transport proteins coordinate drug influx (phase 0) and drug/drug-metabolite efflux (phase III). Genes involved in drug metabolism and disposition are induced by xenobiotic-activated nuclear receptors (NRs), i.e. PXR (pregnane X receptor) and CAR (constitutive androstane receptor), and by the 1α, 25-dihydroxy vitamin D3-activated vitamin D recep...

  20. Effects of two amino acid substitutions in the capsid proteins on the interaction of two cell-adapted PanAsia-1 strains of foot-and-mouth disease virus serotype O with heparan sulfate receptor

    Science.gov (United States)

    2014-01-01

    Background Some cell-adapted strains of foot-and-mouth disease virus (FMDV) can utilize heparan sulfate (HS) as a receptor to facilitate viral infection in cultured cells. A number of independent sites on the capsid that might be involved in FMDV-HS interaction have been studied. However, the previously reported residues do not adequately explain HS-dependent infection of two cell-adapted PanAsia-1 strains (O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc) of FMDV serotype O. To identify the molecular determinant(s) for the interaction of O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc with HS receptor, several chimeric viruses and site-directed mutants were generated by using an infectious cDNA of a non-HS-utilizing rescued virus (Cathay topotype) as the genomic backbone. Phenotypic properties of these viruses were determined by plaque assays and virus adsorption and penetration assays in cultured cells. Results Only two of the rescued viruses encoding VP0 of O/Tibet/CHA/6/99tc or VP1 of O/Fujian/CHA/9/99tc formed plaques on wild-type Chinese hamster ovary (WT-CHO; HS+) cells, but not on HS-negative pgsD-677 cells. The formation of plaques by these two chimeric viruses on WT-CHO cells could be abolished by the introduction of single amino acid mutations Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc and Lys-1083 → Glu in VP1 of O/Fujian/CHA/9/99tc, respectively. Nonetheless, the introduced mutation Leu-2080 → Gln in VP2 of O/Fujian/CHA/9/99tc for the construction of expectant recombinant plasmid led to non-infectious progeny virus in baby hamster kidney 21 (BHK-21) cells, and the site-directed mutant encoding Glu-1083 → Lys in VP1 of O/Tibet/CHA/6/99tc did not acquire the ability to produce plaques on WT-CHO cells. Significant differences in the inhibition of the infectivity of four HS-utilizing viruses by heparin and RGD-containing peptide were observed in BHK-21 cells. Interestingly, the chimeric virus encoding VP0 of O/Fujian/CHA/9/99tc, and the site

  1. Protein-protein interaction based on pairwise similarity

    Directory of Open Access Journals (Sweden)

    Zaki Nazar

    2009-05-01

    Full Text Available Abstract Background Protein-protein interaction (PPI is essential to most biological processes. Abnormal interactions may have implications in a number of neurological syndromes. Given that the association and dissociation of protein molecules is crucial, computational tools capable of effectively identifying PPI are desirable. In this paper, we propose a simple yet effective method to detect PPI based on pairwise similarity and using only the primary structure of the protein. The PPI based on Pairwise Similarity (PPI-PS method consists of a representation of each protein sequence by a vector of pairwise similarities against large subsequences of amino acids created by a shifting window which passes over concatenated protein training sequences. Each coordinate of this vector is typically the E-value of the Smith-Waterman score. These vectors are then used to compute the kernel matrix which will be exploited in conjunction with support vector machines. Results To assess the ability of the proposed method to recognize the difference between "interacted" and "non-interacted" proteins pairs, we applied it on different datasets from the available yeast saccharomyces cerevisiae protein interaction. The proposed method achieved reasonable improvement over the existing state-of-the-art methods for PPI prediction. Conclusion Pairwise similarity score provides a relevant measure of similarity between protein sequences. This similarity incorporates biological knowledge about proteins and it is extremely powerful when combined with support vector machine to predict PPI.

  2. Yeast Interacting Proteins Database: YGR239C, YDR142C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available PEX21 Peroxin required for targeting of peroxisomal matrix proteins containing PTS2; interacts with Pex7p;...N-terminal nonapeptide signal (PTS2) of peroxisomal matrix proteins; WD repeat protein; defects in human homolog...description Peroxin required for targeting of peroxisomal matrix proteins containing PTS2; interacts with Pex7p;...N-terminal nonapeptide signal (PTS2) of peroxisomal matrix proteins; WD repeat protein; defects in human homolog

  3. Evolutionarily conserved herpesviral protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Even Fossum

    2009-09-01

    Full Text Available Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV and Kaposi's sarcoma-associated herpesvirus (KSHV. In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1, murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H, and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species.

  4. Interactions between Intracellular Domains as Key Determinants of the Quaternary Structure and Function of Receptor Heteromers*

    Science.gov (United States)

    Navarro, Gemma; Ferré, Sergi; Cordomi, Arnau; Moreno, Estefania; Mallol, Josefa; Casadó, Vicent; Cortés, Antoni; Hoffmann, Hanne; Ortiz, Jordi; Canela, Enric I.; Lluís, Carme; Pardo, Leonardo; Franco, Rafael; Woods, Amina S.

    2010-01-01

    G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In this study, using resonance energy transfer techniques in experiments with mutated receptors, we provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A2A, cannabinoid CB1, and dopamine D2 receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A2A, CB1, and D2) was found to include two evolutionarily conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB1 receptor with A2A and D2 receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A2A-CB1-D2 receptor heteromers. Analysis of MAPK signaling in striatal slices of CB1 receptor KO mice and wild-type littermates supported the existence of A1-CB1-D2 receptor heteromer in the brain. These findings allowed us to propose the first molecular model of the quaternary structure of a receptor heteromultimer. PMID:20562103

  5. Integrative computational modeling of protein interactions

    NARCIS (Netherlands)

    Garcia Lopes Maia Rodrigues, João; Bonvin, Alexandre M J J

    2014-01-01

    Protein interactions define the homeostatic state of the cell. Our ability to understand these interactions and their role in both health and disease is tied to our knowledge of the 3D atomic structure of the interacting partners and their complexes. Despite advances in experimental method of

  6. Soluble Extracellular Domain of Death Receptor 5 Inhibits TRAIL-Induced Apoptosis by Disrupting Receptor-Receptor Interactions.

    Science.gov (United States)

    Vunnam, Nagamani; Lo, Chih Hung; Grant, Benjamin D; Thomas, David D; Sachs, Jonathan N

    2017-09-15

    Dysregulation of tumor necrosis factor (TNF) receptor signaling is a key feature of various inflammatory disorders. Current treatments for TNF-related diseases function either by sequestering ligand or blocking ligand-receptor interactions, which can cause dangerous side effects by inhibiting the receptors that are not involved in the disease condition. Thus, alternate strategies that target receptor-receptor interactions are needed. We hypothesized that the soluble extracellular domain (ECD) of long isoform of death receptor 5 (DR5) could block endogenous receptor assembly, mimicking the biological effect of decoy receptors that lack the death domain to trigger apoptosis. Using live-cell fluorescence resonance energy transfer studies, we demonstrated that soluble ECD disrupts endogenous DR5-DR5 interactions. Cell viability assays were used to demonstrate the complete inhibition of TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by the ECD, although TRAIL is still able to bind to the receptor. Importantly, we used mutagenesis to prove that the inhibition of TRAIL-induced apoptosis by the ECD predominantly comes from the disruption of DR5 oligomerization and not ligand sequestration. Inhibition of death receptor activation should have important therapeutic applications in diseases such as nonalcoholic fatty liver disease. More generally, this approach should be generalized to enable the inhibition of other TNF receptor signaling mechanisms that are associated in a wide range of clinical conditions. Copyright © 2017. Published by Elsevier Ltd.

  7. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions.

    Directory of Open Access Journals (Sweden)

    Jessica B Hostetler

    2015-12-01

    Full Text Available A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC, and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion.We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further suggesting that the proteins

  8. Protein-protein interaction predictions using text mining methods.

    Science.gov (United States)

    Papanikolaou, Nikolas; Pavlopoulos, Georgios A; Theodosiou, Theodosios; Iliopoulos, Ioannis

    2015-03-01

    It is beyond any doubt that proteins and their interactions play an essential role in most complex biological processes. The understanding of their function individually, but also in the form of protein complexes is of a great importance. Nowadays, despite the plethora of various high-throughput experimental approaches for detecting protein-protein interactions, many computational methods aiming to predict new interactions have appeared and gained interest. In this review, we focus on text-mining based computational methodologies, aiming to extract information for proteins and their interactions from public repositories such as literature and various biological databases. We discuss their strengths, their weaknesses and how they complement existing experimental techniques by simultaneously commenting on the biological databases which hold such information and the benchmark datasets that can be used for evaluating new tools. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. REEPs are membrane shaping adapter proteins that modulate specific g protein-coupled receptor trafficking by affecting ER cargo capacity.

    Directory of Open Access Journals (Sweden)

    Susann Björk

    Full Text Available Receptor expression enhancing proteins (REEPs were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs, specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6 and model GPCRs (α2A and α2C adrenergic receptors, we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31 lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo

  10. REEPs are membrane shaping adapter proteins that modulate specific g protein-coupled receptor trafficking by affecting ER cargo capacity.

    Science.gov (United States)

    Björk, Susann; Hurt, Carl M; Ho, Vincent K; Angelotti, Timothy

    2013-01-01

    Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins

  11. Mapping Protein-Protein Interactions by Quantitative Proteomics

    DEFF Research Database (Denmark)

    Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy

    2010-01-01

    Proteins exert their function inside a cell generally in multiprotein complexes. These complexes are highly dynamic structures changing their composition over time and cell state. The same protein may thereby fulfill different functions depending on its binding partners. Quantitative mass...... spectrometry (MS)-based proteomics in combination with affinity purification protocols has become the method of choice to map and track the dynamic changes in protein-protein interactions, including the ones occurring during cellular signaling events. Different quantitative MS strategies have been used...... to characterize protein interaction networks. In this chapter we describe in detail the use of stable isotope labeling by amino acids in cell culture (SILAC) for the quantitative analysis of stimulus-dependent dynamic protein interactions....

  12. Interaction between plate make and protein in protein crystallisation screening.

    Directory of Open Access Journals (Sweden)

    Gordon J King

    Full Text Available BACKGROUND: Protein crystallisation screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate. METHODOLOGY/PRINCIPAL FINDINGS: We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised. CONCLUSIONS/SIGNIFICANCE: Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallize, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make.

  13. Differences in the interaction of acetylcholine receptor antibodies with receptor from normal, denervated and myasthenic human muscle.

    OpenAIRE

    Lefvert, A. K.

    1982-01-01

    The interaction of acetylcholine receptor antibodies with different kinds of human skeletal muscle receptor was investigated. The reaction of most receptor antibodies was strongest with receptor from a patient with myasthenia gravis and with receptor from denervated muscle. Results obtained with these receptors were well correlated. The binding of most receptor antibodies to receptor from functionally normal muscle was much weaker and also qualitatively different. In a few patients with moder...

  14. Neuronal low-density lipoprotein receptor-related protein 1 binds and endocytoses prion fibrils via receptor cluster 4

    DEFF Research Database (Denmark)

    Jen, Angela; Parkyn, Celia J; Mootoosamy, Roy C

    2010-01-01

    For infectious prion protein (designated PrP(Sc)) to act as a template to convert normal cellular protein (PrP(C)) to its distinctive pathogenic conformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrP(C) is the low-density lipop......For infectious prion protein (designated PrP(Sc)) to act as a template to convert normal cellular protein (PrP(C)) to its distinctive pathogenic conformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrP(C) is the low...... clusters 2 and 4, PrP(C) and PrP(Sc) fibrils bind only to receptor cluster 4. PrP(Sc) fibrils out-compete PrP(C) for internalization. When endocytosed, PrP(Sc) fibrils are routed to lysosomes, rather than recycled to the cell surface with PrP(C). Thus, although LRP1 binds both forms of PrP, it traffics...

  15. G protein-coupled receptor kinase 2 promotes cardiac hypertrophy

    Science.gov (United States)

    Tscheschner, Henrike; Gao, Erhe; Schumacher, Sarah M.; Yuan, Ancai; Backs, Johannes; Most, Patrick; Wieland, Thomas; Koch, Walter J.; Katus, Hugo A.; Raake, Philip W.

    2017-01-01

    The increase in protein activity and upregulation of G-protein coupled receptor kinase 2 (GRK2) is a hallmark of cardiac stress and heart failure. Inhibition of GRK2 improved cardiac function and survival and diminished cardiac remodeling in various animal heart failure models. The aim of the present study was to investigate the effects of GRK2 on cardiac hypertrophy and dissect potential molecular mechanisms. In mice we observed increased GRK2 mRNA and protein levels following transverse aortic constriction (TAC). Conditional GRK2 knockout mice showed attenuated hypertrophic response with preserved ventricular geometry 6 weeks after TAC operation compared to wild-type animals. In isolated neonatal rat ventricular cardiac myocytes stimulation with angiotensin II and phenylephrine enhanced GRK2 expression leading to enhanced signaling via protein kinase B (PKB or Akt), consecutively inhibiting glycogen synthase kinase 3 beta (GSK3β), such promoting nuclear accumulation and activation of nuclear factor of activated T-cells (NFAT). Cardiac myocyte hypertrophy induced by in vitro GRK2 overexpression increased the cytosolic interaction of GRK2 and phosphoinositide 3-kinase γ (PI3Kγ). Moreover, inhibition of PI3Kγ as well as GRK2 knock down prevented Akt activation resulting in halted NFAT activity and reduced cardiac myocyte hypertrophy. Our data show that enhanced GRK2 expression triggers cardiac hypertrophy by GRK2-PI3Kγ mediated Akt phosphorylation and subsequent inactivation of GSK3β, resulting in enhanced NFAT activity. PMID:28759639

  16. Yeast Interacting Proteins Database: YOR124C, YGR268C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available that of Type I J-proteins; computational analysis of large-scale protein-protein interaction data suggests a...plasmic protein containing a zinc finger domain with sequence similarity to that of Type I J-proteins; computational

  17. Characterization of protein-protein interactions by isothermal titration calorimetry.

    Science.gov (United States)

    Velazquez-Campoy, Adrian; Leavitt, Stephanie A; Freire, Ernesto

    2004-01-01

    Isothermal titration calorimetry (ITC) is a powerful technique to study both protein-ligand and protein-protein interactions. This methods chapter is devoted to describing protein-protein interactions, in particular, the association between two different proteins and the self-association of a protein into homodimers. ITC is the only technique that determines directly the thermodynamic parameters of a given reaction: DeltaG, DeltaH, DeltaS, and DeltaCP. Isothermal titration calorimeters have evolved over the years and one of the latest models is the VP-ITC produced by Microcal, Inc. In this chapter we will be describing the general procedure for performing an ITC experiment as well as for the specific cases of porcine pancreatic trypsin binding to soybean trypsin inhibitor and the dissociation of bovine pancreatic alpha-chymotrypsin.

  18. Yeast Interacting Proteins Database: YGL161C, YGL198W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YGL161C YIP5 Protein that interacts with Rab GTPases, localized to late Golgi vesicles; computational...that interacts with Rab GTPases, localized to late Golgi vesicles; computational ...eracts with Rab GTPases, localized to late Golgi vesicles; computational analysis of large-scale protein-pro...ized to late Golgi vesicles; computational analysis of large-scale protein-protein interaction data suggests

  19. Yeast Interacting Proteins Database: YGL198W, YGL161C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YGL198W YIP4 Protein that interacts with Rab GTPases, localized to late Golgi vesicles; computational...that interacts with Rab GTPases, localized to late Golgi vesicles; computational ...eracts with Rab GTPases, localized to late Golgi vesicles; computational analysis of large-scale protein-pro...ized to late Golgi vesicles; computational analysis of large-scale protein-protein interaction data suggests

  20. Data management of protein interaction networks

    CERN Document Server

    Cannataro, Mario

    2012-01-01

    Interactomics: a complete survey from data generation to knowledge extraction With the increasing use of high-throughput experimental assays, more and more protein interaction databases are becoming available. As a result, computational analysis of protein-to-protein interaction (PPI) data and networks, now known as interactomics, has become an essential tool to determine functionally associated proteins. From wet lab technologies to data management to knowledge extraction, this timely book guides readers through the new science of interactomics, giving them the tools needed to: Generate

  1. Receptor-interacting protein 2 (RIP2) gene polymorphisms are associated with increased risk of subclinical atherosclerosis and clinical and metabolic parameters. The Genetics of Atherosclerotic Disease (GEA) Mexican study.

    Science.gov (United States)

    Posadas-Sánchez, Rosalinda; Ángeles-Martínez, Javier; Pérez-Hernández, Nonanzit; Rodríguez-Pérez, José Manuel; López-Bautista, Fabiola; Villarreal-Molina, Teresa; Fragoso, José Manuel; Posadas-Romero, Carlos; Vargas-Alarcón, Gilberto

    2017-02-01

    The receptor-interacting protein 2 (Rip2) is a serine/threonine kinase involved in multiple nuclear factor-κB (NFκB) activation pathways and is a key regulator of cellular lipid metabolism and cardiovascular disease. The aim of the present study was to evaluate the role of RIP2 gene polymorphisms as susceptibility markers for subclinical atherosclerosis (SA). Using an informatics analysis, four RIP2 gene polymorphisms with predicted functional effects (rs2293808, rs43133, rs431264, and rs16900627) were selected. The polymorphisms were genotyped in 405 individuals with SA (calcium score>0 assessed by computed tomography) and 1099 controls (calcium score=0). Clinical, anthropometric, tomographic and biochemical traits were measured. The association between the RIP2 polymorphisms and SA was evaluated using logistic regression analyses. Pair wise linkage disequilibrium (LD, D') estimations between polymorphisms and haplotype reconstruction were performed with Haploview version 4:1. Under different models adjusted by age, gender, body mass index, hypertension, diabetes mellitus, smoking habit, total cholesterol, HDL-cholesterol, LDL-cholesterol and triglyceride levels, rs43133 (OR=1.43, 95% CI: 1.05-1.94, P=0.022), and rs16900627 (OR=1.59, 95% CI: 1.00-2.54, Pdom=0.048 and OR=1.60, 95% CI: 1.05-2.54, Padd=0.028) were associated with increased risk of developing SA. Moreover, rs2293808, and rs431264 were associated with clinical or metabolic parameters in SA individuals and in healthy controls. The four polymorphisms were in high linkage disequilibrium and the GAAG haplotype was associated with increased risk of developing SA (OR=1.47, P=0.027). This study shows for the first time, that RIP2 polymorphisms are associated with increased risk of SA and with some clinical and metabolic parameters. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Computational Analysis of the CB1 Carboxyl-terminus in the Receptor-G Protein Complex

    OpenAIRE

    Shim, Joong-Youn; Khurana, Leepakshi; Kendall, Debra A.

