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Sample records for domain interaction partners

  1. Characterization of the Connexin45 Carboxyl-Terminal Domain Structure and Interactions with Molecular Partners

    PubMed Central

    Kopanic, Jennifer L.; Al-mugotir, Mona H.; Kieken, Fabien; Zach, Sydney; Trease, Andrew J.; Sorgen, Paul L.

    2014-01-01

    interacted within one intrinsically disordered region (P278-P285). This domain has similarities with other cardiac connexins, and we propose they constitute a master regulatory domain, which contains overlapping molecular partner binding, cis-trans proline isomerization, and phosphorylation sites. PMID:24853747

  2. Discovery of novel interacting partners of PSMD9, a proteasomal chaperone: Role of an Atypical and versatile PDZ-domain motif interaction and identification of putative functional modules

    PubMed Central

    Sangith, Nikhil; Srinivasaraghavan, Kannan; Sahu, Indrajit; Desai, Ankita; Medipally, Spandana; Somavarappu, Arun Kumar; Verma, Chandra; Venkatraman, Prasanna

    2014-01-01

    PSMD9 (Proteasome Macropain non-ATPase subunit 9), a proteasomal assembly chaperone, harbors an uncharacterized PDZ-like domain. Here we report the identification of five novel interacting partners of PSMD9 and provide the first glimpse at the structure of the PDZ-domain, including the molecular details of the interaction. We based our strategy on two propositions: (a) proteins with conserved C-termini may share common functions and (b) PDZ domains interact with C-terminal residues of proteins. Screening of C-terminal peptides followed by interactions using full-length recombinant proteins, we discovered hnRNPA1 (an RNA binding protein), S14 (a ribosomal protein), CSH1 (a growth hormone), E12 (a transcription factor) and IL6 receptor as novel PSMD9-interacting partners. Through multiple techniques and structural insights, we clearly demonstrate for the first time that human PDZ domain interacts with the predicted Short Linear Sequence Motif (SLIM) at the C-termini of the client proteins. These interactions are also recapitulated in mammalian cells. Together, these results are suggestive of the role of PSMD9 in transcriptional regulation, mRNA processing and editing, hormone and receptor activity and protein translation. Our proof-of-principle experiments endorse a novel and quick method for the identification of putative interacting partners of similar PDZ-domain proteins from the proteome and for discovering novel functions. PMID:25009770

  3. Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners.

    PubMed

    Gemperle, Jakub; Hexnerová, Rozálie; Lepšík, Martin; Tesina, Petr; Dibus, Michal; Novotný, Marian; Brábek, Jan; Veverka, Václav; Rosel, Daniel

    2017-08-14

    CAS is a docking protein downstream of the proto-oncogene Src with a role in invasion and metastasis of cancer cells. The CAS SH3 domain is indispensable for CAS-mediated signaling, but structural aspects of CAS SH3 ligand binding and regulation are not well understood. Here, we identified the consensus CAS SH3 binding motif and structurally characterized the CAS SH3 domain in complex with ligand. We revealed the requirement for an uncommon centrally localized lysine residue at position +2 of CAS SH3 ligands and two rather dissimilar optional anchoring residues, leucine and arginine, at position +5. We further expanded the knowledge of CAS SH3 ligand binding regulation by manipulating tyrosine 12 phosphorylation and confirmed the negative role of this phosphorylation on CAS SH3 ligand binding. Finally, by exploiting the newly identified binding requirements of the CAS SH3 domain, we predicted and experimentally verified two novel CAS SH3 binding partners, DOK7 and GLIS2.

  4. Preferred SH3 Domain Partners of ADAM Metalloproteases Include Shared and ADAM-Specific SH3 Interactions

    PubMed Central

    Kleino, Iivari; Järviluoma, Annika; Hepojoki, Jussi; Huovila, Ari Pekka; Saksela, Kalle

    2015-01-01

    A disintegrin and metalloproteinases (ADAMs) constitute a protein family essential for extracellular signaling and regulation of cell adhesion. Catalytic activity of ADAMs and their predicted potential for Src-homology 3 (SH3) domain binding show a strong correlation. Here we present a comprehensive characterization of SH3 binding capacity and preferences of the catalytically active ADAMs 8, 9, 10, 12, 15, 17, and 19. Our results revealed several novel interactions, and also confirmed many previously reported ones. Many of the identified SH3 interaction partners were shared by several ADAMs, whereas some were ADAM-specific. Most of the ADAM-interacting SH3 proteins were adapter proteins or kinases, typically associated with sorting and endocytosis. Novel SH3 interactions revealed in this study include TOCA1 and CIP4 as preferred partners of ADAM8, and RIMBP1 as a partner of ADAM19. Our results suggest that common as well as distinct mechanisms are involved in regulation and execution of ADAM signaling, and provide a useful framework for addressing the pathways that connect ADAMs to normal and aberrant cell behavior. PMID:25825872

  5. Mechanism for the Selective Interaction of C-terminal Eps15 Homology Domain Proteins with Specific Asn-Pro-Phe-containing Partners*

    PubMed Central

    Kieken, Fabien; Sharma, Mahak; Jović, Marko; Giridharan, Sai Srinivas Panapakkam; Naslavsky, Naava; Caplan, Steve; Sorgen, Paul L.

    2010-01-01

    Epidermal growth factor receptor tyrosine kinase substrate 15 (Eps15) homology (EH)-domain proteins can be divided into two classes: those with an N-terminal EH-domain(s), and the C-terminal Eps15 homology domain-containing proteins (EHDs). Whereas many N-terminal EH-domain proteins regulate internalization events, the best characterized C-terminal EHD, EHD1, regulates endocytic recycling. Because EH-domains interact with the tripeptide Asn-Pro-Phe (NPF), it is of critical importance to elucidate the molecular mechanisms that allow EHD1 and its paralogs to interact selectively with a subset of the hundreds of NPF-containing proteins expressed in mammalian cells. Here, we capitalize on our findings that C-terminal EH-domains possess highly positively charged interaction surfaces and that many NPF-containing proteins that interact with C-terminal (but not N-terminal) EH-domains are followed by acidic residues. Using the recently identified EHD1 interaction partner molecule interacting with CasL (MICAL)-Like 1 (MICAL-L1) as a model, we have demonstrated that only the first of its two NPF motifs is required for EHD1 binding. Because only this first NPF is followed by acidic residues, we have utilized glutathione S-transferase pulldowns, two-hybrid analysis, and NMR to demonstrate that the flanking acidic residues “fine tune” the binding affinity to EHD1. Indeed, our NMR solution structure of the EHD1 EH-domain in complex with the MICAL-L1 NPFEEEEED peptide indicates that the first two flanking Glu residues lie in a position favorable to form salt bridges with Lys residues within the EH-domain. Our data provide a novel explanation for the selective interaction of C-terminal EH-domains with specific NPF-containing proteins and allow for the prediction of new interaction partners with C-terminal EHDs. PMID:20106972

  6. AIP and its interacting partners.

    PubMed

    Trivellin, Giampaolo; Korbonits, Márta

    2011-08-01

    Germline mutations in the aryl hydrocarbon receptor-interacting protein gene (AIP) predispose to young-onset pituitary tumours, most often to GH- or prolactin-secreting adenomas, and most of these patients belong to familial isolated pituitary adenoma families. The molecular pathway initiated by the loss-of-function AIP mutations leading to pituitary tumour formation is unknown. AIP, a co-chaperone of heat-shock protein 90 and various nuclear receptors, belongs to the family of tetratricopeptide repeat (TPR)-containing proteins. It has three antiparallel α-helix motifs (TPR domains) that mediate the interaction of AIP with most of its partners. In this review, we summarise the known interactions of AIP described so far. The identification of AIP partners and the understanding of how AIP interacts with these proteins might help to explain the specific phenotype of the families with heterozygous AIP mutations, to gain deeper insight into the pathological process of pituitary tumour formation and to identify novel drug targets.

  7. Sequence Analysis of LRPPRC and Its SEC1 Domain Interaction Partners Suggests Roles in Cytoskeletal Organization, Vesicular Trafficking, Nucleocytosolic Shuttling and Chromosome Activity

    PubMed Central

    Liu, Leyuan; McKeehan, Wallace L.

    2011-01-01

    LRPPRC (originally called LRP130) is an intracellular 130-kDa leucine-rich protein that co-purifies with the FGF receptor from liver cell extracts and has been detected in diverse multi-protein complexes from the cell membrane, cytoskeleton and nucleus. Here we report results of a sequence homology analysis of LRPPRC and its SEC1 domain interactive partners. Twenty-three copies of tandem repeats that are similar to PPR, TPR and HEAT repeats characterize the LRPPRC sequence. The N-terminus exhibits multiple copies of leucine-rich nuclear transport signals followed by ENTH, DUF28 and SEC1 homology domains. We used the SEC1 domain to trap interactive partners expressed from a human liver cDNA library. Interactive C19ORF5 (XP_038600) exhibited a strong homology to microtubule-associated proteins (MAP) and a potential arginine-rich mRNA binding motif. UXT (XP_033860) exhibited α-helical properties homologous to the actin-associated spectrin repeat and L/I heptad repeats in mobile transcription factors. C6ORF34 (XP_004305) was homologous to the non-DNA binding C-terminus of the E. coli Rob transcription factor. CECR2 (AAK15343) exhibited a transcription factor AT-hook motif next to two bromodomains and a homology to guanylate-binding protein 1. Taken together these features suggest a regulatory role of LRPPRC and its SEC1 domain-interactive partners in integration of cytoskeletal networks with vesicular trafficking, nucleocytosolic shuttling, chromosome remodeling and transcription. PMID:11827465

  8. The SAM domain of ANKS6 has different interacting partners and mutations can induce different cystic phenotypes.

    PubMed

    Bakey, Zeineb; Bihoreau, Marie-Thérèse; Piedagnel, Rémi; Delestré, Laure; Arnould, Catherine; de Villiers, Alexandre d'Hotman; Devuyst, Olivier; Hoffmann, Sigrid; Ronco, Pierre; Gauguier, Dominique; Lelongt, Brigitte