    2016-01-01

    Despite the important role of the carboxyl-terminus (Ct) of the activated brain cannabinoid receptor one (CB1) in the regulation of G protein signaling, a structural understanding of interactions with G proteins is lacking. This is largely due to the highly flexible nature of the CB1 Ct that dynamically adapts its conformation to the presence of G proteins. In the present study, we explored how the CB1 Ct can interact with the G protein by building on our prior modeling of the CB1-Gi complex ...

  3. AAV serotypes have distinctive interactions with domains of the cellular receptor AAVR.

    Science.gov (United States)

    Pillay, Sirika; Zou, Wei; Cheng, Fang; Puschnik, Andreas S; Meyer, Nancy L; Ganaie, Safder S; Deng, Xuefeng; Wosen, Jonathan E; Davulcu, Omar; Yan, Ziying; Engelhardt, John F; Brown, Kevin E; Chapman, Michael S; Qiu, Jianming; Carette, Jan E

    2017-07-05

    Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and proteinaceous receptor(s). Adeno-associated virus receptor (AAVR; also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes including the evolutionary distant serotypes, AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. Using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a 150-kDa molecular mass. By establishing a purification procedure, performing further protein separation through two-dimensional electrophoresis and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is N-linked glycosylated, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and viral overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like PKD repeat domain (PKD2) present in the ectodomain of AAVR. By contrast, AAV5 interacts primarily through the first, most membrane distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes including AAV1 and 8 require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor.IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. Yet, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors

  4. Van der Waals Interactions Involving Proteins

    Science.gov (United States)

    Roth, Charles M.; Neal, Brian L.; Lenhoff, Abraham M.

    1996-01-01

    Van der Waals (dispersion) forces contribute to interactions of proteins with other molecules or with surfaces, but because of the structural complexity of protein molecules, the magnitude of these effects is usually estimated based on idealized models of the molecular geometry, e.g., spheres or spheroids. The calculations reported here seek to account for both the geometric irregularity of protein molecules and the material properties of the interacting media. Whereas the latter are found to fall in the generally accepted range, the molecular shape is shown to cause the magnitudes of the interactions to differ significantly from those calculated using idealized models. with important consequences. First, the roughness of the molecular surface leads to much lower average interaction energies for both protein-protein and protein-surface cases relative to calculations in which the protein molecule is approximated as a sphere. These results indicate that a form of steric stabilization may be an important effect in protein solutions. Underlying this behavior is appreciable orientational dependence, one reflection of which is that molecules of complementary shape are found to exhibit very strong attractive dispersion interactions. Although this has been widely discussed previously in the context of molecular recognition processes, the broader implications of these phenomena may also be important at larger molecular separations, e.g., in the dynamics of aggregation, precipitation, and crystal growth.

  5. Protein-Protein Interaction Detection: Methods and Analysis

    Directory of Open Access Journals (Sweden)

    V. Srinivasa Rao

    2014-01-01

    Full Text Available Protein-protein interaction plays key role in predicting the protein function of target protein and drug ability of molecules. The majority of genes and proteins realize resulting phenotype functions as a set of interactions. The in vitro and in vivo methods like affinity purification, Y2H (yeast 2 hybrid, TAP (tandem affinity purification, and so forth have their own limitations like cost, time, and so forth, and the resultant data sets are noisy and have more false positives to annotate the function of drug molecules. Thus, in silico methods which include sequence-based approaches, structure-based approaches, chromosome proximity, gene fusion, in silico 2 hybrid, phylogenetic tree, phylogenetic profile, and gene expression-based approaches were developed. Elucidation of protein interaction networks also contributes greatly to the analysis of signal transduction pathways. Recent developments have also led to the construction of networks having all the protein-protein interactions using computational methods for signaling pathways and protein complex identification in specific diseases.

  6. Essential domain of receptor tyrosine phosphatase beta (RPTPbeta) for interaction with Helicobacter pylori vacuolating cytotoxin

    DEFF Research Database (Denmark)

    Yahiro, Kinnosuke; Wada, Akihiro; Yamasaki, Eiki

    2004-01-01

    Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, RPTPbeta and RPTPalpha, RPTPbeta was found to be responsible for gastric damage caused...

  7. A simple dependence between protein evolution rate and the number of protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Hirsh Aaron E

    2003-05-01

    Full Text Available Abstract Background It has been shown for an evolutionarily distant genomic comparison that the number of protein-protein interactions a protein has correlates negatively with their rates of evolution. However, the generality of this observation has recently been challenged. Here we examine the problem using protein-protein interaction data from the yeast Saccharomyces cerevisiae and genome sequences from two other yeast species. Results In contrast to a previous study that used an incomplete set of protein-protein interactions, we observed a highly significant correlation between number of interactions and evolutionary distance to either Candida albicans or Schizosaccharomyces pombe. This study differs from the previous one in that it includes all known protein interactions from S. cerevisiae, and a larger set of protein evolutionary rates. In both evolutionary comparisons, a simple monotonic relationship was found across the entire range of the number of protein-protein interactions. In agreement with our earlier findings, this relationship cannot be explained by the fact that proteins with many interactions tend to be important to yeast. The generality of these correlations in other kingdoms of life unfortunately cannot be addressed at this time, due to the incompleteness of protein-protein interaction data from organisms other than S. cerevisiae. Conclusions Protein-protein interactions tend to slow the rate at which proteins evolve. This may be due to structural constraints that must be met to maintain interactions, but more work is needed to definitively establish the mechanism(s behind the correlations we have observed.

  8. Yeast Interacting Proteins Database: YMR047C, YOR112W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available repetitive GLFG motif that interacts with mRNA export factor Mex67p and with karyopherin Kap95p; homologous...tRNA export pathway; interacts with nuclear pore component Nup116p; copurifies with tRNA export receptors...repetitive GLFG motif that interacts with mRNA export factor Mex67p and with karyopherin Kap95p; homologous...tRNA export pathway; interacts with nuclear pore component Nup116p; copurifies with tRNA export receptors

  9. Molecular principles of human virus protein-protein interactions.

    Science.gov (United States)

    Halehalli, Rachita Ramachandra; Nagarajaram, Hampapathalu Adimurthy

    2015-04-01

    Viruses, from the human protein-protein interaction network perspective, target hubs, bottlenecks and interconnected nodes enriched in certain biological pathways. However, not much is known about the general characteristic features of the human proteins interacting with viral proteins (referred to as hVIPs) as well as the motifs and domains utilized by human-virus protein-protein interactions (referred to as Hu-Vir PPIs). Our study has revealed that hVIPs are mostly disordered proteins, whereas viral proteins are mostly ordered proteins. Protein disorder in viral proteins and hVIPs varies from one subcellular location to another. In any given viral-human PPI pair, at least one of the two proteins is structurally disordered suggesting that disorder associated conformational flexibility as one of the characteristic features of virus-host interaction. Further analyses reveal that hVIPs are (i) slowly evolving proteins, (ii) associated with high centrality scores in human-PPI network, (iii) involved in multiple pathways, (iv) enriched in eukaryotic linear motifs (ELMs) associated with protein modification, degradation and regulatory processes, (v) associated with high number of splice variants and (vi) expressed abundantly across multiple tissues. These aforementioned findings suggest that conformational flexibility, spatial diversity, abundance and slow evolution are the characteristic features of the human proteins targeted by viral proteins. Hu-Vir PPIs are mostly mediated via domain-motif interactions (DMIs) where viral proteins employ motifs that mimic host ELMs to bind to domains in human proteins. DMIs are shared among viruses belonging to different families indicating a possible convergent evolution of these motifs to help viruses to adopt common strategies to subvert host cellular pathways. Hu-Vir PPI data, DDI and DMI data for human-virus PPI can be downloaded from http://cdfd.org.in/labpages/computational_biology_datasets.html. Supplementary data are

  10. Yeast Interacting Proteins Database: YGL237C, YOR047C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ene expression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding prote... expression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein

  11. Non-interacting surface solvation and dynamics in protein-protein interactions

    NARCIS (Netherlands)

    Visscher, Koen M.; Kastritis, Panagiotis L.|info:eu-repo/dai/nl/315886668; Bonvin, Alexandre M J J|info:eu-repo/dai/nl/113691238

    2015-01-01

    Protein-protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure-based drug design methods, as well as design of de novo

  12. Yeast Interacting Proteins Database: YKL002W, YOR047C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ene expression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding prote...xpression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Sp

  13. Duchenne Muscular Dystrophy (DMD) Protein-Protein Interaction Mapping.

    Science.gov (United States)

    Rezaei Tavirani, Mostafa; OkHOVATIAN, Farshad; Zamanian Azodi, Mona; Rezaei Tavirani, Majid

    2017-01-01

    Duchenne muscular dystrophy (DMD) is one of the mortal diseases, subjected to study in terms of molecular investigation. In this study, the protein interaction map of this muscle-wasting condition was generated to gain a better knowledge of interactome profile of DMD. Applying Cytoscape and String Database, the protein-protein interaction network was constructed and the gene ontology of the constructed network was analyzed for biological process, molecular function, and cellular component annotations. Among 100 proteins related to DMD, dystrophin, utrophin, caveolin 3, and myogenic differentiation 1 play key roles in DMD network. In addition, the gene ontology analysis showed that regulation processes, kinase activity, and sarcoplasmic reticulum were the highlighted biological processes, molecular function, and cell component enrichments respectively for the proteins related to DMD. The central proteins and the enriched ontologies can be suggested as possible prominent agents in DMD; however, the validation studies may be required.

  14. On the role of electrostatics on protein-protein interactions

    Science.gov (United States)

    Zhang, Zhe; Witham, Shawn; Alexov, Emil

    2011-01-01

    The role of electrostatics on protein-protein interactions and binding is reviewed in this article. A brief outline of the computational modeling, in the framework of continuum electrostatics, is presented and basic electrostatic effects occurring upon the formation of the complex are discussed. The role of the salt concentration and pH of the water phase on protein-protein binding free energy is demonstrated and indicates that the increase of the salt concentration tends to weaken the binding, an observation that is attributed to the optimization of the charge-charge interactions across the interface. It is pointed out that the pH-optimum (pH of optimal binding affinity) varies among the protein-protein complexes, and perhaps is a result of their adaptation to particular subcellular compartment. At the end, the similarities and differences between hetero- and homo-complexes are outlined and discussed with respect to the binding mode and charge complementarity. PMID:21572182

  15. Iterative cluster analysis of protein interaction data.

    Science.gov (United States)

    Arnau, Vicente; Mars, Sergio; Marín, Ignacio

    2005-02-01

    Generation of fast tools of hierarchical clustering to be applied when distances among elements of a set are constrained, causing frequent distance ties, as happens in protein interaction data. We present in this work the program UVCLUSTER, that iteratively explores distance datasets using hierarchical clustering. Once the user selects a group of proteins, UVCLUSTER converts the set of primary distances among them (i.e. the minimum number of steps, or interactions, required to connect two proteins) into secondary distances that measure the strength of the connection between each pair of proteins when the interactions for all the proteins in the group are considered. We show that this novel strategy has advantages over conventional clustering methods to explore protein-protein interaction data. UVCLUSTER easily incorporates the information of the largest available interaction datasets to generate comprehensive primary distance tables. The versatility, simplicity of use and high speed of UVCLUSTER on standard personal computers suggest that it can be a benchmark analytical tool for interactome data analysis. The program is available upon request from the authors, free for academic users. Additional information available at http://www.uv.es/genomica/UVCLUSTER.

  16. Website on Protein Interaction and Protein Structure Related Work

    Science.gov (United States)

    Samanta, Manoj; Liang, Shoudan; Biegel, Bryan (Technical Monitor)

    2003-01-01

    In today's world, three seemingly diverse fields - computer information technology, nanotechnology and biotechnology are joining forces to enlarge our scientific knowledge and solve complex technological problems. Our group is dedicated to conduct theoretical research exploring the challenges in this area. The major areas of research include: 1) Yeast Protein Interactions; 2) Protein Structures; and 3) Current Transport through Small Molecules.

  17. Eukaryotic LYR Proteins Interact with Mitochondrial Protein Complexes

    Directory of Open Access Journals (Sweden)

    Heike Angerer

    2015-02-01

    Full Text Available In eukaryotic cells, mitochondria host ancient essential bioenergetic and biosynthetic pathways. LYR (leucine/tyrosine/arginine motif proteins (LYRMs of the Complex1_LYR-like superfamily interact with protein complexes of bacterial origin. Many LYR proteins function as extra subunits (LYRM3 and LYRM6 or novel assembly factors (LYRM7, LYRM8, ACN9 and FMC1 of the oxidative phosphorylation (OXPHOS core complexes. Structural insights into complex I accessory subunits LYRM6 and LYRM3 have been provided by analyses of EM and X-ray structures of complex I from bovine and the yeast Yarrowia lipolytica, respectively. Combined structural and biochemical studies revealed that LYRM6 resides at the matrix arm close to the ubiquinone reduction site. For LYRM3, a position at the distal proton-pumping membrane arm facing the matrix space is suggested. Both LYRMs are supposed to anchor an acyl-carrier protein (ACPM independently to complex I. The function of this duplicated protein interaction of ACPM with respiratory complex I is still unknown. Analysis of protein-protein interaction screens, genetic analyses and predicted multi-domain LYRMs offer further clues on an interaction network and adaptor-like function of LYR proteins in mitochondria.

  18. Protein-Protein Interactions (PPI) reagents: | Office of Cancer Genomics

    Science.gov (United States)

    The CTD2 Center at Emory University has a library of genes used to study protein-protein interactions in mammalian cells. These genes are cloned in different mammalian expression vectors. A list of available cancer-associated genes can be accessed below.

  19. Protein-Protein Interaction Reagents | Office of Cancer Genomics

    Science.gov (United States)

    The CTD2 Center at Emory University has a library of genes used to study protein-protein interactions in mammalian cells. These genes are cloned in different mammalian expression vectors. A list of available cancer-associated genes can be accessed below. Emory_CTD^2_PPI_Reagents.xlsx Contact: Haian Fu

  20. New factors influencing G protein coupled receptors' system functions

    African Journals Online (AJOL)

    New factors such as the G protein coupled receptor (GPCR) surrounding's chemical environment, cell membrane constituents, the existent gap junction, endogenous receptor affinity status and animal species have been shown to influence the GPCR physiology and variations of those factors can modify the functions of the ...

  1. Yeast Interacting Proteins Database: YOR158W, YLR423C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YOR158W PET123 Mitochondrial ribosomal protein of the small subunit; PET123 exhibits genetic interactions...23 Bait description Mitochondrial ribosomal protein of the small subunit; PET123 exhibits genetic interact...ions with PET122, which encodes a COX3 mRNA-specific translational activator Rows w

  2. Bidirectional allosteric interactions between cannabinoid receptor 1 (CB1) and dopamine receptor 2 long (D2L) heterotetramers.

    Science.gov (United States)

    Bagher, Amina M; Laprairie, Robert B; Toguri, J Thomas; Kelly, Melanie E M; Denovan-Wright, Eileen M

    2017-10-15

    Type 1 cannabinoid (CB1) and dopamine 2 long form (D2L) receptors can physically interact to form heteromers that display unique pharmacology in vitro compared to homomeric complexes. Co-expression of CB1 and D2L and co-application of CB1 and D2 agonists increases cAMP levels while administration of either agonist alone decreases cAMP levels. To understand the observed co-agonist response, our first goal of the current study was to define the stoichiometry of CB1/D2L/Gα protein complexes. Using bioluminescence resonance energy transfer 2 (BRET(2)), we confirmed that, CB1 homodimers, D2L homodimers, and CB1/D2L heteromers are formed. By using sequential resonance energy transfer 2 (SRET(2)) combined with bimolecular fluorescence complementation (BiFC), we were able to demonstrate that CB1/D2L form heterotetramers consisting of CB1 and D2L homodimers. We demonstrated that CB1/D2L heterotetramers are coupled to at least two Gα proteins. The second aim of the study was to investigate allosteric effects of a D2L agonist (quinpirole) on CB1 receptor function and to investigate the effects of a CB1 agonist [arachidonyl-2-chloroethylamide (ACEA)] on D2L receptor function within CB1/D2L heterotetramers. Treating cells co-expressing CB1 and D2L with both ACEA and quinpirole switched CB1 and D2L receptor coupling and signaling from Gαi to Gαs proteins, enhanced β-arrestin1 recruitment and receptor co-internalization. The concept of bidirectional allosteric interaction within CB1/D2 heterotetramers has important implications for understanding the activity of receptor complexes in native tissues and under pathological conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Concentration dependent model of protein-protein interaction networks

    CERN Document Server

    Zhang, Jingshan

    2007-01-01

    The scale free structure p(k)~k^{-gamma} of protein-protein interaction networks can be produced by a static physical model. We find the earlier study of deterministic threshold models with exponential fitness distributions can be generalized to explain the apparent scale free degree distribution of the physical model, and this explanation provides a generic mechanism of "scale free" networks. We predict the dependence of gamma on experimental protein concentrations. The clustering coefficient distribution of the model is also studied.

  4. HCVpro: Hepatitis C virus protein interaction database

    KAUST Repository

    Kwofie, Samuel K.

    2011-12-01

    It is essential to catalog characterized hepatitis C virus (HCV) protein-protein interaction (PPI) data and the associated plethora of vital functional information to augment the search for therapies, vaccines and diagnostic biomarkers. In furtherance of these goals, we have developed the hepatitis C virus protein interaction database (HCVpro) by integrating manually verified hepatitis C virus-virus and virus-human protein interactions curated from literature and databases. HCVpro is a comprehensive and integrated HCV-specific knowledgebase housing consolidated information on PPIs, functional genomics and molecular data obtained from a variety of virus databases (VirHostNet, VirusMint, HCVdb and euHCVdb), and from BIND and other relevant biology repositories. HCVpro is further populated with information on hepatocellular carcinoma (HCC) related genes that are mapped onto their encoded cellular proteins. Incorporated proteins have been mapped onto Gene Ontologies, canonical pathways, Online Mendelian Inheritance in Man (OMIM) and extensively cross-referenced to other essential annotations. The database is enriched with exhaustive reviews on structure and functions of HCV proteins, current state of drug and vaccine development and links to recommended journal articles. Users can query the database using specific protein identifiers (IDs), chromosomal locations of a gene, interaction detection methods, indexed PubMed sources as well as HCVpro, BIND and VirusMint IDs. The use of HCVpro is free and the resource can be accessed via http://apps.sanbi.ac.za/hcvpro/ or http://cbrc.kaust.edu.sa/hcvpro/. © 2011 Elsevier B.V.

  5. Length, protein–protein interactions, and complexity

    NARCIS (Netherlands)

    Tan, T.; Frenkel, D.; Gupta, V.; Deem, M.W.