    2015-08-01

    The ankyrin repeat and sterile α motif (SAM) domain-containing six gene (Anks6) is a candidate for polycystic kidney disease (PKD). Originally identified in the PKD/Mhm(cy/+) rat model of PKD, the disease is caused by a mutation (R823W) in the SAM domain of the encoded protein. Recent studies support the etiological role of the ANKS6 SAM domain in human cystic diseases, but its function in kidney remains unknown. To investigate the role of ANKS6 in cyst formation, we screened an archive of N-ethyl-N-nitrosourea-treated mice and derived a strain carrying a missense mutation (I747N) within the SAM domain of ANKS6. This mutation is only six amino acids away from the PKD-causing mutation (R823W) in cy/+ rats. Evidence of renal cysts in these mice confirmed the crucial role of the SAM domain of ANKS6 in kidney function. Comparative phenotype analysis in cy/+ rats and our Anks6(I747N) mice further showed that the two models display noticeably different PKD phenotypes and that there is a defective interaction between ANKS6 with ANKS3 in the rat and between ANKS6 and BICC1 (bicaudal C homolog 1) in the mouse. Thus, our data demonstrate the importance of ANKS6 for kidney structure integrity and the essential mediating role of its SAM domain in the formation of protein complexes.

  9. Cloning, expression, purification, crystallization and preliminary crystallographic analysis of pseudo death-effector domain of HIPPI, a molecular partner of Huntingtin-interacting protein HIP-1

    SciTech Connect

    Banerjee, Manisha; Majumder, Pritha; Bhattacharyya, Nitai P.; Dattagupta, Jiban K.; Sen, Udayaditya

    2006-12-01

    A pseudo death-effector domain (pDED) of HIPPI, a partner of Huntingtin-interacting protein HIP1, has been cloned, overexpressed and crystallized. The crystals of pDED-HIPPI diffracted to 2.2 Å. The formation of a heterodimer between Huntingtin-interacting protein-1 (HIP-1) and its novel partner HIPPI (HIP-1 protein interactor) through their pseudo death-effector domains (pDEDs) is a key step that recruits caspase-8 and initiates apoptosis. This could be one of the pathways by which apoptosis is increased in Huntington’s disease (HD). A construct consisting of the pDED of HIPPI has been cloned and overexpressed as 6NH-tagged protein and purified by Ni–NTA affinity chromatography. Crystals of the pDED of HIPPI were grown in space group P4{sub 1}, with unit-cell parameters a = b = 77.42, c = 33.31 Å and a calculated Matthews coefficient of 1.88 Å{sup 3} Da{sup −1} (33% solvent content) with two molecules per asymmetric unit.

  10. Activity of a Bacterial Cell Envelope Stress Response Is Controlled by the Interaction of a Protein Binding Domain with Different Partners*

    PubMed Central

    Flores-Kim, Josué; Darwin, Andrew J.

    2015-01-01

    The bacterial phage shock protein (Psp) system is a highly conserved cell envelope stress response required for virulence in Yersinia enterocolitica and Salmonella enterica. In non-inducing conditions the transcription factor PspF is inhibited by an interaction with PspA. In contrast, PspA associates with the cytoplasmic membrane proteins PspBC during inducing conditions. This has led to the proposal that PspBC exists in an OFF state, which cannot recruit PspA, or an ON state, which can. However, nothing was known about the difference between these two states. Here, we provide evidence that it is the C-terminal domain of Y. enterocolitica PspC (PspCCT) that interacts directly with PspA, both in vivo and in vitro. Site-specific photocross-linking revealed that this interaction occurred only during Psp-inducing conditions in vivo. Importantly, we have also discovered that PspCCT can interact with the C-terminal domain of PspB (PspCCT·PspBCT). However, the PspCCT·PspBCT and PspCCT·PspA interactions were mutually exclusive in vitro. Furthermore, in vivo, PspCCT contacted PspBCT in the OFF state, whereas it contacted PspA in the ON state. These findings provide the first description of the previously proposed PspBC OFF and ON states and reveal that the regulatory switch is centered on a PspCCT partner-switching mechanism. PMID:25802329

  11. Forkhead-Associated Domain of Yeast Xrs2, a Homolog of Human Nbs1, Promotes Nonhomologous End Joining Through Interaction With a Ligase IV Partner Protein, Lif1

    PubMed Central

    Matsuzaki, Kenichiro; Shinohara, Akira; Shinohara, Miki

    2008-01-01

    DNA double-strand breaks (DSB) are repaired through two different pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ). Yeast Xrs2, a homolog of human Nbs1, is a component of the Mre11-Rad50-Xrs2 (MRX) complex required for both HR and NHEJ. Previous studies showed that the N-terminal forkhead-associated (FHA) domain of Xrs2/Nbs1 in yeast is not involved in HR, but is likely to be in NHEJ. In this study, we showed that the FHA domain of Xrs2 plays a critical role in efficient DSB repair by NHEJ. The FHA domain of Xrs2 specifically interacts with Lif1, a component of the ligase IV complex, Dnl4-Nej1-Lif1 (DNL). Lif1, which is phosphorylated in vivo, contains two Xrs2-binding regions. Serine 383 of Lif1 plays an important role in the interaction with Xrs2 as well as in NHEJ. Interestingly, the phospho-mimetic substitutions of serine 383 enhance the NHEJ activity of Lif1. Our results suggest that the phosphorylation of Lif1 at serine 383 is recognized by the Xrs2 FHA domain, which in turn may promote recruitment of the DNL complex to DSB for NHEJ. The interaction between Xrs2 and Lif1 through the FHA domain is conserved in humans; the FHA domain Nbs1 interacts with Xrcc4, a Lif1 homolog of human. PMID:18458108

  12. Evolutionary history exposes radical diversification among classes of interaction partners of the MLLE domain of plant poly(A)-binding proteins.