    2005-01-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein–protein interactions has driven the selection of longer proteins, as they

  6. Cell-based, bioluminescent assay for monitoring the interaction between PCSK9 and the LDL receptor.

    Science.gov (United States)

    Duellman, Sarah J; Machleidt, Thomas; Cali, James J; Vidugiriene, Jolanta

    2017-08-01

    Monitoring the expression of cell-surface receptors, their interaction with extracellular ligands, and their fate upon ligand binding is important for understanding receptor function and developing new therapies. We describe a cell-based method that utilizes bioluminescent protein complementation technology to interrogate binding of a cellular receptor with its extracellular protein ligand, specifically LDL receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9). Purified, full-length tagged PCSK9 is added to assay wells containing cells that stably express LDLR with an extracellular complementary tag. When the tagged PCSK9 binds the receptor, a bright luminescence signal is generated. The interaction is detected at the cell membrane with add-and-read simplicity, no wash steps, and flexibility, allowing data to be collected in endpoint format, kinetically, or with bioluminescent imaging. The assay is flexible, is rapid, and reports accurate biology. It is amenable to 96-well and 384-well formats, and the robustness allows for screening of new drug candidates (Z' = 0.83). The assay reports correct potencies for antibody titrations across a 50%-150% potency range and detects potency changes due to heat stress, suggesting that it may be useful during drug development. This assay technology can be broadly applied when studying other receptors with their extracellular ligands, whether protein or small-molecule binding partners. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  7. Stabilization of the angiotensin-(1-7) receptor Mas through interaction with PSD95.

    Science.gov (United States)

    Bian, Weihua; Sun, Licui; Yang, Longyan; Li, Ji-Feng; Hu, Jia; Zheng, Shuai; Guo, Ruihan; Feng, Duiping; Ma, Qian; Shi, Xiaocui; Xiong, Ying; Yang, Xiaomei; Song, Ran; Xu, Jianguo; Wang, Songlin; He, Junqi

    2013-08-01

    The functions and signalling mechanisms of the Ang-(1-7) [angiotensin-(1-7)] receptor Mas have been studied extensively. However, less attention has been paid to the intracellular regulation of Mas protein. In the present study, PSD95 (postsynaptic density 95), a novel binding protein of Mas receptor, was identified, and their association was characterized further. Mas specifically interacts with PDZ1-2, but not the PDZ3, domain of PSD95 via Mas-CT (Mas C-terminus), and the last four amino acids [ETVV (Glu-Thr-Val-Val)] of Mas-CT were determined to be essential for this interaction, as shown by GST pull-down, co-immunoprecipitation and confocal co-localization experiments. Gain-of-function and loss-of-function studies indicated that PSD95 enhanced Mas protein expression by increasing the stabilization of the receptor. Mas degradation was robustly inhibited by the proteasome inhibitor MG132 in time- and dose-dependent manners, and the expression of PSD95 impaired Mas ubiquitination, indicating that the PSD95-Mas association inhibits Mas receptor degradation via the ubiquitin-proteasome proteolytic pathway. These findings reveal a novel mechanism of Mas receptor regulation by which its expression is modulated at the post-translational level by ubiquitination, and clarify the role of PSD95, which binds directly to Mas, blocking the ubiquitination and subsequent degradation of the receptor via the ubiquitin-proteasome proteolytic pathway.

  8. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

    Science.gov (United States)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

    2015-11-12

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

  9. Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation.

    Science.gov (United States)

    Penela, P

    2016-01-01

    G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Synergistic Regulation of Coregulator/Nuclear Receptor Interaction by Ligand and DNA.

    Science.gov (United States)

    de Vera, Ian Mitchelle S; Zheng, Jie; Novick, Scott; Shang, Jinsai; Hughes, Travis S; Brust, Richard; Munoz-Tello, Paola; Gardner, William J; Marciano, David P; Kong, Xiangming; Griffin, Patrick R; Kojetin, Douglas J

    2017-10-03

    Nuclear receptor (NR) transcription factors bind various coreceptors, small-molecule ligands, DNA response element sequences, and transcriptional coregulator proteins to affect gene transcription. Small-molecule ligands and DNA are known to influence receptor structure, coregulator protein interaction, and function; however, little is known on the mechanism of synergy between ligand and DNA. Using quantitative biochemical, biophysical, and solution structural methods, including 13C-detected nuclear magnetic resonance and hydrogen/deuterium exchange (HDX) mass spectrometry, we show that ligand and DNA cooperatively recruit the intrinsically disordered steroid receptor coactivator-2 (SRC-2/TIF2/GRIP1/NCoA-2) receptor interaction domain to peroxisome proliferator-activated receptor gamma-retinoid X receptor alpha (PPARγ-RXRα) heterodimer and reveal the binding determinants of the complex. Our data reveal a thermodynamic mechanism by which DNA binding propagates a conformational change in PPARγ-RXRα, stabilizes the receptor ligand binding domain dimer interface, and impacts ligand potency and cooperativity in NR coactivator recruitment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Protein complexes predictions within protein interaction networks using genetic algorithms.

    Science.gov (United States)

    Ramadan, Emad; Naef, Ahmed; Ahmed, Moataz

    2016-07-25

    Protein-protein interaction networks are receiving increased attention due to their importance in understanding life at the cellular level. A major challenge in systems biology is to understand the modular structure of such biological networks. Although clustering techniques have been proposed for clustering protein-protein interaction networks, those techniques suffer from some drawbacks. The application of earlier clustering techniques to protein-protein interaction networks in order to predict protein complexes within the networks does not yield good results due to the small-world and power-law properties of these networks. In this paper, we construct a new clustering algorithm for predicting protein complexes through the use of genetic algorithms. We design an objective function for exclusive clustering and overlapping clustering. We assess the quality of our proposed clustering algorithm using two gold-standard data sets. Our algorithm can identify protein complexes that are significantly enriched in the gold-standard data sets. Furthermore, our method surpasses three competing methods: MCL, ClusterOne, and MCODE in terms of the quality of the predicted complexes. The source code and accompanying examples are freely available at http://faculty.kfupm.edu.sa/ics/eramadan/GACluster.zip .

  12. Water-Protein Interactions: The Secret of Protein Dynamics

    Directory of Open Access Journals (Sweden)

    Silvia Martini

    2013-01-01

    Full Text Available Water-protein interactions help to maintain flexible conformation conditions which are required for multifunctional protein recognition processes. The intimate relationship between the protein surface and hydration water can be analyzed by studying experimental water properties measured in protein systems in solution. In particular, proteins in solution modify the structure and the dynamics of the bulk water at the solute-solvent interface. The ordering effects of proteins on hydration water are extended for several angstroms. In this paper we propose a method for analyzing the dynamical properties of the water molecules present in the hydration shells of proteins. The approach is based on the analysis of the effects of protein-solvent interactions on water protons NMR relaxation parameters. NMR relaxation parameters, especially the nonselective (R1NS and selective (R1SE spin-lattice relaxation rates of water protons, are useful for investigating the solvent dynamics at the macromolecule-solvent interfaces as well as the perturbation effects caused by the water-macromolecule interactions on the solvent dynamical properties. In this paper we demonstrate that Nuclear Magnetic Resonance Spectroscopy can be used to determine the dynamical contributions of proteins to the water molecules belonging to their hydration shells.

  13. Structural–Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work

    Science.gov (United States)

    Kleinau, Gunnar; Worth, Catherine L.; Kreuchwig, Annika; Biebermann, Heike; Marcinkowski, Patrick; Scheerer, Patrick; Krause, Gerd

    2017-01-01

    The thyroid-stimulating hormone receptor (TSHR) is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs). TSHR and its endogenous ligand thyrotropin (TSH) are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy) or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016) concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other class A GPCRs to

  14. Inferring domain-domain interactions from protein-protein interactions with formal concept analysis.

    Directory of Open Access Journals (Sweden)

    Susan Khor

    Full Text Available Identifying reliable domain-domain interactions will increase our ability to predict novel protein-protein interactions, to unravel interactions in protein complexes, and thus gain more information about the function and behavior of genes. One of the challenges of identifying reliable domain-domain interactions is domain promiscuity. Promiscuous domains are domains that can occur in many domain architectures and are therefore found in many proteins. This becomes a problem for a method where the score of a domain-pair is the ratio between observed and expected frequencies because the protein-protein interaction network is sparse. As such, many protein-pairs will be non-interacting and domain-pairs with promiscuous domains will be penalized. This domain promiscuity challenge to the problem of inferring reliable domain-domain interactions from protein-protein interactions has been recognized, and a number of work-arounds have been proposed. This paper reports on an application of Formal Concept Analysis to this problem. It is found that the relationship between formal concepts provides a natural way for rare domains to elevate the rank of promiscuous domain-pairs and enrich highly ranked domain-pairs with reliable domain-domain interactions. This piggybacking of promiscuous domain-pairs onto less promiscuous domain-pairs is possible only with concept lattices whose attribute-labels are not reduced and is enhanced by the presence of proteins that comprise both promiscuous and rare domains.

  15. Yeast Interacting Proteins Database: YEL017W, YEL017W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available Bait description Protein of unknown function with a possible role in glutathione metabolism, as suggested by computational...ion Protein of unknown function with a possible role in glutathione metabolism, as suggested by computational...putational analysis of large-scale protein-protein interaction data; GFP-fusion pro...tational analysis of large-scale protein-protein interaction data; GFP-fusion prote...17W GTT3 Protein of unknown function with a possible role in glutathione metabolism, as suggested by compu

  16. Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics

    Directory of Open Access Journals (Sweden)

    María S. Aymerich

    2011-01-01

    Full Text Available Understanding the trafficking of G-protein-coupled receptors (GPCRs and their regulation by agonists and antagonists is fundamental to develop more effective drugs. Optical methods using fluorescent-tagged receptors and spinning disk confocal microscopy are useful tools to investigate membrane receptor dynamics in living cells. The aim of this study was to develop a method to characterize receptor dynamics using this system which offers the advantage of very fast image acquisition with minimal cell perturbation. However, in short-term assays photobleaching was still a problem. Thus, we developed a procedure to perform a photobleaching-corrected image analysis. A study of short-term dynamics of the long isoform of the dopamine type 2 receptor revealed an agonist-induced increase in the mobile fraction of receptors with a rate of movement of 0.08 μm/s For long-term assays, the ratio between the relative fluorescence intensity at the cell surface versus that in the intracellular compartment indicated that receptor internalization only occurred in cells co-expressing G protein-coupled receptor kinase 2. These results indicate that the lateral movement of receptors and receptor internalization are not directly coupled. Thus, we believe that live imaging of GPCRs using spinning disk confocal image analysis constitutes a powerful tool to study of receptor dynamics.

  17. Interactions of Rodent Coronaviruses with Cellular Receptors

    Science.gov (United States)

    2016-05-08

    canine coronavirus, CCV), cats (feline coronavirus, FIPV and FeCV), cattle (bovine coronavirus, BCV), and rats 11 (rat coronavirus, PRCV, SDAV and...porcine transmissible gastroenteris vi rus; Cell , canine coronavi rus; FECI/ , feline enteric coronavlrus; FIPV. fel ine infectious peritonitis...different types of cells viral replication may be blocked at any stage of the virus life cycle . Therefore, cell receptors are not the only

  18. Direct Interaction of GABAB Receptors with M2 Muscarinic Receptors Enhances Muscarinic Signaling

    OpenAIRE

    Boyer, Stephanie B.; Clancy, Sinead M.; Terunuma, Miho; Revilla-Sanchez, Raquel; Thomas, Steven M.; Moss, Stephen J.; Slesinger, Paul A.

    2009-01-01

    Down-regulation of G protein coupled receptors (GPCR) provides an important mechanism for reducing neurotransmitter signaling during sustained stimulation. Chronic stimulation of M2 muscarinic receptors (M2R) causes internalization of M2R and G protein-activated inwardly rectifying potassium (GIRK) channels in neuronal PC12 cells, resulting in loss of function. Here, we show that co-expression of GABAB R2 receptors (GBR2) rescues both surface expression and function of M2R, including M2R-indu...

  19. Design and Synthesis of High-Affinity Dimeric Inhibitors Targeting the Interactions between Gephyrin and Inhibitory Neurotransmitter Receptors

    DEFF Research Database (Denmark)

    Maric, Hans-Michael; Kasaragod, Vikram Babu; Kedström, Linda Maria Haugaard

    2015-01-01

    Gephyrin is the central scaffolding protein for inhibitory neurotransmitter receptors in the brain. Here we describe the development of dimeric peptides that inhibit the interaction between gephyrin and these receptors, a process which is fundamental to numerous synaptic functions and diseases...

  20. Nuclear localization of bradykinin B(2) receptors reflects binding to the nuclear envelope protein lamin C.

    Science.gov (United States)

    Takano, Masaoki; Kanoh, Akira; Amako, Katsumi; Otani, Mieko; Sano, Keiji; Kanazawa-Hamada, Michiko; Matsuyama, Shogo

    2014-01-15

    The mechanism of action of bradykinin (BK), a pro-inflammatory mediator, is thought to be mediated by specific cell surface membrane bradykinin B2 receptors. Some evidence suggests that there are both intracellular and nuclear bradykinin B2 receptors. This study identified proteins that interact with the C-terminus of the bradykinin B2 receptor (in particular, the nuclear membrane protein lamin C), using the yeast two-hybrid system. The motif of the C-terminal domain (CT) mutant 303-320 in bradykinin B2 receptor was identified as a lamin C protein binding motif. Immunohistochemistry revealed colocalization of FLAG- bradykinin B2 receptor with HA-lamin C in the nucleus of HEK 293T cells. In situ proximity ligation assay (PLA) showed that FLAG-bradykinin B2 receptor formed heterodimers with HA-lamin C in the nucleus. In addition, live cell fluorescence imaging showed that bradykinin B2 receptor-EGFP was located in the nucleus and co-localized with HcRed-lamin C. Interestingly, neither BK addition nor bradykinin B2 receptor CT mutation reduced the binding to lamin C or changed the distribution of bradykinin B2 receptor. Taken together, these findings demonstrate that bradykinin B2 receptor-lamin C heterodimers form in the nucleus independent of BK stimulation and CT mutation. We propose that heterodimerization of bradykinin B2 receptor with lamin C is essential to nuclear localization of bradykinin B2 receptor and plays an important role in cell signaling and function. © 2013 Elsevier B.V. All rights reserved.

  1. Regulation of dopamine transporter function by protein-protein interactions: new discoveries and methodological challenges

    DEFF Research Database (Denmark)

    Eriksen, Jacob; Jørgensen, Trine Nygaard; Gether, Ulrik

    2010-01-01

    -synaptic neurons. This has led to the identification of a plethora of different kinases, receptors and scaffolding proteins that interact with DAT and hereby either modulate the catalytic activity of the transporter or regulate its trafficking and degradation. Several new tools for studying DAT regulation in live...... cells have also recently become available such as fluorescently tagged cocaine analogues and fluorescent substrates. Here we review the current knowledge about the role of protein-protein interactions in DAT regulation as well as we describe the most recent methodological developments that have been......The dopamine transporter (DAT) plays a key role in regulating dopaminergic signalling in the brain by mediating rapid clearance of dopamine from the synaptic clefts. The psychostimulatory actions of cocaine and amphetamine are primarily the result of a direct interaction of these compounds with DAT...

  2. A protein-protein interaction dictates Borrelial infectivity.

    Science.gov (United States)

    Thakur, Meghna; Sharma, Kavita; Chao, Kinlin; Smith, Alexis A; Herzberg, Osnat; Pal, Utpal

    2017-06-07

    Two Borrelia burgdorferi interacting proteins, BB0238 and BB0323, play distinct roles in pathogen biology and infectivity although a significance of their interaction remained enigmatic. Here we identified the polypeptide segment essential for BB0238-BB0323 interaction and examined how it supports spirochete infectivity. We show that the interaction region in BB0323 requires amino acid residues 22-200, suggesting that the binding encompasses discontinuous protein segments. In contrast, the interaction region in BB0238 spans only 11 amino acids, residues 120-130. A deletion of these 11 amino acids neither alters the overall secondary structure of the protein, nor affects its stability or oligomerization property, however, it reduces the post-translational stability of the binding partner, BB0323. Mutant B. burgdorferi isolates producing BB0238 lacking the 11-amino acid interaction region were able to persist in ticks but failed to transmit to mice or to establish infection. These results suggest that BB0238-BB0323 interaction is critical for post-translational stability of BB0323, and that this interaction is important for mammalian infectivity and transmission of B. burgdorferi. We show that saturation or inhibition of BB0238-BB0323 interaction could be studied in a luciferase assay, which could be amenable for future identification of small molecule inhibitors to combat B. burgdorferi infection.

  3. Effect of Interaction between Polymorphisms in Insulin Receptor ...

    African Journals Online (AJOL)

    Effect of Interaction between Polymorphisms in Insulin Receptor Substrate Genes in Type 2 Diabetes Mellitus Patients with Severe/Acute Hyperglycemia. ... Methods: Testing Haplotype EffectS in Association Studies (THESIAS) software was used to investigate allelic and haplotype interactions between the polymorphisms in ...

  4. Cellular prion protein and NMDA receptor modulation: protecting against excitotoxicity

    Directory of Open Access Journals (Sweden)

    Stefanie A.G. Black

    2014-08-01

    Full Text Available Although it is well established that misfolding of the cellular prion protein (PrPC into the beta-sheet-rich, aggregated scrapie conformation (PrPSc causes a variety of transmissible spongiform encephalopathies (TSEs, the physiological roles of PrPC are still incompletely understood. There is accumulating evidence describing the roles of PrPC in neurodegeneration and neuroinflammation. Recently, we identified a functional regulation of NMDA receptors by PrPC that involves formation of a physical protein complex between these proteins. Excessive NMDA receptor activity during conditions such as ischemia mediates enhanced Ca2+ entry into cells and contributes to excitotoxic neuronal death. In addition, NMDA receptors and/or PrPC play critical roles in neuroinflammation and glial cell toxicity. Inhibition of NMDA receptor activity protects against PrPSc-induced neuronal death. Moreover, in mice lacking PrPC, infarct size is increased after focal cerebral ischemia, and absence of PrPC increases susceptibility of neurons to NMDA receptor-dependent death. Recently, PrPC was found to be a receptor for oligomeric beta-amyloid (Abeta peptides, suggesting a role for PrPC in Alzheimer’s disease. Our recent findings suggest that Abeta peptides enhance NMDA receptor current by perturbing the normal copper- and PrPC-dependent regulation of these receptors. Here, we review evidence highlighting a role for PrPC in preventing NMDA receptor-mediated excitotoxicity and inflammation. There is a need for more detailed molecular characterization of PrPC-mediated regulation of NMDA receptors, such as determining which NMDA receptor subunits mediate pathogenic effects upon loss of PrPC-mediated regulation and identifying PrPC binding site(s on the receptor. This knowledge will allow development of novel therapeutic interventions for not only TSEs, but also for Alzheimer’s disease and other neurodegenerative disorders involving dysfunction of PrPC.

  5. Negation of protein-protein interactions: analysis and extraction.

    Science.gov (United States)

    Sanchez-Graillet, Olivia; Poesio, Massimo

    2007-07-01

    Negative information about protein-protein interactions--from uncertainty about the occurrence of an interaction to knowledge that it did not occur--is often of great use to biologists and could lead to important discoveries. Yet, to our knowledge, no proposals focusing on extracting such information have been proposed in the text mining literature. In this work, we present an analysis of the types of negative information that is reported, and a heuristic-based system using a full dependency parser to extract such information. We performed a preliminary evaluation study that shows encouraging results of our system. Finally, we have obtained an initial corpus of negative protein-protein interactions as basis for the construction of larger ones. The corpus is available by request from the authors.

  6. Bioluminescence resonance energy transfer methods to study G protein-coupled receptor-receptor tyrosine kinase heteroreceptor complexes.

    Science.gov (United States)

    Borroto-Escuela, Dasiel O; Flajolet, Marc; Agnati, Luigi F; Greengard, Paul; Fuxe, Kjell

    2013-01-01

    A large body of evidence indicates that G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) can form heteroreceptor complexes. In these complexes, the signaling from each interacting protomer is modulated to produce an integrated and therefore novel response upon agonist(s) activation. In the GPCR-RTK heteroreceptor complexes, GPCRs can activate RTK in the absence of added growth factor through the use of RTK signaling molecules. This integrative phenomenon is reciprocal and can place also RTK signaling downstream of GPCR. Formation of either stable or transient complexes by these two important classes of membrane receptors is involved in regulating all aspects of receptor function, from ligand binding to signal transduction, trafficking, desensitization, and downregulation among others. Functional phenomena can be modulated with conformation-specific inhibitors that stabilize defined GPCR states to abrogate both GPCR agonist- and growth factor-stimulated cell responses or by means of small interfering heteroreceptor complex interface peptides. The bioluminescence resonance energy transfer (BRET) technology has emerged as a powerful method to study the structure of heteroreceptor complexes closely associated with the study of receptor-receptor interactions in such complexes. In this chapter, we provide an overview of different BRET(2) assays that can be used to study the structure of GPCR-RTK heteroreceptor complexes and their functions. Various experimental designs for optimization of these experiments are also described. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach.

    Directory of Open Access Journals (Sweden)

    Kalyan C Tirupula

    Full Text Available Propagation of signals from G protein-coupled receptors (GPCRs in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM. We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A 'cardiac-specific finger print' of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS, cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a 'MAS-signalosome' model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of 'signalosome' components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor.

  8. MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach.

    Science.gov (United States)

    Tirupula, Kalyan C; Zhang, Dongmei; Osbourne, Appledene; Chatterjee, Arunachal; Desnoyer, Russ; Willard, Belinda; Karnik, Sadashiva S

    2015-01-01

    Propagation of signals from G protein-coupled receptors (GPCRs) in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct) is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM). We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A 'cardiac-specific finger print' of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS), cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a 'MAS-signalosome' model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of 'signalosome' components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor.

  9. Quantitative study of protein-protein interactions by quartz nanopipettes.

    Science.gov (United States)

    Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin

    2014-09-07

    In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.

  10. The role of insulin receptor substrate (IRS) proteins in oncogenic transformation.

    Science.gov (United States)

    Gorgisen, G; Gulacar, I M; Ozes, O N

    2017-01-30

    Insulin Receptor Substrate (IRS) proteins are the main cytoplasmic adaptor molecules involved in transducing extracellular signals from receptors to downstream proteins. This protein family have pivotal roles on maintenance, distribution and regulation of signaling networks. Since IRS1/2 interact with and transmits signals from the receptors of insulin, Insulin Like Growth Factor 1 (IGF1), prolactin, growth hormone (GH), leptin, Vascular Endothelial Growth Factor (VEGF), TrkB, ALK and integrins this promoted scientist to think that IRS1 may have functions in cell proliferation, tumorigenesis and metastasis. Therefore, over the past decade, studies on IRS proteins and their functions in cancer has been increased and these studies provided valuable results claiming the involvement of IRS1/2 in cancer development. In this review, we discuss the function and contributions of IRS1 and IRS2 in development of  breast cancer.

  11. Nectin-like interactions between poliovirus and its receptor trigger conformational changes associated with cell entry.

    Science.gov (United States)

    Strauss, Mike; Filman, David J; Belnap, David M; Cheng, Naiqian; Noel, Roane T; Hogle, James M

    2015-04-01

    Poliovirus infection is initiated by attachment to a receptor on the cell surface called Pvr or CD155. At physiological temperatures, the receptor catalyzes an irreversible expansion of the virus to form an expanded form of the capsid called the 135S particle. This expansion results in the externalization of the myristoylated capsid protein VP4 and the N-terminal extension of the capsid protein VP1, both of which become inserted into the cell membrane. Structures of the expanded forms of poliovirus and of several related viruses have recently been reported. However, until now, it has been unclear how receptor binding triggers viral expansion at physiological temperature. Here, we report poliovirus in complex with an enzymatically partially deglycosylated form of the 3-domain ectodomain of Pvr at a 4-Å resolution, as determined by cryo-electron microscopy. The interaction of the receptor with the virus in this structure is reminiscent of the interactions of Pvr with its natural ligands. At a low temperature, the receptor induces very few changes in the structure of the virus, with the largest changes occurring within the footprint of the receptor, and in a loop of the internal protein VP4. Changes in the vicinity of the receptor include the displacement of a natural lipid ligand (called "pocket factor"), demonstrating that the loss of this ligand, alone, is not sufficient to induce particle expansion. Finally, analogies with naturally occurring ligand binding in the nectin family suggest which specific structural rearrangements in the virus-receptor complex could help to trigger the irreversible expansion of the capsid. The cell-surface receptor (Pvr) catalyzes a large structural change in the virus that exposes membrane-binding protein chains. We fitted known atomic models of the virus and Pvr into three-dimensional experimental maps of the receptor-virus complex. The molecular interactions we see between poliovirus and its receptor are reminiscent of the nectin

  12. Nectin-Like Interactions between Poliovirus and Its Receptor Trigger Conformational Changes Associated with Cell Entry

    Science.gov (United States)

    Strauss, Mike; Filman, David J.; Belnap, David M.; Cheng, Naiqian; Noel, Roane T.

    2015-01-01

    ABSTRACT Poliovirus infection is initiated by attachment to a receptor on the cell surface called Pvr or CD155. At physiological temperatures, the receptor catalyzes an irreversible expansion of the virus to form an expanded form of the capsid called the 135S particle. This expansion results in the externalization of the myristoylated capsid protein VP4 and the N-terminal extension of the capsid protein VP1, both of which become inserted into the cell membrane. Structures of the expanded forms of poliovirus and of several related viruses have recently been reported. However, until now, it has been unclear how receptor binding triggers viral expansion at physiological temperature. Here, we report poliovirus in complex with an enzymatically partially deglycosylated form of the 3-domain ectodomain of Pvr at a 4-Å resolution, as determined by cryo-electron microscopy. The interaction of the receptor with the virus in this structure is reminiscent of the interactions of Pvr with its natural ligands. At a low temperature, the receptor induces very few changes in the structure of the virus, with the largest changes occurring within the footprint of the receptor, and in a loop of the internal protein VP4. Changes in the vicinity of the receptor include the displacement of a natural lipid ligand (called “pocket factor”), demonstrating that the loss of this ligand, alone, is not sufficient to induce particle expansion. Finally, analogies with naturally occurring ligand binding in the nectin family suggest which specific structural rearrangements in the virus-receptor complex could help to trigger the irreversible expansion of the capsid. IMPORTANCE The cell-surface receptor (Pvr) catalyzes a large structural change in the virus that exposes membrane-binding protein chains. We fitted known atomic models of the virus and Pvr into three-dimensional experimental maps of the receptor-virus complex. The molecular interactions we see between poliovirus and its receptor are

  13. Yeast Interacting Proteins Database: YDL226C, YJL151C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available s bait as prey (0) YJL151C SNA3 Integral membrane protein localized to vacuolar intralumenal vesicles, computational...intralumenal vesicles, computational analysis of large-scale protein-protein interaction data suggests a pos... gene name SNA3 Prey description Integral membrane protein localized to vacuolar

  14. Yeast Interacting Proteins Database: YML064C, YOR284W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available th this bait as prey (0) YOR284W HUA2 Cytoplasmic protein of unknown function; computational analysis of lar...Rows with this bait as prey (0) Prey ORF YOR284W Prey gene name HUA2 Prey description Cytoplasmic protein of unknown function; comput...ational analysis of large-scale protein-protein interact

  15. Hydrophobic interactions of sucralose with protein structures.

    Science.gov (United States)

    Shukla, Nimesh; Pomarico, Enrico; Hecht, Cody J S; Taylor, Erika A; Chergui, Majed; Othon, Christina M

    2018-02-01

    Sucralose is a commonly employed artificial sweetener that appears to destabilize protein native structures. This is in direct contrast to the bio-preservative nature of its natural counterpart, sucrose, which enhances the stability of biomolecules against environmental stress. We have further explored the molecular interactions of sucralose as compared to sucrose to illuminate the origin of the differences in their bio-preservative efficacy. We show that the mode of interactions of sucralose and sucrose in bulk solution differ subtly through the use of hydration dynamics measurement and computational simulation. Sucralose does not appear to disturb the native state of proteins for moderate concentrations (sucralose appears to differ in its interactions with protein leading to the reduction of native state stability. This difference in interaction appears weak. We explored the difference in the preferential exclusion model using time-resolved spectroscopic techniques and observed that both molecules appear to be effective reducers of bulk hydration dynamics. However, the chlorination of sucralose appears to slightly enhance the hydrophobicity of the molecule, which reduces the preferential exclusion of sucralose from the protein-water interface. The weak interaction of sucralose with hydrophobic pockets on the protein surface differs from the behavior of sucrose. We experimentally followed up upon the extent of this weak interaction using isothermal titration calorimetry (ITC) measurements. We propose this as a possible origin for the difference in their bio-preservative properties. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Binding interactions of niclosamide with serum proteins

    Directory of Open Access Journals (Sweden)

    Esra Maltas

    2014-12-01

    Full Text Available A study of the binding of niclosamide (NC to serum proteins such as human serum albumin, hemoglobin, and globulin was carried out using fluorescence and UV-visible spectroscopy. Interactions between NC and these proteins were estimated by Stern–Volmer and van't Hoff equations. The binding constants and the thermodynamic parameters, ΔH, ΔS, and ΔG at different temperatures were also determined by using these equations. Data showed that NC may exhibit a static quenching mechanism with all proteins. The thermodynamic parameters were calculated. Data showed that van der Waals interactions and hydrogen bonds are the main forces for human serum albumin and hemoglobin. Globulin, however, bound to NC via hydrophobic interaction. The spectral changes of synchronous fluorescence suggested that both the microenvironment of NC and the conformation of the proteins changed in relation to their concentrations during NC's binding.

  17. Geometric de-noising of protein-protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Oleksii Kuchaiev

    2009-08-01

    Full Text Available Understanding complex networks of protein-protein interactions (PPIs is one of the foremost challenges of the post-genomic era. Due to the recent advances in experimental bio-technology, including yeast-2-hybrid (Y2H, tandem affinity purification (TAP and other high-throughput methods for protein-protein interaction (PPI detection, huge amounts of PPI network data are becoming available. Of major concern, however, are the levels of noise and incompleteness. For example, for Y2H screens, it is thought that the false positive rate could be as high as 64%, and the false negative rate may range from 43% to 71%. TAP experiments are believed to have comparable levels of noise.We present a novel technique to assess the confidence levels of interactions in PPI networks obtained from experimental studies. We use it for predicting new interactions and thus for guiding future biological experiments. This technique is the first to utilize currently the best fitting network model for PPI networks, geometric graphs. Our approach achieves specificity of 85% and sensitivity of 90%. We use it to assign confidence scores to physical protein-protein interactions in the human PPI network downloaded from BioGRID. Using our approach, we predict 251 interactions in the human PPI network, a statistically significant fraction of which correspond to protein pairs sharing common GO terms. Moreover, we validate a statistically significant portion of our predicted interactions in the HPRD database and the newer release of BioGRID. The data and Matlab code implementing the methods are freely available from the web site: http://www.kuchaev.com/Denoising.

  18. Geometric de-noising of protein-protein interaction networks.

    Science.gov (United States)

    Kuchaiev, Oleksii; Rasajski, Marija; Higham, Desmond J; Przulj, Natasa

    2009-08-01

    Understanding complex networks of protein-protein interactions (PPIs) is one of the foremost challenges of the post-genomic era. Due to the recent advances in experimental bio-technology, including yeast-2-hybrid (Y2H), tandem affinity purification (TAP) and other high-throughput methods for protein-protein interaction (PPI) detection, huge amounts of PPI network data are becoming available. Of major concern, however, are the levels of noise and incompleteness. For example, for Y2H screens, it is thought that the false positive rate could be as high as 64%, and the false negative rate may range from 43% to 71%. TAP experiments are believed to have comparable levels of noise.We present a novel technique to assess the confidence levels of interactions in PPI networks obtained from experimental studies. We use it for predicting new interactions and thus for guiding future biological experiments. This technique is the first to utilize currently the best fitting network model for PPI networks, geometric graphs. Our approach achieves specificity of 85% and sensitivity of 90%. We use it to assign confidence scores to physical protein-protein interactions in the human PPI network downloaded from BioGRID. Using our approach, we predict 251 interactions in the human PPI network, a statistically significant fraction of which correspond to protein pairs sharing common GO terms. Moreover, we validate a statistically significant portion of our predicted interactions in the HPRD database and the newer release of BioGRID. The data and Matlab code implementing the methods are freely available from the web site: http://www.kuchaev.com/Denoising.

  19. Nuclear Receptors in Drug Metabolism, Drug Response and Drug Interactions

    Directory of Open Access Journals (Sweden)

    Chandra Prakash

    2015-12-01

    Full Text Available Orally delivered small-molecule therapeutics are metabolized in the liver and intestine by phase I and phase II drug-metabolizing enzymes (DMEs, and transport proteins coordinate drug influx (phase 0 and drug/drug-metabolite efflux (phase III. Genes involved in drug metabolism and disposition are induced by xenobiotic-activated nuclear receptors (NRs, i.e. PXR (pregnane X receptor and CAR (constitutive androstane receptor, and by the 1α, 25-dihydroxy vitamin D3-activated vitamin D receptor (VDR, due to transactivation of xenobiotic-response elements (XREs present in phase 0-III genes. Additional NRs, like HNF4-α, FXR, LXR-α play important roles in drug metabolism in certain settings, such as in relation to cholesterol and bile acid metabolism. The phase I enzymes CYP3A4/A5, CYP2D6, CYP2B6, CYP2C9, CYP2C19, CYP1A2, CYP2C8, CYP2A6, CYP2J2, and CYP2E1 metabolize >90% of all prescription drugs, and phase II conjugation of hydrophilic functional groups (with/without phase I modification facilitates drug clearance. The conjugation step is mediated by broad-specificity transferases like UGTs, SULTs, GSTs. This review delves into our current understanding of PXR/CAR/VDR-mediated regulation of DME and transporter expression, as well as effects of single nucleotide polymorphism (SNP and epigenome (specified by promoter methylation, histone modification, microRNAs, long non coding RNAs on the expression of PXR/CAR/VDR and phase 0-III mediators, and their impacts on variable drug response. Therapeutic agents that target epigenetic regulation and the molecular basis and consequences (overdosing, underdosing, or beneficial outcome of drug-drug/drug-food/drug-herb interactions are also discussed. Precision medicine requires understanding of a drug's impact on DME and transporter activity and their NR-regulated expression in order to achieve optimal drug efficacy without adverse drug reactions. In future drug screening, new tools such as humanized mouse

  20. Interaction of melanosomal proteins with melanin.

    Science.gov (United States)

    Donatien, P D; Orlow, S J

    1995-08-15

    Melanin is deposited in melanosomes upon a proteinaceous matrix enveloped by a melanosomal membrane. Since melanin is highly detergent insoluble, we hypothesized that the detergent solubility of proteins of the melanosomal matrix might be inversely related to the state of melanosomal melanization. Immunoblotting analyses were performed on extracts of albino and black melanocytes to test this hypothesis. The protein products of the silver (si) and the pink-eyed-dilution (p) loci as well as other matrix constituents were present at twofold higher levels in extracts of albino cells. When black cells were rendered amelanotic by growing cultures in the presence of the tyrosinase inhibitor phenylthiourea, the apparent levels of these proteins were also increased. To obviate the potential role of different levels of synthesis in contributing to these differences, we developed a cell-free melanosomal melanization assay. Upon incubation of a melanosome-rich fraction with the melanin precursor L-3,4-dihydroxyphenylalanine (Dopa) followed by immunoblot analysis, the si locus protein, the p locus protein, and other putative matrix constituents became rapidly insoluble in SDS when compared with the members of the tyrosinase-related family of melanosomal membrane proteins. Our results suggest that melanosomal proteins that interact with melanin may be identified by their relative insolubility in SDS under conditions of increasing melanization. In addition to the si locus protein and other putative melanosomal matrix proteins, the membrane-bound p locus protein may also interact closely with melanin.

  1. Data on overlapping brain disorders and emerging drug targets in human Dopamine Receptors Interaction Network

    Directory of Open Access Journals (Sweden)

    Avijit Podder

    2017-06-01

    Full Text Available Intercommunication of Dopamine Receptors (DRs with their associate protein partners is crucial to maintain regular brain function in human. Majority of the brain disorders arise due to malfunctioning of such communication process. Hence, contributions of genetic factors, as well as phenotypic indications for various neurological and psychiatric disorders are often attributed as sharing in nature. In our earlier research article entitled “Human Dopamine Receptors Interaction Network (DRIN: a systems biology perspective on topology, stability and functionality of the network” (Podder et al., 2014 [1], we had depicted a holistic interaction map of human Dopamine Receptors. Given emphasis on the topological parameters, we had characterized the functionality along with the vulnerable properties of the network. In support of this, we hereby provide an additional data highlighting the genetic overlapping of various brain disorders in the network. The data indicates the sharing nature of disease genes for various neurological and psychiatric disorders in dopamine receptors connecting protein-protein interactions network. The data also indicates toward an alternative approach to prioritize proteins for overlapping brain disorders as valuable drug targets in the network.