    PubMed

    Jiménez-López, Domingo; Bravo, Jaime; Guzmán, Plinio

    2015-09-16

    Poly(A)-binding proteins (PABPs) are evolutionarily conserved proteins that have important functions in the regulation of translation and the control of mRNA stability in eukaryotes. Most PABPs encode a C-terminal domain known as the MLLE domain (previously PABC or CTC), which can mediate protein interactions. In earlier work we identified and predicted that four classes of MLLE-interacting proteins were present in Arabidopsis thaliana, which we named CID A, B, C, and D. These proteins encode transcription-activating domains (CID A), the Lsm and LsmAD domains of ataxin-2 (CID B), the CUE and small MutS-related domains (CID C), and two RNA recognition domains (CID D). We recently found that a novel class that lacks the LsmAD domain is present in CID B proteins. We extended our analysis to other classes of CIDs present in the viridiplantae. We found that novel variants also evolved in classes CID A and CID C. A specific transcription factor domain is present in a distinct lineage in class A, and a variant that lacks at least two distinct domains was also identified in a divergent lineage in class C. We did not detect any variants in Class D CIDs. This class often consists of four to six highly conserved RNA-binding proteins, which suggests that major redundancy is present in this class. CIDs are likely to operate as components of posttranscriptional regulatory assemblies. The evident diversification of CIDs may be neutral or may be important for plant adaptation to the environment and for acquisition of specific traits during evolution. The fact that CIDs subclasses are maintained in early lineages suggest that a presumed interference between duplicates was resolved, and a defined function for each subclass was achieved.

  13. Identification of candidates for interacting partners of the tail domain of DcNMCP1, a major component of the Daucus carota nuclear lamina-like structure.

    PubMed

    Mochizuki, Ryota; Tsugama, Daisuke; Yamazaki, Michihiro; Fujino, Kaien; Masuda, Kiyoshi

    2017-02-01

    NMCP/CRWN (NUCLEAR MATRIX CONSTITUENT PROTEIN/CROWDED NUCLEI) is a major component of a protein fibrous meshwork (lamina-like structure) on the plant inner nuclear membrane. NMCP/CRWN contributes to regulating nuclear shape and nuclear functions. An NMCP/CRWN protein in Daucus carota (DcNMCP1) is localized to the nuclear periphery in interphase cells, and surrounds chromosomes in cells in metaphase and anaphase. The N-terminal region and the C-terminal region of DcNMCP1 are both necessary for localizing DcNMCP1 to the nuclear periphery. Here candidate interacting partners of the amino acid position 975-1053 of DcNMCP1 (T975-1053), which is present in the C-terminal region and contains a conserved sequence that plays a role in localizing DcNMCP1 to the nuclear periphery, are screened for. Arabidopsis thaliana nuclear proteins were subjected to far-Western blotting with GST-fused T975-1053 as a probe, and signals were detected at the positions corresponding to ∼70, ∼40, and ∼18 kDa. These ∼70, ∼40, and ∼18 kDa nuclear proteins were identified by mass spectrometry, and subjected to a yeast 2-hybrid (Y2H) analysis with T975-1053 as bait. In this analysis, the ∼40 kDa protein ARP7, which is a nuclear actin-related protein possibly involved in regulating chromatin structures, was confirmed to interact with T975-1053. Independently of the far-Western blotting, a Y2H screen was performed using T975-1053 as bait. Targeted Y2H assays confirmed that 3 proteins identified in the screen, MYB3, SINAT1, and BIM1, interact with T975-1053. These proteins might have roles in NMCP/CRWN protein-mediated biologic processes.

  14. Identification of cofilin and LIM-domain-containing protein kinase 1 as novel interaction partners of 14-3-3 zeta.

    PubMed Central

    Birkenfeld, Jörg; Betz, Heinrich; Roth, Dagmar

    2003-01-01

    Proteins of the 14-3-3 family have been implicated in various physiological processes, and are thought to function as adaptors in various signal transduction pathways. In addition, 14-3-3 proteins may contribute to the reorganization of the actin cytoskeleton by interacting with as yet unidentified actin-binding proteins. Here we show that the 14-3-3 zeta isoform interacts with both the actin-depolymerizing factor cofilin and its regulatory kinase, LIM (Lin-11/Isl-1/Mec-3)-domain-containing protein kinase 1 (LIMK1). In both yeast two-hybrid assays and glutathione S-transferase pull-down experiments, these proteins bound efficiently to 14-3-3 zeta. Deletion analysis revealed consensus 14-3-3 binding sites on both cofilin and LIMK1. Furthermore, the C-terminal region of 14-3-3 zeta inhibited the binding of cofilin to actin in co-sedimentation experiments. Upon co-transfection into COS-7 cells, 14-3-3 zeta-specific immunoreactivity was redistributed into characteristic LIMK1-induced actin aggregations. Our data are consistent with 14-3-3-protein-induced changes to the actin cytoskeleton resulting from interactions with cofilin and/or LIMK1. PMID:12323073

  15. Inferring interaction partners from protein sequences

    PubMed Central

    Bitbol, Anne-Florence; Dwyer, Robert S.; 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 multiprotein complexes and to enable signal transduction in various pathways. Functional interactions between proteins result in coevolution between the interaction partners, causing their sequences to be correlated. Here we exploit these correlations to accurately identify, from sequence data alone, which proteins are specific interaction partners. Our general approach, which employs a pairwise maximum entropy model to infer couplings between residues, has been successfully used to predict the 3D structures of proteins from sequences. Thus inspired, we introduce an iterative algorithm to predict specific interaction partners from two protein families whose members are known to interact. We first assess the algorithm’s performance on histidine kinases and response regulators from bacterial two-component signaling systems. We obtain a striking 0.93 true positive fraction on our complete dataset without any a priori knowledge of interaction partners, and we uncover the origin of this success. We then apply the algorithm to proteins from ATP-binding cassette (ABC) transporter complexes, and obtain accurate predictions in these systems as well. Finally, we present two metrics that accurately distinguish interacting protein families from noninteracting ones, using only sequence data. PMID:27663738

  16. Domain Structure of the Redβ Single-Strand Annealing Protein: the C-terminal Domain is Required for Fine-Tuning DNA-binding Properties, Interaction with the Exonuclease Partner, and Recombination in vivo.

    PubMed

    Smith, Christopher E; Bell, Charles E

    2016-02-13

    Redβ is a component of the Red recombination system of bacteriophage λ that promotes a single strand annealing (SSA) reaction to generate end-to-end concatemers of the phage genome for packaging. Redβ interacts with λ exonuclease (λexo), the other component of the Red system, to form a "synaptosome" complex that somehow integrates the end resection and annealing steps of the reaction. Previous work using limited proteolysis and chemical modification revealed that Redβ consists of an N-terminal DNA binding domain, residues 1-177, and a flexible C-terminal "tail", residues 178-261. Here, we quantitatively compare the binding of the full-length protein (Redβ(FL)) and the N-terminal domain (Redβ(177)) to different lengths of ssDNA substrate and annealed duplex product. We find that in general, Redβ(FL) binds more tightly to annealed duplex product than to ssDNA substrate, while Redβ(177) binds more tightly to ssDNA. In addition, the C-terminal region of Redβ corresponding to residues 182-261 was purified and found to fold into an α-helical domain that is required for the interaction with λexo to form the synaptosome complex. Deletion analysis of Redβ revealed that removal of just eleven residues from the C-terminus disrupts the interaction with λexo as well as ssDNA and dsDNA recombination in vivo. By contrast, the determinants for self-oligomerization of Redβ appear to reside solely within the N-terminal domain. The subtle but significant differences in the relative binding of Redβ(FL) and Redβ(177) to ssDNA substrate and annealed duplex product may be important for Redβ to function as a SSA protein in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Unique structure of Ascaris suum b5-type cytochrome: an additional α-helix and positively charged residues on the surface domain interact with redox partners

    PubMed Central

    Yokota, Takehiro; Nakajima, Yoshitaka; Yamakura, Fumiyuki; Sugio, Shigetoshi; Hashimoto, Muneaki; Takamiya, Shinzaburo

    2005-01-01

    Cytochrome b5 of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a soluble protein that lacks a C-terminal membrane-anchoring domain, but possesses an N-terminal pre-sequence of 30 amino acids. During the maturation of cytochrome b5, the N-terminal pre-sequence is proteolytically cleaved to form the mature protein of 82 amino acid residues. A. suum cytochrome b5 is a basic protein containing more lysine residues and exhibiting a higher midpoint redox potential than its mammalian counterparts. We developed an expression system for the production of the recombinant nematode cytochrome b5, which is chemically and functionally identical with the native protein. Using this recombinant protein, we have determined the X-ray crystal structure of A. suum cytochrome b5 at 1.8 Å (1 Å=0.1 nm) resolution, and we have shown that this protein is involved in the reduction of nematode body-wall metmyoglobin. The crystal structure of A. suum cytochrome b5 consists of six α-helices and five β-strands. It differs from its mammalian counterparts by having a head-to-tail disulphide bridge, as well as a four-residue insertion in the vicinity of the sixth ligating histidine, which forms an additional α-helix, α4A, between helices α4 and α5. A. suum cytochrome b5 exists predominantly as a haem-orientation B isomer. Furthermore, the haem plane is rotated approx. 80° relative to the axis formed by haem-Fe and Nϵ atoms of the two histidine residues that are ligated to haem-Fe. The charge distribution around the haem crevice of A. suum cytochrome b5 is remarkably different from that of mammalian cytochrome b5 in that the nematode protein bears positively charged lysine residues surrounding the haem crevice. Using immunohistochemistry, we found that A. suum cytochrome b5 is present in the nematode hypodermis. Based on this histochemical and structural information, the physiological function of A. suum cytochrome b5 and its interaction with nematode