  2. Yeast Interacting Proteins Database: YBR135W, YBR252W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available tes proteolysis of M-phase targets through interactions with the proteasome; role in transcriptional regulat...yclin-dependent protein kinase regulatory subunit and adaptor; modulates proteolysis of M-phase targets through interactions

  3. Receptor Quaternary Organization Explains G Protein-Coupled Receptor Family Structure

    Directory of Open Access Journals (Sweden)

    James H. Felce

    2017-09-01

    Full Text Available The organization of Rhodopsin-family G protein-coupled receptors (GPCRs at the cell surface is controversial. Support both for and against the existence of dimers has been obtained in studies of mostly individual receptors. Here, we use a large-scale comparative study to examine the stoichiometric signatures of 60 receptors expressed by a single human cell line. Using bioluminescence resonance energy transfer- and single-molecule microscopy-based assays, we found that a relatively small fraction of Rhodopsin-family GPCRs behaved as dimers and that these receptors otherwise appear to be monomeric. Overall, the analysis predicted that fewer than 20% of ∼700 Rhodopsin-family receptors form dimers. The clustered distribution of the dimers in our sample and a striking correlation between receptor organization and GPCR family size that we also uncover each suggest that receptor stoichiometry might have profoundly influenced GPCR expansion and diversification.

  4. Biased signaling of G protein-coupled receptors - From a chemokine receptor CCR7 perspective

    DEFF Research Database (Denmark)

    Jørgensen, Astrid Sissel; Rosenkilde, Mette M; Hjortø, Gertrud M

    2018-01-01

    Chemokines (chemotactic cytokines) and their associated G protein-coupled receptors (GPCRs) work in a concerted manner to govern immune cell positioning in time and space. Promiscuity of both ligands and receptors, but also biased signaling within the chemokine system, adds to the complexity of how...

  5. Research advances in interactions related to Toxoplasma gondii microneme proteins.

    Science.gov (United States)

    Liu, Qing; Li, Fa-Cai; Zhou, Chun-Xue; Zhu, Xing-Quan

    2017-05-01

    Toxoplasma gondii microneme proteins (TgMICs), secreted by micronemes upon contact with host cells, are reported to play important roles in multiple stages of the T. gondii life cycle, including parasite motility, invasion, intracellular survival, and egress from host cells. Meanwhile, during these processes, TgMICs participate in many protein-protein and protein-carbohydrate interactions, such as undergoing proteolytic maturation, binding to aldolase, engaging the host cell receptors and forming the moving junction (MJ), relying on different types of ectodomains, transmembrane (TM) domains and cytoplasmic domains (CDs). In this review, we summarize the research advances in protein-protein and protein-carbohydrate interactions related to TgMICs, and their intimate associations with corresponding biological processes during T. gondii infection, which will contribute to an improved understanding of the molecular pathogenesis of T. gondii infection, and provide a basis for developing effective control strategies against T. gondii. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. G-Protein Sensitivity of Ligand Binding to Human Dopamine D2 and D3 Receptors Expressed in Escherichia coli : Clues for a Constrained D3 Receptor Structure

    NARCIS (Netherlands)

    Vanhauwe, Jurgen F.M.; Josson, Katty; Luyten, Walter H.M.L.; Driessen, Arnold J.; Leysen, Josée E.

    2000-01-01

    Human dopamine D2 and D3 receptors were expressed in Chinese hamster ovary (CHO) and Escherichia coli cells to compare their ligand binding properties in the presence or absence of G-proteins and to analyze their ability to interact with Gi/o-proteins. Binding affinities of agonists (dopamine,

  7. Mapping of protein-protein interaction network of Alexander disease.

    Science.gov (United States)

    Saxena, A K; Saxena, V L; Dixit, S

    2016-05-30

    Alexander disease (ALXD) is slowly progressive neurodegenerative disorder which affects white matter of the central nervous system. The main cause of disorder is mutation in GFAP gene and mutation in some other genes were also reported. This study was aimed at getting a better insight into ALXD pathogenesis and identifying the important functional and highly interconnected nodes in human protein interaction network, identifying the important sub-networks in the system could be helpful in understanding the underlying molecular mechanism. The topological analysis of human protein interaction network strategy to identify highly interconnected sub-network modules from which six proteins are found i.e. GFAP, PLEC, CRYAB, NDUFV1, CASP3 and MAPK14 plays important role in disease. Further, the enrichment analysis of interaction network identifies crucial pathways in which most of the diseased proteins overlaps. Through system biology approach, the undirected human protein interaction network of ALXD is buildup with the help of Cytoscape tool and its various plugins helps to investigate network further. The systematic approach suggests the finding of previously known proteins, GFAP, PLEC, CRYAB, NDUFV1, CASP3 and MAPK14 can be used as a drug targets and potential treatment discovered also enrichment analysis will provide guidance for the future study on Alexander disease.

  8. SOCS proteins in regulation of receptor tyrosine kinase signaling

    DEFF Research Database (Denmark)

    Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar

    2014-01-01

    proteins, SOCS1-7, and cytokine-inducible SH2-containing protein (CIS). A key feature of this family of proteins is the presence of an SH2 domain and a SOCS box. Recent studies suggest that SOCS proteins also play a role in RTK signaling. Activation of RTK results in transcriptional activation of SOCS......-encoding genes. These proteins associate with RTKs through their SH2 domains and subsequently recruit the E3 ubiquitin machinery through the SOCS box, and thereby limit receptor stability by inducing ubiquitination. In a similar fashion, SOCS proteins negatively regulate mitogenic signaling by RTKs. It is also...

  9. NMR Studies of Protein Hydration and Protein-Ligand Interactions

    Science.gov (United States)

    Chong, Yuan

    Water on the surface of a protein is called hydration water. Hydration water is known to play a crucial role in a variety of biological processes including protein folding, enzymatic activation, and drug binding. Although the significance of hydration water has been recognized, the underlying mechanism remains far from being understood. This dissertation employs a unique in-situ nuclear magnetic resonance (NMR) technique to study the mechanism of protein hydration and the role of hydration in alcohol-protein interactions. Water isotherms in proteins are measured at different temperatures via the in-situ NMR technique. Water is found to interact differently with hydrophilic and hydrophobic groups on the protein. Water adsorption on hydrophilic groups is hardly affected by the temperature, while water adsorption on hydrophobic groups strongly depends on the temperature around 10 C, below which the adsorption is substantially reduced. This effect is induced by the dramatic decrease in the protein flexibility below 10 C. Furthermore, nanosecond to microsecond protein dynamics and the free energy, enthalpy, and entropy of protein hydration are studied as a function of hydration level and temperature. A crossover at 10 C in protein dynamics and thermodynamics is revealed. The effect of water at hydrophilic groups on protein dynamics and thermodynamics shows little temperature dependence, whereas water at hydrophobic groups has stronger effect above 10 C. In addition, I investigate the role of water in alcohol binding to the protein using the in-situ NMR detection. The isotherms of alcohols are first measured on dry proteins, then on proteins with a series of controlled hydration levels. The free energy, enthalpy, and entropy of alcohol binding are also determined. Two distinct types of alcohol binding are identified. On the one hand, alcohols can directly bind to a few specific sites on the protein. This type of binding is independent of temperature and can be

  10. A monoclonal antibody for G protein-coupled receptor crystallography

    DEFF Research Database (Denmark)

    Day, Peter W; Rasmussen, Søren Gøgsig Faarup; Parnot, Charles

    2007-01-01

    G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural...

  11. Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Tian, He; Naganathan, Saranga; Kazmi, Manija A

    2014-01-01

    Novel methods are required for site-specific, quantitative fluorescent labeling of G-protein-coupled receptors (GPCRs) and other difficult-to-express membrane proteins. Ideally, fluorescent probes should perturb the native structure and function as little as possible. We evaluated bioorthogonal...

  12. Endothelial protein C receptor in renal tubular epithelial cells and ...

    African Journals Online (AJOL)

    The endothelial protein C receptor (EPCR) plays an important role within the protein C pathway in regulating coagulation and inflammation. It was reported that EPCR was expressed in large vessels, placenta, heart, liver and lung endothelial cell. However, there are a few studies concerned about renal epithelial cells.

  13. Two active molecular phenotypes of the tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis

    DEFF Research Database (Denmark)

    Holst, B; Hastrup, H; Raffetseder, U

    2001-01-01

    either Galpha(s) or Galpha(q) and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P...

  14. Fibroblast Growth Factor-21 (FGF21) Regulates Low-density Lipoprotein Receptor (LDLR) Levels in Cells via the E3-ubiquitin Ligase Mylip/Idol and the Canopy2 (Cnpy2)/Mylip-interacting Saposin-like Protein (Msap)

    NARCIS (Netherlands)

    Do, Hai Thi; Tselykh, Timofey V.; Mäkelä, Johanna; Ho, Tho Huu; Olkkonen, Vesa M.; Bornhauser, Beat C.; Korhonen, Laura; Zelcer, Noam; Lindholm, Dan

    2012-01-01

    The LDLR is a critical factor in the regulation of blood cholesterol levels that are altered in different human diseases. The level of LDLR in the cell is regulated by both transcriptional and post-transcriptional events. The E3 ubiquitin ligase, myosin regulatory light chain-interacting protein

  15. Emerging issues in receptor protein tyrosine phosphatase function: lifting fog or simply shifting?

    DEFF Research Database (Denmark)

    Petrone, A; Sap, J

    2000-01-01

    Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions...... with signaling pathways involving SRC family kinases, which result from their ability to control phosphorylation of both activating and inhibitory sites in these kinases and possibly also their substrates. Similarly, integrin signaling illustrates how phosphorylation of a single protein, or the activity...

  16. Protein-protein interactions within late pre-40S ribosomes.

    Directory of Open Access Journals (Sweden)

    Melody G Campbell

    2011-01-01

    Full Text Available Ribosome assembly in eukaryotic organisms requires more than 200 assembly factors to facilitate and coordinate rRNA transcription, processing, and folding with the binding of the ribosomal proteins. Many of these assembly factors bind and dissociate at defined times giving rise to discrete assembly intermediates, some of which have been partially characterized with regards to their protein and RNA composition. Here, we have analyzed the protein-protein interactions between the seven assembly factors bound to late cytoplasmic pre-40S ribosomes using recombinant proteins in binding assays. Our data show that these factors form two modules: one comprising Enp1 and the export adaptor Ltv1 near the beak structure, and the second comprising the kinase Rio2, the nuclease Nob1, and a regulatory RNA binding protein Dim2/Pno1 on the front of the head. The GTPase-like Tsr1 and the universally conserved methylase Dim1 are also peripherally connected to this second module. Additionally, in an effort to further define the locations for these essential proteins, we have analyzed the interactions between these assembly factors and six ribosomal proteins: Rps0, Rps3, Rps5, Rps14, Rps15 and Rps29. Together, these results and previous RNA-protein crosslinking data allow us to propose a model for the binding sites of these seven assembly factors. Furthermore, our data show that the essential kinase Rio2 is located at the center of the pre-ribosomal particle and interacts, directly or indirectly, with every other assembly factor, as well as three ribosomal proteins required for cytoplasmic 40S maturation. These data suggest that Rio2 could play a central role in regulating cytoplasmic maturation steps.

  17. Yeast Interacting Proteins Database: YFR049W, YOR047C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; acts as a regulator... (0) YOR047C STD1 Protein involved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sens...ors Snf3p and Rgt2p, and TATA-binding protein Spt15p; ac

  18. Yeast Interacting Proteins Database: YOR047C, YKL038W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available racts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; acts as a...Bait description Protein involved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose senso...rs Snf3p and Rgt2p, and TATA-binding protein Spt15p; acts as a regulator of the tra

  19. KFC Server: interactive forecasting of protein interaction hot spots.

    Science.gov (United States)

    Darnell, Steven J; LeGault, Laura; Mitchell, Julie C

    2008-07-01

    The KFC Server is a web-based implementation of the KFC (Knowledge-based FADE and Contacts) model-a machine learning approach for the prediction of binding hot spots, or the subset of residues that account for most of a protein interface's; binding free energy. The server facilitates the automated analysis of a user submitted protein-protein or protein-DNA interface and the visualization of its hot spot predictions. For each residue in the interface, the KFC Server characterizes its local structural environment, compares that environment to the environments of experimentally determined hot spots and predicts if the interface residue is a hot spot. After the computational analysis, the user can visualize the results using an interactive job viewer able to quickly highlight predicted hot spots and surrounding structural features within the protein structure. The KFC Server is accessible at http://kfc.mitchell-lab.org.

  20. HIV-1 Nef binds with human GCC185 protein and regulates mannose 6 phosphate receptor recycling

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manjeet; Kaur, Supinder; Nazir, Aamir; Tripathi, Raj Kamal, E-mail: rajkamalcdri@gmail.com

    2016-05-20

    HIV-1 Nef modulates cellular function that enhances viral replication in vivo which culminate into AIDS pathogenesis. With no enzymatic activity, Nef regulates cellular function through host protein interaction. Interestingly, trans-cellular introduction of recombinant Nef protein in Caenorhabditis elegans results in AIDS like pathogenesis which might share common pathophysiology because the gene sequence of C. elegans and humans share considerable homology. Therefore employing C. elegans based initial screen complemented with sequence based homology search we identified GCC185 as novel host protein interacting with HIV-1 Nef. The detailed molecular characterization revealed N-terminal EEEE{sub 65} acidic domain of Nef as key region for interaction. GCC185 is a tethering protein that binds with Rab9 transport vesicles. Our results show that Nef-GCC185 interaction disrupts Rab9 interaction resulting in delocalization of CI-MPR (cation independent Mannose 6 phosphate receptor) resulting in elevated secretion of hexosaminidase. In agreement with this, our studies identified novel host GCC185 protein that interacts with Nef EEEE65 acidic domain interfering GCC185-Rab9 vesicle membrane fusion responsible for retrograde vesicular transport of CI-MPR from late endosomes to TGN. In light of existing report suggesting critical role of Nef-GCC185 interaction reveals valuable mechanistic insights affecting specific protein transport pathway in docking of late endosome derived Rab9 bearing transport vesicle at TGN elucidating role of Nef during viral pathogenesis. -- Highlights: •Nef, an accessory protein of HIV-1 interacts with host factor and culminates into AIDS pathogenesis. •Using Caenorhabditis elegans based screen system, novel Nef interacting cellular protein GCC185 was identified. •Molecular characterization of Nef and human protein GCC185 revealed Nef EEEE{sub 65} key region interacted with full length GCC185. •Nef impeded the GCC185-Rab 9 interaction and

  1. Deciphering peculiar protein-protein interacting modules in Deinococcus radiodurans

    Directory of Open Access Journals (Sweden)

    Barkallah Insaf

    2009-04-01

    Full Text Available Abstract Interactomes of proteins under positive selection from ionizing-radiation-resistant bacteria (IRRB might be a part of the answer to the question as to how IRRB, particularly Deinococcus radiodurans R1 (Deira, resist ionizing radiation. Here, using the Database of Interacting Proteins (DIP and the Protein Structural Interactome (PSI-base server for PSI map, we have predicted novel interactions of orthologs of the 58 proteins under positive selection in Deira and other IRRB, but which are absent in IRSB. Among these, 18 domains and their interactomes have been identified in DNA checkpoint and repair; kinases pathways; energy and nucleotide metabolisms were the important biological processes that were found to be involved. This finding provides new clues to the cellular pathways that can to be important for ionizing-radiation resistance in Deira.

  2. The significance of G protein-coupled receptor crystallography for drug discovery.

    Science.gov (United States)

    Salon, John A; Lodowski, David T; Palczewski, Krzysztof

    2011-12-01

    Crucial as molecular sensors for many vital physiological processes, seven-transmembrane domain G protein-coupled receptors (GPCRs) comprise the largest family of proteins targeted by drug discovery. Together with structures of the prototypical GPCR rhodopsin, solved structures of other liganded GPCRs promise to provide insights into the structural basis of the superfamily's biochemical functions and assist in the development of new therapeutic modalities and drugs. One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions. Such conformational changes can be subtle and triggered by relatively small binding energy effects, leading to full or partial efficacy in the activation or inactivation of the receptor system at large. Pharmacological dogma generally dictates that these changes manifest themselves through kinetic modulation of the receptor's G protein partners. Atomic resolution information derived from increasingly available receptor structures provides an entrée to the understanding of these events and practically applying it to drug design. Supported by structure-activity relationship information arising from empirical screening, a unified structural model of GPCR activation/inactivation promises to both accelerate drug discovery in this field and improve our fundamental understanding of structure-based drug design in general. This review discusses fundamental problems that persist in drug design and GPCR structural determination.

  3. Delayed toxicity associated with soluble anthrax toxin receptor decoy-Ig fusion protein treatment.

    Directory of Open Access Journals (Sweden)

    Diane Thomas

    Full Text Available Soluble receptor decoy inhibitors, including receptor-immunogloubulin (Ig fusion proteins, have shown promise as candidate anthrax toxin therapeutics. These agents act by binding to the receptor-interaction site on the protective antigen (PA toxin subunit, thereby blocking toxin binding to cell surface receptors. Here we have made the surprising observation that co-administration of receptor decoy-Ig fusion proteins significantly delayed, but did not protect, rats challenged with anthrax lethal toxin. The delayed toxicity was associated with the in vivo assembly of a long-lived complex comprised of anthrax lethal toxin and the receptor decoy-Ig inhibitor. Intoxication in this system presumably results from the slow dissociation of the toxin complex from the inhibitor following their prolonged circulation. We conclude that while receptor decoy-Ig proteins represent promising candidates for the early treatment of B. anthracis infection, they may not be suitable for therapeutic use at later stages when fatal levels of toxin have already accumulated in the bloodstream.

  4. Mimicking protein-protein interactions through peptide-peptide interactions: HIV-1 gp120 and CXCR4

    Directory of Open Access Journals (Sweden)

    Andrea eGross

    2013-09-01

    Full Text Available We have recently designed a soluble synthetic peptide that functionally mimics the HIV-1 coreceptor CXCR4, which is a chemokine receptor that belongs to the family of seven-transmembrane GPCRs. This CXCR4 mimetic peptide, termed CX4-M1, presents the three extracellular loops (ECLs of the receptor. In binding assays involving recombinant proteins, as well as in cellular infection assays, CX4-M1 was found to selectively recognize gp120 from HIV-1 strains that use CXCR4 for cell entry (X4 tropic HIV-1. Furthermore, anti-HIV-1 antibodies modulate this interaction in a molecular mechanism related to that of their impact on the gp120-CXCR4 interaction. We could now show that the selectivity of CX4-M1 pertains not only to gp120 from X4 tropic HIV-1, but also to synthetic peptides presenting the V3 loops of these gp120 proteins. The V3 loop is thought to be an essential part of the coreceptor binding site of gp120 that contacts the second ECL of the coreceptor. We were able to experimentally confirm this notion in binding assays using substitution analogs of CX4-M1 and the V3 loop peptides, respectively, as well as in cellular infection assays. These results indicate that interactions of the HIV-1 Env with coreceptors can be mimicked by synthetic peptides, which may be useful to explore these interactions at the molecular level in more detail.

  5. PIWI Proteins and PIWI-Interacting RNA

    DEFF Research Database (Denmark)

    Han, Yi Neng; Li, Yuan; Xia, Sheng Qiang

    2017-01-01

    P-Element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a type of noncoding RNAs (ncRNAs) and interact with PIWI proteins. piRNAs were primarily described in the germline, but emerging evidence revealed that piRNAs are expressed in a tissue-specific manner among multiple human somatic......-cell maintenance, self-renewal, retrotransposons silencing and the male germline mobility control. A growing number of studies have demonstrated that several piRNA and PIWI proteins are aberrantly expressed in various kinds of cancers and may probably serve as a novel biomarker and therapeutic target for cancer...... treatment. Nevertheless, their specific mechanisms and functions need further investigation. In this review, we discuss about the biogenesis, functions and the emerging role of piRNAs and PIWI proteins in cancer, providing novel insights into the possible applications of piRNAs and PIWI proteins in cancer...

  6. PIWI Proteins and PIWI-Interacting RNA

    DEFF Research Database (Denmark)

    Han, Yi Neng; Li, Yuan; Xia, Sheng Qiang

    2017-01-01

    P-Element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a type of noncoding RNAs (ncRNAs) and interact with PIWI proteins. piRNAs were primarily described in the germline, but emerging evidence revealed that piRNAs are expressed in a tissue-specific manner among multiple human somatic...... tissue types as well and play important roles in transposon silencing, epigenetic regulation, gene and protein regulation, genome rearrangement, spermatogenesis and germ stem-cell maintenance. PIWI proteins were first discovered in Drosophila and they play roles in spermatogenesis, germline stem......-cell maintenance, self-renewal, retrotransposons silencing and the male germline mobility control. A growing number of studies have demonstrated that several piRNA and PIWI proteins are aberrantly expressed in various kinds of cancers and may probably serve as a novel biomarker and therapeutic target for cancer...

  7. Potential disruption of protein-protein interactions by graphene oxide

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Mei [Department of Physics, Institute of Quantitative Biology, Zhejiang University, Hangzhou 310027 (China); Kang, Hongsuk; Luan, Binquan [Computational Biological Center, IBM Thomas J. Watson Research Center, Yorktown Heights, New York 10598 (United States); Yang, Zaixing [Institute of Quantitative Biology and Medicine, SRMP and RAD-X, and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123 (China); Zhou, Ruhong, E-mail: ruhong@us.ibm.com [Department of Physics, Institute of Quantitative Biology, Zhejiang University, Hangzhou 310027 (China); Computational Biological Center, IBM Thomas J. Watson Research Center, Yorktown Heights, New York 10598 (United States); Department of Chemistry, Columbia University, New York, New York 10027 (United States)

    2016-06-14

    Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions and eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications.

  8. Potential disruption of protein-protein interactions by graphene oxide

    Science.gov (United States)

    Feng, Mei; Kang, Hongsuk; Yang, Zaixing; Luan, Binquan; Zhou, Ruhong

    2016-06-01

    Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions and eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications.

  9. Melanocortin receptor binding determinants in the agouti protein.

    Science.gov (United States)

    Kiefer, L L; Veal, J M; Mountjoy, K G; Wilkison, W O

    1998-01-27

    The agouti protein plays an important role in the development of diabetes and obesity in rodents and has been shown to be a potent antagonist of melanocortin receptors. For this reason alanine-scanning mutagenesis was performed on the agouti protein carboxyl terminus to locate residues important for melanocortin receptor binding inhibition. When agouti residues Arg116 and Phe118 are changed to alanine, very large decreases in agouti affinity for melanocortin receptor 1, 3, and 4 result. Mutation of Phe117 to alanine causes a similar increase in agouti KI app at melanocortin receptor 4. Substitution of agouti residue Asp108 with alanine results in large increases in KI app for all three melanocortin receptors examined. All of these residues are conserved in the agouti-related transcript, ART, whose expression is up-regulated in animal models of obesity. The three-dimensional structure of the agouti carboxyl terminus was modeled, and residues which decrease receptor binding by a factor of > or = 15 when mutated to alanine localize to one side of the structure. These agouti variants with altered receptor selectivity may be useful in determining the role of melanocortin receptors in diabetes and obesity.

  10. A method for predicting protein-protein interaction types.

    Directory of Open Access Journals (Sweden)

    Yael Silberberg

    Full Text Available Protein-protein interactions (PPIs govern basic cellular processes through signal transduction and complex formation. The diversity of those processes gives rise to a remarkable diversity of interactions types, ranging from transient phosphorylation interactions to stable covalent bonding. Despite our increasing knowledge on PPIs in humans and other species, their types remain relatively unexplored and few annotations of types exist in public databases. Here, we propose the first method for systematic prediction of PPI type based solely on the techniques by which the interaction was detected. We show that different detection methods are better suited for detecting specific types. We apply our method to ten interaction types on a large scale human PPI dataset. We evaluate the performance of the method using both internal cross validation and external data sources. In cross validation, we obtain an area under receiver operating characteristic (ROC curve ranging from 0.65 to 0.97 with an average of 0.84 across the predicted types. Comparing the predicted interaction types to external data sources, we obtained significant agreements for phosphorylation and ubiquitination interactions, with hypergeometric p-value = 2.3e(-54 and 5.6e(-28 respectively. We examine the biological relevance of our predictions using known signaling pathways and chart the abundance of interaction types in cell processes. Finally, we investigate the cross-relations between different interaction types within the network and characterize the discovered patterns, or motifs. We expect the resulting annotated network to facilitate the reconstruction of process-specific subnetworks and assist in predicting protein function or interaction.

  11. Prediction of Protein-Protein Interacting Sites: How to Bridge Molecular Events to Large Scale Protein Interaction Networks

    Science.gov (United States)

    Bartoli, Lisa; Martelli, Pier Luigi; Rossi, Ivan; Fariselli, Piero; Casadio, Rita

    Most of the cellular functions are the result of the concerted action of protein complexes forming pathways and networks. For this reason, efforts were devoted to the study of protein-protein interactions. Large-scale experiments on whole genomes allowed the identification of interacting protein pairs. However residues involved in the interaction are generally not known and the majority of the interactions still lack a structural characterization. A crucial step towards the deciphering of the interaction mechanism of proteins is the recognition of their interacting surfaces, particularly in those structures for which also the most recent interaction network resources do not contain information. To this purpose, we developed a neural network-based method that is able to characterize protein complexes, by predicting amino acid residues that mediate the interactions. All the Protein Data Bank (PDB) chains, both in the unbound and in the complexed form, are predicted and the results are stored in a database of interaction surfaces (http://gpcr.biocomp.unibo.it/zenpatches). Finally, we performed a survey on the different computational methods for protein-protein interaction prediction and on their training/testing sets in order to highlight the most informative properties of protein interfaces.

  12. Yeast Interacting Proteins Database: YOR097C, YML008C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YOR097C - Putative protein of unknown function; identified as interacting with Hsp82p in a high-throughpu... description Putative protein of unknown function; identified as interacting with... Hsp82p in a high-throughput two-hybrid screen; YOR097C is not an essential gene Rows with this bait as bait

  13. Yeast Interacting Proteins Database: YLR223C, YOR247W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YLR223C IFH1 Essential protein with a highly acidic N-terminal domain; IFH1 exhibits genetic interactions...ion Essential protein with a highly acidic N-terminal domain; IFH1 exhibits genetic interactions with FHL1,

  14. Yeast Interacting Proteins Database: YBR187W, YNR032W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available null mutant; GFP-fusion protein localizes to the vacuole; expression pattern and physical interactions sugge...expression is reduced in a gcr1 null mutant; GFP-fusion protein localizes to the vacuole; expression pattern and physical interaction

  15. Yeast Interacting Proteins Database: YOR180C, YGL153W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available central component of the peroxisomal protein import machinery; interacts with both PTS1 (Pex5p) and PTS2...central component of the peroxisomal protein import machinery; interacts with both PTS1 (Pex5p) and PTS2

  16. Yeast Interacting Proteins Database: YCR036W, YGL153W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available central component of the peroxisomal protein import machinery; interacts with both PTS1 (Pex5p) and PTS2...central component of the peroxisomal protein import machinery; interacts with both PTS1 (Pex5p) and PTS2

  17. Yeast Interacting Proteins Database: YDR256C, YGL153W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available central component of the peroxisomal protein import machinery; interacts with both PTS1 (Pex5p) and PTS2...central component of the peroxisomal protein import machinery; interacts with both PTS1 (Pex5p) and PTS2

  18. Yeast Interacting Proteins Database: YMR280C, YOR047C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available olved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sensor... glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, an

  19. Yeast Interacting Proteins Database: YGR173W, YDR152W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available plasmic RWD domain-containing protein of unknown function; interacts with Rbg1p and Gcn1p; associates with translating...slating ribosomes; putative intrinsically unstructured p...ion Highly-acidic cytoplasmic RWD domain-containing protein of unknown function; interacts with Rbg1p and Gcn1p; associates with tran

  20. Yeast Interacting Proteins Database: YHR114W, YDR422C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available substrate specificity; vacuolar protein containing KIS (Kinase-Interacting Sequence) and ASC (Association w...strate specificity; vacuolar protein containing KIS (Kinase-Interacting Sequence) and ASC (Association with ...e 4 CuraGen (0 or 1) 0 S. Fields (0 or 1) 0 Association (0 or 1,YPD) 0 Complex (0

  1. Yeast Interacting Proteins Database: YDR026C, YDL030W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available -purification experiments; Myb-like DNA-binding protein that may bind to the Ter region of rDNA; interacts physically...n experiments; Myb-like DNA-binding protein that may bind to the Ter region of rDNA; interacts physically wi

  2. Yeast Interacting Proteins Database: YMR047C, YNL078W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available L078W NIS1 Protein localized in the bud neck at G2/M phase; physically interacts ...ene name NIS1 Prey description Protein localized in the bud neck at G2/M phase; physically interacts with se

  3. Yeast Interacting Proteins Database: YPR040W, YNR032W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YPR040W TIP41 Protein that interacts physically and genetically with Tap42p, which ... 0 0 0 0 0 - - - - - 0 0 3 - Show YPR040W Bait ORF YPR040W Bait gene name TIP41 Bait description Protein that interacts physically

  4. Yeast Interacting Proteins Database: YBR108W, YGR136W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YBR108W AIM3 Protein interacting with Rvs167p; null mutant is viable and displays e...w YBR108W Bait ORF YBR108W Bait gene name AIM3 Bait description Protein interacting with Rvs167p; null mutant is viable and display

  5. G protein-coupled receptor signaling complexity in neuronal tissue: implications for novel therapeutics.

    Science.gov (United States)

    Maudsley, Stuart; Martin, Bronwen; Luttrell, Louis M

    2007-02-01

    The manipulation of transmembrane signaling by G protein-coupled receptors (GPCRs) constitutes perhaps the single most important therapeutic target in medicine. Therapeutics acting on GPCRs have traditionally been classified as agonists, partial agonists, or antagonists based on a two state model of receptor function embodied in the ternary complex model. Over the past decade, however, many lines of investigation have shown that GPCR signaling exhibits greater diversity and 'texture' than previously appreciated. Signal diversity arises from numerous factors, among them the ability of receptors to adopt multiple 'active' states with different effector coupling profiles, the formation of receptor dimers that exhibit unique pharmacology, signaling, and trafficking, the dissociation of receptor 'activation' from desensitization and internalization, and the discovery that non-G protein effectors mediate some aspects of GPCR signaling. At the same time, clustering of GPCRs with their downstream effectors in membrane microdomains, and interactions between receptors and a plethora of multidomain scaffolding proteins and accessory/chaperone molecules confers signal preorganization, efficiency, and specificity. More importantly it is likely that alteration in the interactions of these proteins with GPCRs may occur in aging or neurodegenerative disorders, thus defining a distinct 'pharmacology' from that seen in young organisms or normal physiology. In this context, the concept of agonist selective trafficking of receptor signaling, which recognizes that a bound ligand may select between a menu of 'active' receptor conformations and induce only a subset of the possible response profile, presents the opportunity to develop drugs that change the quality as well as the quantity of efficacy and enhance these qualities for specific disorders or other paradigms. As a more comprehensive understanding of the complexity of GPCR signaling is developed, the rational design of ligands

  6. Peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands

    Energy Technology Data Exchange (ETDEWEB)

    Snyder, S.H.; Verma, A.; Trifiletti, R.R.

    1987-10-01

    The peripheral-type benzodiazepine receptor is a site identified by its nanomolar affinity for (/sup 3/H)diazepam, similar to the affinity of diazepam for the central-type benzodiazepine receptor in the brain. The peripheral type benzodiazepine receptor occurs in many peripheral tissues but has discrete localizations as indicated by autoradiographic studies showing uniquely high densities of the receptors in the adrenal cortex and in Leydig cells of the testes. Subcellular localization studies reveal a selective association of the receptors with the outer membrane of mitochondria. Photoaffinity labeling of the mitochondrial receptor with (/sup 3/H)flunitrazepam reveals two discrete labeled protein bands of 30 and 35 kDa, respectively. The 35-kDa band appears to be identical with the voltage-dependent anion channel protein porin. Fractionation of numerous peripheral tissues reveals a single principal endogenous ligand for the receptor, consisting of porphyrins, which display nanomolar affinity. Interactions of porphyrins with the mitochondrial receptor may clarify its physiological role and account for many pharmacological actions of benzodiazepines.

  7. Disruption of the Interaction of the Androgen Receptor with Chromatin: A Novel Therapeutic Approach in Prostate Cancer

    Science.gov (United States)

    2016-10-01

    level. KEYWORDS: Prostate cancer, Androgen Receptor, FOXA1, androgen receptor co- regulators , Protein-protein interactions, peptides...peptidomimetics ACCOMPLISHMENTS: What were the major goals of the project? Aim 1: Select and evaluate peptides/peptidomimetics in models of PCa. Based on...will be tested for its stability, pharmacologic properties, toxicity, efficacy in disrupting AR/FoxA1 PPI, and for activity on AR- driven gene

  8. HCVpro: hepatitis C virus protein interaction database.

    Science.gov (United States)

    Kwofie, Samuel K; Schaefer, Ulf; Sundararajan, Vijayaraghava S; Bajic, Vladimir B; Christoffels, Alan

    2011-12-01

    It is essential to catalog characterized hepatitis C virus (HCV) protein-protein interaction (PPI) data and the associated plethora of vital functional information to augment the search for therapies, vaccines and diagnostic biomarkers. In furtherance of these goals, we have developed the hepatitis C virus protein interaction database (HCVpro) by integrating manually verified hepatitis C virus-virus and virus-human protein interactions curated from literature and databases. HCVpro is a comprehensive and integrated HCV-specific knowledgebase housing consolidated information on PPIs, functional genomics and molecular data obtained from a variety of virus databases (VirHostNet, VirusMint, HCVdb and euHCVdb), and from BIND and other relevant biology repositories. HCVpro is further populated with information on hepatocellular carcinoma (HCC) related genes that are mapped onto their encoded cellular proteins. Incorporated proteins have been mapped onto Gene Ontologies, canonical pathways, Online Mendelian Inheritance in Man (OMIM) and extensively cross-referenced to other essential annotations. The database is enriched with exhaustive reviews on structure and functions of HCV proteins, current state of drug and vaccine development and links to recommended journal articles. Users can query the database using specific protein identifiers (IDs), chromosomal locations of a gene, interaction detection methods, indexed PubMed sources as well as HCVpro, BIND and VirusMint IDs. The use of HCVpro is free and the resource can be accessed via http://apps.sanbi.ac.za/hcvpro/ or http://cbrc.kaust.edu.sa/hcvpro/. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Quantitative Interaction Proteomics of Neurodegenerative Disease Proteins

    Directory of Open Access Journals (Sweden)

    Fabian Hosp

    2015-05-01

    Full Text Available Several proteins have been linked to neurodegenerative disorders (NDDs, but their molecular function is not completely understood. Here, we used quantitative interaction proteomics to identify binding partners of Amyloid beta precursor protein (APP and Presenilin-1 (PSEN1 for Alzheimer’s disease (AD, Huntingtin (HTT for Huntington’s disease, Parkin (PARK2 for Parkinson’s disease, and Ataxin-1 (ATXN1 for spinocerebellar ataxia type 1. Our network reveals common signatures of protein degradation and misfolding and recapitulates known biology. Toxicity modifier screens and comparison to genome-wide association studies show that interaction partners are significantly linked to disease phenotypes in vivo. Direct comparison of wild-type proteins and disease-associated variants identified binders involved in pathogenesis, highlighting the value of differential interactome mapping. Finally, we show that the mitochondrial protein LRPPRC interacts preferentially with an early-onset AD variant of APP. This interaction appears to induce mitochondrial dysfunction, which is an early phenotype of AD.

  10. The effect of protein-protein and protein-membrane interactions on membrane fouling in ultrafiltration

    NARCIS (Netherlands)

    Huisman, I.H.; Prádanos, P.; Hernández, A.