  18. Identification of brain-specific angiogenesis inhibitor 2 as an interaction partner of glutaminase interacting protein

    SciTech Connect

    Zencir, Sevil; Ovee, Mohiuddin; Dobson, Melanie J.; Banerjee, Monimoy; Topcu, Zeki; Mohanty, Smita

    2011-08-12

    Highlights: {yields} Brain-specific angiogenesis inhibitor 2 (BAI2) is a new partner protein for GIP. {yields} BAI2 interaction with GIP was revealed by yeast two-hybrid assay. {yields} Binding of BAI2 to GIP was characterized by NMR, CD and fluorescence. {yields} BAI2 and GIP binding was mediated through the C-terminus of BAI2. -- Abstract: The vast majority of physiological processes in living cells are mediated by protein-protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80-100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-1, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase L, {beta}-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kir 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP.

  19. DIMA 3.0: Domain Interaction Map.

    PubMed

    Luo, Qibin; Pagel, Philipp; Vilne, Baiba; Frishman, Dmitrij

    2011-01-01

    Domain Interaction MAp (DIMA, available at http://webclu.bio.wzw.tum.de/dima) is a database of predicted and known interactions between protein domains. It integrates 5807 structurally known interactions imported from the iPfam and 3did databases and 46,900 domain interactions predicted by four computational methods: domain phylogenetic profiling, domain pair exclusion algorithm correlated mutations and domain interaction prediction in a discriminative way. Additionally predictions are filtered to exclude those domain pairs that are reported as non-interacting by the Negatome database. The DIMA Web site allows to calculate domain interaction networks either for a domain of interest or for entire organisms, and to explore them interactively using the Flash-based Cytoscape Web software.

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

    PubMed Central

    Khor, Susan

    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 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. PMID:24586450

  1. The N- and C-terminal domains of MecA recognize different partners in the competence molecular switch.

    PubMed

    Persuh, M; Turgay, K; Mandic-Mulec, I; Dubnau, D

    1999-08-01

    ComK is a transcription factor required for the expression of competence genes in Bacillus subtilis. Binding to MecA targets ComK for degradation by the ClpCP protease. MecA therefore acts as an adapter protein recruiting a regulatory protein for proteolysis. However, when ComS is synthesized, ComK is released from binding by MecA and thereby protected from degradation. MecA binds to three protein partners during these processes: ComK, ClpC and ComS. Using limited proteolysis, we have defined N- and C-terminal structural domains of MecA and evaluated the interactions of these domains with the protein partners of MecA. Using surface plasmon resonance, we have determined that the N-terminal domain of MecA interacts with ComK and ComS and the C-terminal domain with ClpC. MecA is shown to exist as a dimer with dimerization sites on both the N- and C-terminal domains. The C-terminal domain stimulates the ATPase activity of ClpC and is degraded by the ClpCP protease, while the N-terminal domain is inactive in both of these assays. In vivo data were consistent with these findings, as comG-lacZ expression was decreased in a strain overproducing the N-terminal domain, indicating reduced ComK activity. We propose a model in which binding of ClpC to the C-terminal domain of MecA induces a conformational change enabling the N-terminal domain to bind ComK with enhanced affinity. MecA is widespread among Gram-positive organisms and may act generally as an adapter protein, targeting proteins for regulated degradation.

  2. Identification of new interacting partners of the shuttling protein ubinuclein (Ubn-1)

    SciTech Connect

    Lupo, Julien; Conti, Audrey; Sueur, Charlotte; Coly, Pierre-Alain; Coute, Yohann; Hunziker, Walter; Burmeister, Wim P.; Germi, Raphaelle; Manet, Evelyne; Gruffat, Henri; and others

    2012-03-10

    We have previously characterized ubinuclein (Ubn-1) as a NACos (Nuclear and Adherent junction Complex components) protein which interacts with viral or cellular transcription factors and the tight junction (TJ) protein ZO-1. The purpose of the present study was to get more insights on the binding partners of Ubn-1, notably those present in the epithelial junctions. Using an in vivo assay of fluorescent protein-complementation assay (PCA), we demonstrated that the N-terminal domains of the Ubn-1 and ZO-1 proteins triggered a functional interaction inside the cell. Indeed, expression of both complementary fragments of venus fused to the N-terminal parts of Ubn-1 and ZO-1 was able to reconstitute a fluorescent venus protein. Furthermore, nuclear expression of the chimeric Ubn-1 triggered nuclear localization of the chimeric ZO-1. We could localize this interaction to the PDZ2 domain of ZO-1 using an in vitro pull-down assay. More precisely, a 184-amino acid region (from amino acids 39 to 223) at the N-terminal region of Ubn-1 was responsible for the interaction with the PDZ2 domain of ZO-1. Co-imunoprecipitation and confocal microscopy experiments also revealed the tight junction protein cingulin as a new interacting partner of Ubn-1. A proteomic approach based on mass spectrometry analysis (MS) was then undertaken to identify further binding partners of GST-Ubn-1 fusion protein in different subcellular fractions of human epithelial HT29 cells. LYRIC (Lysine-rich CEACAM1-associated protein) and RACK-1 (receptor for activated C-kinase) proteins were validated as bona fide interacting partners of Ubn-1. Altogether, these results suggest that Ubn-1 is a scaffold protein influencing protein subcellular localization and is involved in several processes such as cell-cell contact signalling or modulation of gene activity.