    2000-01-01

    It was studied how protein-protein and protein-membrane interactions influence the filtration performance during the ultrafiltration of protein solutions over polymeric membranes. This was done by measuring flux, streaming potential, and protein transmission during filtration of bovine serum albumin

  11. Targeting protein-protein interactions for parasite control.

    Directory of Open Access Journals (Sweden)

    Christina M Taylor

    2011-04-01

    Full Text Available Finding new drug targets for pathogenic infections would be of great utility for humanity, as there is a large need to develop new drugs to fight infections due to the developing resistance and side effects of current treatments. Current drug targets for pathogen infections involve only a single protein. However, proteins rarely act in isolation, and the majority of biological processes occur via interactions with other proteins, so protein-protein interactions (PPIs offer a realm of unexplored potential drug targets and are thought to be the next-generation of drug targets. Parasitic worms were chosen for this study because they have deleterious effects on human health, livestock, and plants, costing society billions of dollars annually and many sequenced genomes are available. In this study, we present a computational approach that utilizes whole genomes of 6 parasitic and 1 free-living worm species and 2 hosts. The species were placed in orthologous groups, then binned in species-specific orthologous groups. Proteins that are essential and conserved among species that span a phyla are of greatest value, as they provide foundations for developing broad-control strategies. Two PPI databases were used to find PPIs within the species specific bins. PPIs with unique helminth proteins and helminth proteins with unique features relative to the host, such as indels, were prioritized as drug targets. The PPIs were scored based on RNAi phenotype and homology to the PDB (Protein DataBank. EST data for the various life stages, GO annotation, and druggability were also taken into consideration. Several PPIs emerged from this study as potential drug targets. A few interactions were supported by co-localization of expression in M. incognita (plant parasite and B. malayi (H. sapiens parasite, which have extremely different modes of parasitism. As more genomes of pathogens are sequenced and PPI databases expanded, this methodology will become increasingly

  12. Presynaptic G Protein-Coupled Receptors: Gatekeepers of Addiction?

    Directory of Open Access Journals (Sweden)

    Kari A Johnson

    2016-11-01

    Full Text Available Drug abuse and addiction cause widespread social and public health problems, and the neurobiology underlying drug actions and drug use and abuse is an area of intensive research. Drugs of abuse alter synaptic transmission, and these actions contribute to acute intoxication as well as the chronic effects of abused substances. Transmission at most mammalian synapses involves neurotransmitter activation of two receptor subtypes, ligand-gated ion channels that mediate fast synaptic responses, and G protein-coupled receptors (GPCRs that have slower neuromodulatory actions. The GPCRs represent a large proportion of neurotransmitter receptors involved in almost all facets of nervous system function. In addition, these receptors are targets for many pharmacotherapeutic agents. Drugs of abuse directly or indirectly affect neuromodulation mediated by GPCRs, with important consequences for intoxication, drug taking and responses to prolonged drug exposure, withdrawal and addiction. Among the GPCRs are several subtypes involved in presynaptic inhibition, most of which are coupled to the Gi/o class of G protein. There is increasing evidence that these presynaptic Gi/o-coupled GPCRs have important roles in the actions of drugs of abuse, as well as behaviors related to these drugs. This topic will be reviewed, with particular emphasis on receptors for three neurotransmitters, dopamine (D1- and D2-like receptors, endocannabinoids (CB1 receptors and glutamate (group II metabotropic glutamate (mGlu receptors. The focus is on recent evidence from laboratory animal models (and some evidence in humans implicating these receptors in the acute and chronic effects of numerous abused drugs, as well as in the control of drug seeking and taking. The ability of drugs targeting these receptors to modify drug seeking behavior has raised the possibility of using compounds targeting these receptors for addiction pharmacotherapy. This topic is also discussed, with emphasis on

  13. The roles of post-translational modifications in the context of protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Guangyou Duan

    2015-02-01

    Full Text Available Among other effects, post-translational modifications (PTMs have been shown to exert their function via the modulation of protein-protein interactions. For twelve different main PTM-types and associated subtypes and across 9 diverse species, we investigated whether particular PTM-types are associated with proteins with specific and possibly "strategic" placements in the network of all protein interactions by determining informative network-theoretic properties. Proteins undergoing a PTM were observed to engage in more interactions and positioned in more central locations than non-PTM proteins. Among the twelve considered PTM-types, phosphorylated proteins were identified most consistently as being situated in central network locations and with the broadest interaction spectrum to proteins carrying other PTM-types, while glycosylated proteins are preferentially located at the network periphery. For the human interactome, proteins undergoing sumoylation or proteolytic cleavage were found with the most characteristic network properties. PTM-type-specific protein interaction network (PIN properties can be rationalized with regard to the function of the respective PTM-carrying proteins. For example, glycosylation sites were found enriched in proteins with plasma membrane localizations and transporter or receptor activity, which generally have fewer interacting partners. The involvement in disease processes of human proteins undergoing PTMs was also found associated with characteristic PIN properties. By integrating global protein interaction networks and specific PTMs, our study offers a novel approach to unraveling the role of PTMs in cellular processes.

  14. The roles of post-translational modifications in the context of protein interaction networks.

    Science.gov (United States)

    Duan, Guangyou; Walther, Dirk

    2015-02-01

    Among other effects, post-translational modifications (PTMs) have been shown to exert their function via the modulation of protein-protein interactions. For twelve different main PTM-types and associated subtypes and across 9 diverse species, we investigated whether particular PTM-types are associated with proteins with specific and possibly "strategic" placements in the network of all protein interactions by determining informative network-theoretic properties. Proteins undergoing a PTM were observed to engage in more interactions and positioned in more central locations than non-PTM proteins. Among the twelve considered PTM-types, phosphorylated proteins were identified most consistently as being situated in central network locations and with the broadest interaction spectrum to proteins carrying other PTM-types, while glycosylated proteins are preferentially located at the network periphery. For the human interactome, proteins undergoing sumoylation or proteolytic cleavage were found with the most characteristic network properties. PTM-type-specific protein interaction network (PIN) properties can be rationalized with regard to the function of the respective PTM-carrying proteins. For example, glycosylation sites were found enriched in proteins with plasma membrane localizations and transporter or receptor activity, which generally have fewer interacting partners. The involvement in disease processes of human proteins undergoing PTMs was also found associated with characteristic PIN properties. By integrating global protein interaction networks and specific PTMs, our study offers a novel approach to unraveling the role of PTMs in cellular processes.

  15. Mineralocorticoid receptor interaction with SP1 generates a new response element for pathophysiologically relevant gene expression.

    Science.gov (United States)

    Meinel, Sandra; Ruhs, Stefanie; Schumann, Katja; Strätz, Nicole; Trenkmann, Kay; Schreier, Barbara; Grosse, Ivo; Keilwagen, Jens; Gekle, Michael; Grossmann, Claudia

    2013-09-01

    The mineralocorticoid receptor (MR) is a ligand-induced transcription factor belonging to the steroid receptor family and involved in water-electrolyte homeostasis, blood pressure regulation, inflammation and fibrosis in the renocardiovascular system. The MR shares a common hormone-response-element with the glucocorticoid receptor but nevertheless elicits MR-specific effects including enhanced epidermal growth factor receptor (EGFR) expression via unknown mechanisms. The EGFR is a receptor tyrosine kinase that leads to activation of MAP kinases, but that can also function as a signal transducer for other signaling pathways. In the present study, we mechanistically investigate the interaction between a newly discovered MR- but not glucocorticoid receptor- responsive-element (=MRE1) of the EGFR promoter, specificity protein 1 (SP1) and MR to gain general insights into MR-specificity. Biological relevance of the interaction for EGFR expression and consequently for different signaling pathways in general is demonstrated in human, rat and murine vascular smooth muscle cells and cells of EGFR knockout mice. A genome-wide promoter search for identical binding regions followed by quantitative PCR validation suggests that the identified MR-SP1-MRE1 interaction might be applicable to other genes. Overall, a novel principle of MR-specific gene expression is explored that applies to the pathophysiologically relevant expression of the EGFR and potentially also to other genes.

  16. Quantitative study of protein-protein interactions by quartz nanopipettes

    Science.gov (United States)

    Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin

    2014-08-01

    In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with

  17. Interactions affected by arginine methylation in the yeast protein-protein interaction network.

    Science.gov (United States)

    Erce, Melissa A; Abeygunawardena, Dhanushi; Low, Jason K K; Hart-Smith, Gene; Wilkins, Marc R

    2013-11-01

    Protein-protein interactions can be modulated by the methylation of arginine residues. As a means of testing this, we recently described a conditional two-hybrid system, based on the bacterial adenylate cyclase (BACTH) system. Here, we have used this conditional two-hybrid system to explore the effect of arginine methylation in modulating protein-protein interactions in a subset of the Saccharomyces cerevisiae arginine methylproteome network. Interactions between the yeast hub protein Npl3 and yeast proteins Air2, Ded1, Gbp2, Snp1, and Yra1 were first validated in the absence of methylation. The major yeast arginine methyltransferase Hmt1 was subsequently included in the conditional two-hybrid assay, initially to determine the degree of methylation that occurs. Proteins Snp1 and Yra1 were confirmed as Hmt1 substrates, with five and two novel arginine methylation sites mapped by ETD LC-MS/MS on these proteins, respectively. Proteins Ded1 and Gbp2, previously predicted but not confirmed as substrates of Hmt1, were also found to be methylated with five and seven sites mapped respectively. Air2 was found to be a novel substrate of Hmt1 with two sites mapped. Finally, we investigated the interactions of Npl3 with the five interaction partners in the presence of active Hmt1 and in the presence of Hmt1 with a G68R inactivation mutation. We found that the interaction between Npl3 and Air2, and Npl3 and Ded1, were significantly increased in the presence of active Hmt1; the interaction of Npl3 and Snp1 showed a similar degree of increase in interaction but this was not statistically significant. The interactions of Npl3 and Gbp2, along with Npl3 and Yra1, were not significantly increased or decreased by methylation. We conclude that methylarginine may be a widespread means by which the interactions of proteins are modulated.

  18. Yeast Interacting Proteins Database: YGL127C, YOR047C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ith protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; acts as a regula...rotein involved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sensors

  19. Yeast Interacting Proteins Database: YOR358W, YOR047C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; act...rotein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein Spt15p; acts as a regulator o

  20. Fibroblast growth factor-21 (FGF21) regulates low-density lipoprotein receptor (LDLR) levels in cells via the E3-ubiquitin ligase Mylip/Idol and the Canopy2 (Cnpy2)/Mylip-interacting saposin-like protein (Msap).

    Science.gov (United States)

    Do, Hai Thi; Tselykh, Timofey V; Mäkelä, Johanna; Ho, Tho Huu; Olkkonen, Vesa M; Bornhauser, Beat C; Korhonen, Laura; Zelcer, Noam; Lindholm, Dan

    2012-04-13

    The LDLR is a critical factor in the regulation of blood cholesterol levels that are altered in different human diseases. The level of LDLR in the cell is regulated by both transcriptional and post-transcriptional events. The E3 ubiquitin ligase, myosin regulatory light chain-interacting protein (Mylip)/inducible degrader of the LDL-R (Idol) was shown to induce degradation of LDLR via protein ubiquitination. We have here studied novel factors and mechanisms that may regulate Mylip/Idol in human hepatocyte cells and in mouse macrophages. We observed that FGF21 that is present in serum in different conditions reduced Mylip/Idol at the RNA and protein level, and increased LDLR levels and stability in the cells. FGF21 also enhanced expression of Canopy2 (Cnpy2)/MIR-interacting Saposin-like protein (Msap) that is known to interact with Mylip/Idol. Overexpression of Cnpy2/Msap increased LDLRs, and knockdown experiments showed that Cnpy2/Msap is crucial for the FGF21 effect on LDLRs. Experiments using DiI-labeled LDL particles showed that FGF21 increased lipoprotein uptake and the effect of FGF21 was additive to that of statins. Our results are consistent with an important role of FGF21 and Cnpy2/Msap in the regulation of LDLRs in cultured cells, which warrants further studies using human samples.

  1. Fibroblast Growth Factor-21 (FGF21) Regulates Low-density Lipoprotein Receptor (LDLR) Levels in Cells via the E3-ubiquitin Ligase Mylip/Idol and the Canopy2 (Cnpy2)/Mylip-interacting Saposin-like Protein (Msap)*

    Science.gov (United States)

    Do, Hai Thi; Tselykh, Timofey V.; Mäkelä, Johanna; Ho, Tho Huu; Olkkonen, Vesa M.; Bornhauser, Beat C.; Korhonen, Laura; Zelcer, Noam; Lindholm, Dan

    2012-01-01

    The LDLR is a critical factor in the regulation of blood cholesterol levels that are altered in different human diseases. The level of LDLR in the cell is regulated by both transcriptional and post-transcriptional events. The E3 ubiquitin ligase, myosin regulatory light chain-interacting protein (Mylip)/inducible degrader of the LDL-R (Idol) was shown to induce degradation of LDLR via protein ubiquitination. We have here studied novel factors and mechanisms that may regulate Mylip/Idol in human hepatocyte cells and in mouse macrophages. We observed that FGF21 that is present in serum in different conditions reduced Mylip/Idol at the RNA and protein level, and increased LDLR levels and stability in the cells. FGF21 also enhanced expression of Canopy2 (Cnpy2)/MIR-interacting Saposin-like protein (Msap) that is known to interact with Mylip/Idol. Overexpression of Cnpy2/Msap increased LDLRs, and knockdown experiments showed that Cnpy2/Msap is crucial for the FGF21 effect on LDLRs. Experiments using DiI-labeled LDL particles showed that FGF21 increased lipoprotein uptake and the effect of FGF21 was additive to that of statins. Our results are consistent with an important role of FGF21 and Cnpy2/Msap in the regulation of LDLRs in cultured cells, which warrants further studies using human samples. PMID:22378787

  2. Alix facilitates the interaction between c-Cbl and platelet-derived growth factor beta-receptor and thereby modulates receptor down-regulation.

    Science.gov (United States)

    Lennartsson, Johan; Wardega, Piotr; Engström, Ulla; Hellman, Ulf; Heldin, Carl-Henrik

    2006-12-22

    Alix (ALG-2-interacting protein X) is an adaptor protein involved in down-regulation and sorting of cell surface receptors through the endosomal compartments toward the lysosome. In this study, we show that Alix interacts with the C-terminal region of the platelet-derived growth factor (PDGF) beta-receptor (PDGFRbeta) and becomes transiently tyrosine-phosphorylated in response to PDGF-BB stimulation. Increased expression levels of Alix resulted in a reduced rate of PDGFRbeta removal from the cell surface following receptor activation, and this was associated with decreased receptor degradation. Furthermore, Alix was found to co-immunoprecipitate with the ubiquitin ligase c-Cbl, and elevated Alix levels increased the interaction between c-Cbl and PDGFRbeta. Interestingly, Alix interacted constitutively with both c-Cbl and PDGFRbeta. Moreover, c-Cbl was found to be hyperphosphorylated in cells engineered to overexpress Alix compared with control cells. The increased c-Cbl phosphorylation correlated with enhanced proteasomal degradation of c-Cbl, which in turn correlated with a decreased ubiquitination of PDGFRbeta. Our data suggest that Alix inhibits down-regulation of PDGFRbeta by modulating the interaction between c-Cbl and the receptor, thereby affecting the ubiquitination of the receptor.

  3. The interaction between signal regulatory protein alpha (SIRPα) and CD47: structure, function, and therapeutic target

    NARCIS (Netherlands)

    Barclay, A. Neil; van den Berg, Timo K.

    2014-01-01

    CD47 is a broadly expressed membrane protein that interacts with the myeloid inhibitory immunoreceptor SIRPα (also termed CD172a or SHPS-1). SIRPα is the prototypic member of the SIRP paired receptor family of closely related SIRP proteins. Engagement of SIRPα by CD47 provides a downregulatory

  4. Studying protein-protein interactions using peptide arrays

    NARCIS (Netherlands)

    Katz, C.; Levy-Beladev, L.; Rotem-Bamberger, S.; Rito, T.; Rudiger, S.G.D.; Friedler, A.

    2010-01-01

    Screening of arrays and libraries of compounds is well-established as a high-throughput method for detecting and analyzing interactions in both biological and chemical systems. Arrays and libraries can be composed from various types of molecules, ranging from small organic compounds to DNA, proteins

  5. Motif mediated protein-protein interactions as drug targets.

    Science.gov (United States)

    Corbi-Verge, Carles; Kim, Philip M

    2016-03-02

    Protein-protein interactions (PPI) are involved in virtually every cellular process and thus represent an attractive target for therapeutic interventions. A significant number of protein interactions are frequently formed between globular domains and short linear peptide motifs (DMI). Targeting these DMIs has proven challenging and classical approaches to inhibiting such interactions with small molecules have had limited success. However, recent new approaches have led to the discovery of potent inhibitors, some of them, such as Obatoclax, ABT-199, AEG-40826 and SAH-p53-8 are likely to become approved drugs. These novel inhibitors belong to a wide range of different molecule classes, ranging from small molecules to peptidomimetics and biologicals. This article reviews the main reasons for limited success in targeting PPIs, discusses how successful approaches overcome these obstacles to discovery promising inhibitors for human protein double minute 2 (HDM2), B-cell lymphoma 2 (Bcl-2), X-linked inhibitor of apoptosis protein (XIAP), and provides a summary of the promising approaches currently in development that indicate the future potential of PPI inhibitors in drug discovery.

  6. Species difference in the G protein selectivity of the human and bovine A1-adenosine receptor.

    Science.gov (United States)

    Jockers, R; Linder, M E; Hohenegger, M; Nanoff, C; Bertin, B; Strosberg, A D; Marullo, S; Freissmuth, M

    1994-12-23

    The purified bovine brain A1-adenosine receptor has previously been shown to discriminate among closely related G protein alpha-subunits. To obtain analogous information for the human receptor, the cDNA coding for the human A1-adenosine receptor was inserted into a plasmid placing the synthesis of the receptor protein under the control of the MalE promoter. Following induction by maltose, active receptor accumulated in Escherichia coli membranes. Binding of the antagonist 8-[3H]cyclopentyl-1,3-dipropylxanthine to E. coli membranes (KD approximately 2 nM, Bmax approximately 0.2-0.4 pmol/mg) showed the appropriate pharmacological profile. Incubation of E. coli membranes with purified Go,i-reconstituted guanine nucleotide-sensitive high affinity binding of the agonist (-)[125I] N6-3-(iodo-4-hydroxyphenylisopropyl)adenosine to the receptor (KD approximately 1 nM). In the presence of purified beta gamma-subunit, the recombinant receptor interacted equally well with the recombinant G protein alpha-subunits Gi alpha-1, Gi alpha-2, Gi alpha-3; G(o) alpha displayed a lower affinity for the receptor while Gs alpha was inactive. Parallel experiments were carried out in bovine and human brain membranes pretreated with N-ethylmaleimide to inactivate the endogenous G(o)/Gi proteins; Gi alpha-3 was most potent in reconstituting 125I-HPIA binding to bovine membranes, while Gi alpha-1, Gi alpha-2, and G(o) alpha displayed similar affinities. However, in human membranes, Gi alpha-1, Gi alpha-2, and Gi alpha-3, were equipotent and high concentrations of G(o) alpha were required to promote 125I-HPIA binding. These observations show (i) that functional human A1-adenosine receptors were synthesized in E. coli; (ii) that the pattern of G protein coupling is identical for the recombinant human A1-receptor and its counterpart in the native membrane; (iii) and that species differences between bovine and human receptor exist not only in their pharmacological profile but also in their G

  7. The repertoire of trace amine G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Gloriam, David E.; Bjarnadóttir, Thóra K; Yan, Yi-Lin

    2005-01-01

    Trace amines, such as tyramine, beta-phenylethylamine, tryptamine, and octopamine, are present in trace levels in nervous systems and bind a specific family of G-protein-coupled receptors (GPCR), but the function or origin of this system is not well understood. We searched the genomes of several ...... ancestor of vertebrate TA-receptors arose before the split of the ray-finned and lobe-finned fishes. The evolutionary history of the TA-receptors is more complex than for most other GPCR families and here we suggest a mechanism by which they may have arisen....

  8. A framework for protein and membrane interactions

    Directory of Open Access Journals (Sweden)

    Giorgio Bacci

    2009-11-01

    Full Text Available We introduce the BioBeta Framework, a meta-model for both protein-level and membrane-level interactions of living cells. This formalism aims to provide a formal setting where to encode, compare and merge models at different abstraction levels; in particular, higher-level (e.g. membrane activities can be given a formal biological justification in terms of low-level (i.e., protein interactions. A BioBeta specification provides a protein signature together a set of protein reactions, in the spirit of the kappa-calculus. Moreover, the specification describes when a protein configuration triggers one of the only two membrane interaction allowed, that is "pinch" and "fuse". In this paper we define the syntax and semantics of BioBeta, analyse its properties, give it an interpretation as biobigraphical reactive systems, and discuss its expressivity by comparing with kappa-calculus and modelling significant examples. Notably, BioBeta has been designed after a bigraphical metamodel for the same purposes. Hence, each instance of the calculus corresponds to a bigraphical reactive system, and vice versa (almost. Therefore, we can inherith the rich theory of bigraphs, such as the automatic construction of labelled transition systems and behavioural congruences.

  9. PCorral--interactive mining of protein interactions from MEDLINE.

    Science.gov (United States)

    Li, Chen; Jimeno-Yepes, Antonio; Arregui, Miguel; Kirsch, Harald; Rebholz-Schuhmann, Dietrich

    2013-01-01

    The extraction of information from the scientific literature is a complex task-for researchers doing manual curation and for automatic text processing solutions. The identification of protein-protein interactions (PPIs) requires the extraction of protein named entities and their relations. Semi-automatic interactive support is one approach to combine both solutions for efficient working processes to generate reliable database content. In principle, the extraction of PPIs can be achieved with different methods that can be combined to deliver high precision and/or high recall results in different combinations at the same time. Interactive use can be achieved, if the analytical methods are fast enough to process the retrieved documents. PCorral provides interactive mining of PPIs from the scientific literature allowing curators to skim MEDLINE for PPIs at low overheads. The keyword query to PCorral steers the selection of documents, and the subsequent text analysis generates high recall and high precision results for the curator. The underlying components of PCorral process the documents on-the-fly and are available, as well, as web service from the Whatizit infrastructure. The human interface summarizes the identified PPI results, and the involved entities are linked to relevant resources and databases. Altogether, PCorral serves curator at both the beginning and the end of the curation workflow for information retrieval and information extraction. Database URL: http://www.ebi.ac.uk/Rebholz-srv/pcorral.

  10. G protein-coupled receptor accessory proteins and signaling: pharmacogenomic insights.

    Science.gov (United States)

    Thompson, Miles D; Cole, David E C; Jose, Pedro A; Chidiac, Peter

    2014-01-01

    The identification and characterization of the genes encoding G protein-coupled receptors (GPCRs) and the proteins necessary for the processes of ligand binding, GPCR activation, inactivation, and receptor trafficking to the membrane are discussed in the context of human genetic disease. In addition to functional GPCR variants, the identification of genetic disruptions affecting proteins necessary to GPCR functions have provided insights into the function of these pathways. Gsα and Gβ subunit polymorphisms have been found to result in complex phenotypes. Disruptions in accessory proteins that normally modify or organize heterotrimeric G-protein coupling may also result in disease states. These include the contribution of variants of the regulator of G protein signaling (RGS) protein to hypertension; the role variants of the activator of G protein signaling (AGS) proteins to phenotypes (such as the type III AGS8 variant to hypoxia); the contribution of G protein-coupled receptor kinase (GRK) proteins, such as GRK4, in disorders such as hypertension. The role of accessory proteins in GPCR structure and function is discussed in the context of genetic disorders associated with disruption of the genes that encode them. An understanding of the pharmacogenomics of GPCR and accessory protein signaling provides the basis for examining both GPCR pharmacogenetics and the genetics of monogenic disorders that result from disruption of given receptor systems.

  11. Unconventional interaction forces in protein and protein-ligand systems and their impacts to drug design.

    Science.gov (United States)

    Wang, Qing-Yan; Lu, Jian; Liao, Si-Ming; Du, Qi-Shi; Huang, Ri-Bo

    2013-01-01

    In drug design and enzyme engineering, the information of interactions between receptors and ligands is crucially important. In many cases, the protein structures and drug-target complex structures are determined by a delicate balance of several weak molecular interaction types. Among these interaction forces several unconventional interactions play important roles, however, less familiar for researchers. The cation-π interaction is a unique noncovalent interaction only acting between aromatic amino acids and organic cations (protonated amino acids) and inorganic cations (proton and metallic). This article reports new study results in the interaction strength, the behaviors and the structural characters of cation-π interactions between aromatic amino acids (Phe, Tyr, and Trp) and organic and inorganic cations (Lys(+), Arg(+), H(+), H3O(+), Li(+), Na(+), K(+), Ca(2+), and Zn(2+)) in gas phase and in solutions (water, acetonitrile, and cyclohexane). Systematical research revealed that the cation-π interactions are point-to-plane (aromatic group) interactions, distance and orientationdependent, and the interaction energies change in a broad range. In gas phase the cation-π interaction energies between aromatic amino acids (Phe, Tyr, and Trp) and metallic cations (Li(+), Na(+), K(+), Ca(2+), and Zn(2+)) are in the range -12 to -160 kcal/mol, and the interaction energies of protonated amino acids (Arg(+) and Lys(+)) are in the range from -9 to -18 kcal/mol. In solutions the cation-π energies decrease with the dielectric constant ε of solvents. However, in aqueous solution the cation-π energies of H3O(+) and protonated amino acids are less affected by solvation effects. The applications of unconventional interaction forces in drug design and in protein engineering are introduced.

  12. All three LDL receptor homology regions of the LDL receptor-related protein bind multiple ligands.

    Science.gov (United States)

    Croy, Johnny E; Shin, William D; Knauer, Mary F; Knauer, Daniel J; Komives, Elizabeth A

    2003-11-11

    The three complete human LDL receptor homology regions of the LDL receptor-related protein (sLRP2, sLRP3, and sLRP4) have been expressed in Pichia pastoris SMD1168 with constitutive coexpression of the receptor-associated protein (RAP). Each sLRP was purified to homogeneity after deglycosylation using a combination of anion-exchange and size exclusion chromatography. Mass spectrometry and N-terminal sequencing confirmed the identity of each fragment at purified yields of several milligrams per liter. Despite the large number of disulfide linkages and glycosylation sites in each LDL receptor homology region (sLRP), all were shown to be competent for binding to several LRP1 ligands. Each sLRP also bound human RAP, which is thought to be a generalized receptor antagonist, in solution-binding experiments. As expected, sLRP2 bound the receptor-binding domain of alpha(2)-macroglobulin (residues 1304-1451). All three sLRPs bound human apolipoprotein-enriched beta very low density lipoprotein, the canonical ligand for this receptor. All three sLRPs also bound lactoferrin and thrombin-protease nexin 1 complexes. Only sLRP4 bound thrombin-antithrombin III complexes. The results show that binding-competent LDL receptor homology regions (sLRPs) can be produced in high yield in P. pastoris and readily purified. Each sLRP has binding sites for multiple ligands, but not all ligand binding could be competed by RAP.

  13. Interaction between mineralocorticoid receptor and cAMP/CREB signaling.

    Science.gov (United States)

    Grossmann, Claudia; Ruhs, Stefanie; Seiferth, Anja; Gekle, Michael

    2010-01-01

    Besides regulating water and electrolyte homeostasis, the mineralocorticoid receptor (MR) elicits pathophysiological effects in the renocardiovascular system. Although the MR's closest relative, the glucocorticoid receptor (GR), acts as a transcription factor at the same hormone-response-element (HRE), activated glucocorticoid receptor mediates very different effects. One explanation for this discrepancy is that the MR interacts with additional signaling pathways in the cytosol. In the literature, there are several indications for an interaction between aldosterone/MR and the cAMP/CREB signaling. Here we summarize the current knowledge of the cross-talk between the two signaling pathways, including some unpublished observations of our own that indicate that MR/CREB signaling is mediated by calcineurin and has potentially pathophysiological consequences. Copyright 2009 Elsevier Inc. All rights reserved.

  14. Next-Generation Sequencing for Binary Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Bernhard eSuter

    2015-12-01

    Full Text Available The yeast two-hybrid (Y2H system exploits host cell genetics in order to display binary protein-protein interactions (PPIs via defined and selectable phenotypes. Numerous improvements have been made to this method, adapting the screening principle for diverse applications, including drug discovery and the scale-up for proteome wide interaction screens in human and other organisms. Here we discuss a systematic workflow and analysis scheme for screening data generated by Y2H and related assays that includes high-throughput selection procedures, readout of comprehensive results via next-generation sequencing (NGS, and the interpretation of interaction data via quantitative statistics. The novel assays and tools will serve the broader scientific community to harness the power of NGS technology to address PPI networks in health and disease. We discuss examples of how this next-generation platform can be applied to address specific questions in diverse fields of biology and medicine.

  15. Next-Generation Sequencing for Binary Protein-Protein Interactions.

    Science.gov (United States)

    Suter, Bernhard; Zhang, Xinmin; Pesce, C Gustavo; Mendelsohn, Andrew R; Dinesh-Kumar, Savithramma P; Mao, Jian-Hua

    2015-01-01

    The yeast two-hybrid (Y2H) system exploits host cell genetics in order to display binary protein-protein interactions (PPIs) via defined and selectable phenotypes. Numerous improvements have been made to this method, adapting the screening principle for diverse applications, including drug discovery and the scale-up for proteome wide interaction screens in human and other organisms. Here we discuss a systematic workflow and analysis scheme for screening data generated by Y2H and related assays that includes high-throughput selection procedures, readout of comprehensive results via next-generation sequencing (NGS), and the interpretation of interaction data via quantitative statistics. The novel assays and tools will serve the broader scientific community to harness the power of NGS technology to address PPI networks in health and disease. We discuss examples of how this next-generation platform can be applied to address specific questions in diverse fields of biology and medicine.

  16. Heparan sulfate proteoglycans: structure, protein interactions and cell signaling

    Directory of Open Access Journals (Sweden)

    Juliana L. Dreyfuss

    2009-09-01

    Full Text Available Heparan sulfate proteoglycans are ubiquitously found at the cell surface and extracellular matrix in all the animal species. This review will focus on the structural characteristics of the heparan sulfate proteoglycans related to protein interactions leading to cell signaling. The heparan sulfate chains due to their vast structural diversity are able to bind and interact with a wide variety of proteins, such as growth factors, chemokines, morphogens, extracellular matrix components, enzymes, among others. There is a specificity directing the interactions of heparan sulfates and target proteins, regarding both the fine structure of the polysaccharide chain as well precise protein motifs. Heparan sulfates play a role in cellular signaling either as receptor or co-receptor for different ligands, and the activation of downstream pathways is related to phosphorylation of different cytosolic proteins either directly or involving cytoskeleton interactions leading to gene regulation. The role of the heparan sulfate proteoglycans in cellular signaling and endocytic uptake pathways is also discussed.Proteoglicanos de heparam sulfato são encontrados tanto superfície celular quanto na matriz extracelular em todas as espécies animais. Esta revisão tem enfoque nas características estruturais dos proteoglicanos de heparam sulfato e nas interações destes proteoglicanos com proteínas que levam à sinalização celular. As cadeias de heparam sulfato, devido a sua variedade estrutural, são capazes de se ligar e interagir com ampla gama de proteínas, como fatores de crescimento, quimiocinas, morfógenos, componentes da matriz extracelular, enzimas, entreoutros. Existe uma especificidade estrutural que direciona as interações dos heparam sulfatos e proteínas alvo. Esta especificidade está relacionada com a estrutura da cadeia do polissacarídeo e os motivos conservados da cadeia polipeptídica das proteínas envolvidas nesta interação. Os heparam

  17. Interaction of plant polyphenols with salivary proteins.

    Science.gov (United States)

    Bennick, Anders

    2002-01-01

    Tannins are polyphenols that occur widespread in plant-based food. They are considered to be part of the plant defense system against environmental stressors. Tannins have a number of effects on animals, including growth-rate depression and inhibition of digestive enzymes. Tannins also have an effect on humans: They are, for example, the cause of byssinosis, a condition that is due to exposure to airborne tannin. Their biological effect is related to the great efficiency by which tannins precipitate proteins, an interaction that occurs by hydrophobic forces and hydrogen bonding. Two groups of salivary proteins, proline-rich proteins and histatins, are highly effective precipitators of tannin, and there is evidence that at least proline-rich proteins act as a first line of defense against tannins, perhaps by precipitating tannins in food and preventing their absorption from the alimentary canal. Proline plays an important role in the interaction of proline-rich proteins with tannins. In contrast, it is primarily basic residues that are responsible for the binding of histatins to tannin. The high concentration of tannin-binding proteins in human saliva may be related to the fruit and vegetable diet of human ancestors.

  18. Drosophila protein interaction map (DPiM): a paradigm for metazoan protein complex interactions.

    Science.gov (United States)

    Guruharsha, K G; Obar, Robert A; Mintseris, Julian; Aishwarya, K; Krishnan, R T; Vijayraghavan, K; Artavanis-Tsakonas, Spyros

    2012-01-01

    Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when, what and where of potential interactions, is therefore crucial to understanding the cellular function of any protein-especially those that have not been well studied by traditional molecular genetic approaches. We generated a large-scale resource of affinity-tagged expression-ready clones and used co-affinity purification combined with tandem mass-spectrometry to identify protein partners of nearly 5,000 Drosophila melanogaster proteins. The resulting protein complex "map" provided a blueprint of metazoan protein complex organization. Here we describe how the map has provided valuable insights into protein function in addition to generating hundreds of testable hypotheses. We also discuss recent technological advancements that will be critical in addressing the next generation of questions arising from the map.

  19. Specificity and stability of transient protein-protein interactions.

    Science.gov (United States)

    Vishwanath, Sneha; Sukhwal, Anshul; Sowdhamini, Ramanathan; Srinivasan, Narayanaswamy

    2017-06-01

    Remarkable features that are achieved in a protein-protein complex to precise levels are stability and specificity. Deviation from the normal levels of specificity and stability, which is often caused by mutations, could result in disease conditions. Chemical nature, 3-D arrangement and dynamics of interface residues code for both specificity and stability. This article reviews roles of interfacial residues in transient protein-protein complexes. It is proposed that aside from hotspot residues conferring stability to the complex, a small set of 'rigid' residues at the interface that maintain conformation between complexed and uncomplexed forms, play a major role in conferring specificity. Exceptionally, 'super hotspot' residues, which confer both stability and specificity, are attractive sites for interaction with small molecule inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Interaction of chemokines with their receptors--from initial chemokine binding to receptor activating steps

    DEFF Research Database (Denmark)

    Thiele, Stefanie; Rosenkilde, Mette Marie

    2014-01-01

    The human chemokine system comprises 19 seven-transmembrane helix (7TM) receptors and 45 endogenous chemokines that often interact with each other in a promiscuous manner. Due to the chemokine system's primary function in leukocyte migration, it has a central role in immune homeostasis and survei......The human chemokine system comprises 19 seven-transmembrane helix (7TM) receptors and 45 endogenous chemokines that often interact with each other in a promiscuous manner. Due to the chemokine system's primary function in leukocyte migration, it has a central role in immune homeostasis...

  1. A molecular modelling study of the interaction of noradrenaline with the β 2-adrenergic receptor

    Science.gov (United States)

    Mitchell, T. J.; Tute, M. S.; Webb, G. A.

    1989-09-01

    A model of the β 2-adrenergic receptor binding site is built from the primary structure of the receptor, experimental evidence for key binding residues and analogy with a homologous protein of partially determined structure. It is suggested that residues Trp-109, Thr-110 and Asp-113 are involved in ligand binding. Noradrenaline is successfully docked into this model, and the results of an INDO molecular orbital calculation on the complex indicate that a charge transfer interaction between Trp-109 and noradrenaline is possible.

  2. Protein-Protein Interaction Site Predictions with Three-Dimensional Probability Distributions of Interacting Atoms on Protein Surfaces

    Science.gov (United States)

    Chen, Ching-Tai; Peng, Hung-Pin; Jian, Jhih-Wei; Tsai, Keng-Chang; Chang, Jeng-Yih; Yang, Ei-Wen; Chen, Jun-Bo; Ho, Shinn-Ying; Hsu, Wen-Lian; Yang, An-Suei

    2012-01-01

    Protein-protein interactions are key to many biological processes. Computational methodologies devised to predict protein-protein interaction (PPI) sites on protein surfaces are important tools in providing insights into the biological functions of proteins and in developing therapeutics targeting the protein-protein interaction sites. One of the general features of PPI sites is that the core regions from the two interacting protein surfaces are complementary to each other, similar to the interior of proteins in packing density and in the physicochemical nature of the amino acid composition. In this work, we simulated the physicochemical complementarities by constructing three-dimensional probability density maps of non-covalent interacting atoms on the protein surfaces. The interacting probabilities were derived from the interior of known structures. Machine learning algorithms were applied to learn the characteristic patterns of the probability density maps specific to the PPI sites. The trained predictors for PPI sites were cross-validated with the training cases (consisting of 432 proteins) and were tested on an independent dataset (consisting of 142 proteins). The residue-based Matthews correlation coefficient for the independent test set was 0.423; the accuracy, precision, sensitivity, specificity were 0.753, 0.519, 0.677, and 0.779 respectively. The benchmark results indicate that the optimized machine learning models are among the best predictors in identifying PPI sites on protein surfaces. In particular, the PPI site prediction accuracy increases with increasing size of the PPI site and with increasing hydrophobicity in amino acid composition of the PPI interface; the core interface regions are more likely to be recognized with high prediction confidence. The results indicate that the physicochemical complementarity patterns on protein surfaces are important determinants in PPIs, and a substantial portion of the PPI sites can be predicted correctly with

  3. Protein-protein interaction site predictions with three-dimensional probability distributions of interacting atoms on protein surfaces.

    Directory of Open Access Journals (Sweden)

    Ching-Tai Chen

    Full Text Available Protein-protein interactions are key to many biological processes. Computational methodologies devised to predict protein-protein interaction (PPI sites on protein surfaces are important tools in providing insights into the biological functions of proteins and in developing therapeutics targeting the protein-protein interaction sites. One of the general features of PPI sites is that the core regions from the two interacting protein surfaces are complementary to each other, similar to the interior of proteins in packing density and in the physicochemical nature of the amino acid composition. In this work, we simulated the physicochemical complementarities by constructing three-dimensional probability density maps of non-covalent interacting atoms on the protein surfaces. The interacting probabilities were derived from the interior of known structures. Machine learning algorithms we