  3. Identification of new interacting partners of the shuttling protein ubinuclein (Ubn-1).

    PubMed

    Lupo, Julien; Conti, Audrey; Sueur, Charlotte; Coly, Pierre-Alain; Couté, Yohann; Hunziker, Walter; Burmeister, Wim P; Germi, Raphaelle; Manet, Evelyne; Gruffat, Henri; Morand, Patrice; Boyer, Véronique

    2012-03-10

    We have previously characterized ubinuclein (Ubn-1) as a NACos (Nuclear and Adherent junction Complex components) protein which interacts with viral or cellular transcription factors and the tight junction (TJ) protein ZO-1. The purpose of the present study was to get more insights on the binding partners of Ubn-1, notably those present in the epithelial junctions. Using an in vivo assay of fluorescent protein-complementation assay (PCA), we demonstrated that the N-terminal domains of the Ubn-1 and ZO-1 proteins triggered a functional interaction inside the cell. Indeed, expression of both complementary fragments of venus fused to the N-terminal parts of Ubn-1 and ZO-1 was able to reconstitute a fluorescent venus protein. Furthermore, nuclear expression of the chimeric Ubn-1 triggered nuclear localization of the chimeric ZO-1. We could localize this interaction to the PDZ2 domain of ZO-1 using an in vitro pull-down assay. More precisely, a 184-amino acid region (from amino acids 39 to 223) at the N-terminal region of Ubn-1 was responsible for the interaction with the PDZ2 domain of ZO-1. Co-imunoprecipitation and confocal microscopy experiments also revealed the tight junction protein cingulin as a new interacting partner of Ubn-1. A proteomic approach based on mass spectrometry analysis (MS) was then undertaken to identify further binding partners of GST-Ubn-1 fusion protein in different subcellular fractions of human epithelial HT29 cells. LYRIC (Lysine-rich CEACAM1-associated protein) and RACK-1 (receptor for activated C-kinase) proteins were validated as bona fide interacting partners of Ubn-1. Altogether, these results suggest that Ubn-1 is a scaffold protein influencing protein subcellular localization and is involved in several processes such as cell-cell contact signalling or modulation of gene activity. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Virtual Partner Interaction (VPI): Exploring Novel Behaviors via Coordination Dynamics

    PubMed Central

    Kelso, J. A. Scott; de Guzman, Gonzalo C.; Reveley, Colin; Tognoli, Emmanuelle

    2009-01-01

    Inspired by the dynamic clamp of cellular neuroscience, this paper introduces VPI—Virtual Partner Interaction—a coupled dynamical system for studying real time interaction between a human and a machine. In this proof of concept study, human subjects coordinate hand movements with a virtual partner, an avatar of a hand whose movements are driven by a computerized version of the Haken-Kelso-Bunz (HKB) equations that have been shown to govern basic forms of human coordination. As a surrogate system for human social coordination, VPI allows one to examine regions of the parameter space not typically explored during live interactions. A number of novel behaviors never previously observed are uncovered and accounted for. Having its basis in an empirically derived theory of human coordination, VPI offers a principled approach to human-machine interaction and opens up new ways to understand how humans interact with human-like machines including identification of underlying neural mechanisms. PMID:19492044

  5. Nonergodic subdiffusion from transient interactions with heterogeneous partners

    NASA Astrophysics Data System (ADS)

    Charalambous, C.; Muñoz-Gil, G.; Celi, A.; Garcia-Parajo, M. F.; Lewenstein, M.; Manzo, C.; García-March, M. A.

    2017-03-01

    Spatiotemporal disorder has been recently associated to the occurrence of anomalous nonergodic diffusion of molecular components in biological systems, but the underlying microscopic mechanism is still unclear. We introduce a model in which a particle performs continuous Brownian motion with changes of diffusion coefficients induced by transient molecular interactions with diffusive binding partners. In spite of the exponential distribution of waiting times, the model shows subdiffusion and nonergodicity similar to the heavy-tailed continuous time random walk. The dependence of these properties on the density of binding partners is analyzed and discussed. Our work provides an experimentally testable microscopic model to investigate the nature of nonergodicity in disordered media.

  6. Identification of novel MYO18A interaction partners required for myoblast adhesion and muscle integrity

    PubMed Central

    Cao, Jian-Meng; Cheng, Xiao-Ning; Li, Shang-Qi; Heller, Stefan; Xu, Zhi-Gang; Shi, De-Li

    2016-01-01

    The unconventional myosin MYO18A that contains a PDZ domain is required for muscle integrity during zebrafish development. However, the mechanism by which it functions in myofibers is not clear. The presence of a PDZ domain suggests that MYO18A may interact with other partners to perform muscle-specific functions. Here we performed double-hybrid screening and co-immunoprecipitation to identify MYO18A-interacting proteins, and have identified p190RhoGEF and Golgin45 as novel partners for the MYO18A PDZ domain. We have also identified Lurap1, which was previously shown to bind MYO18A. Functional analyses indicate that, similarly as myo18a, knockdown of lurap1, p190RhoGEF and Golgin45 by morpholino oligonucleotides disrupts dystrophin localization at the sarcolemma and produces muscle lesions. Simultaneous knockdown of myo18a with either of these genes severely disrupts myofiber integrity and dystrophin localization, suggesting that they may function similarly to maintain myofiber integrity. We further show that MYO18A and its interaction partners are required for adhesion of myoblasts to extracellular matrix, and for the formation of the Golgi apparatus and organization of F-actin bundles in myoblast cells. These findings suggest that MYO18A has the potential to form a multiprotein complex that links the Golgi apparatus to F-actin, which regulates muscle integrity and function during early development. PMID:27824130

  7. Domains of concern of intimate partners of sudden cardiac arrest survivors after ICD implantation.

    PubMed

    Dougherty, Cynthia M; Pyper, Gail P; Benoliel, Jeanne Q

    2004-01-01

    There is limited research that describes the experiences of intimate partners of sudden cardiac arrest (SCA) survivors. The purposes of this article are to (1) describe the domains of concern of intimate partners of SCA survivors during the first year after internal cardioverter defibrillator (ICD) implantation and (2) outline strategies used by partners of SCA survivors in dealing with the concerns and demands of recovery in the first year after ICD implantation. This is a secondary analysis of interview data collected for the primary study "Family Experiences Following Sudden Cardiac Arrest." A grounded theory method was used to identify experiences of SCA survivors and their family members from hospitalization through the first year after ICD implantation. Data were collected from the SCA survivor and one intimate partner at 5 times: hospital discharge, and at 1, 3, 6, and 12 months postdischarge. Eight Domains of Concern were identified for intimate partners following SCA and ICD implantation during the first year. These included (1) Care of the survivor, (2) My (partner) self-care, (3) Relationship, (4) ICD, (5) Money, (6) Uncertain future, (7) Health care providers, and (8) Family. Five categories of strategies to deal with the Domains of Concerns were identified (1) Care of the survivor, (2) My (partner) self-care, (3) Relationship, (4) Uncertain future, and (5) Controlling the environment. Nursing intervention programs should include the intimate partner of SCA survivors and contain education and support in the following areas: (1) information on the function of the ICD, (2) normal progression of physical and emotional recovery experiences, (3) safety and maintenance of the ICD, (4) activities of daily living after an ICD, (5) strategies to assist with the survivors care, and (6) strategies to assist with partner self care.

  8. Phage display library screening for identification of interacting protein partners.

    PubMed

    Addepalli, Balasubrahmanyam; Rao, Suryadevara; Hunt, Arthur G

    2015-01-01

    Phage display is a versatile high-throughput screening method employed to understand and improve the chemical biology, be it production of human monoclonal antibodies or identification of interacting protein partners. A majority of cell proteins operate in a concerted fashion either by stable or transient interactions. Such interactions can be mediated by recognition of small amino acid sequence motifs on the protein surface. Phage display can play a crucial role in identification of such motifs. This report describes the use of phage display for the identification of high affinity sequence motifs that could be responsible for interactions with a target (bait) protein.

  9. Polycomb Group Targeting through Different Binding Partners of RING1B C-Terminal Domain

    PubMed Central

    Wang, Renjing; Taylor, Alexander B.; Leal, Belinda Z.; Chadwell, Linda V.; Ilangovan, Udayar; Robinson, Angela K.; Schirf, Virgil; Hart, P. John; Lafer, Eileen M.; Demeler, Borries; Hinck, Andrew P.; McEwen, Donald G.; Kim, Chongwoo A.

    2015-01-01

    SUMMARY RING1B, a Polycomb Group (PcG) protein, binds methylated chromatin through its association with another PcG protein called Polycomb (Pc). However, RING1B can associate with nonmethylated chromatin suggesting an alternate mechanism for RING1B interaction with chromatin. Here, we demonstrate that two proteins with little sequence identity between them, the Pc cbox domain and RYBP, bind the same surface on the C-terminal domain of RING1B (C-RING1B). Pc cbox and RYBP each fold into a nearly identical, intermolecular beta sheet with C-RING1B and a loop structure which are completely different in the two proteins. Both the beta sheet and loop are required for stable binding and transcription repression. Further, a mutation engineered to disrupt binding on the Drosophila dRING1 protein prevents chromatin association and PcG function in vivo. These results suggest that PcG targeting to different chromatin locations relies, in part, on binding partners of C-RING1B that are diverse in sequence and structure. PMID:20696397

  10. Discovering interacting domains and motifs in protein-protein interactions.

    PubMed

    Hugo, Willy; Sung, Wing-Kin; Ng, See-Kiong

    2013-01-01

    Many important biological processes, such as the signaling pathways, require protein-protein interactions (PPIs) that are designed for fast response to stimuli. These interactions are usually transient, easily formed, and disrupted, yet specific. Many of these transient interactions involve the binding of a protein domain to a short stretch (3-10) of amino acid residues, which can be characterized by a sequence pattern, i.e., a short linear motif (SLiM). We call these interacting domains and motifs domain-SLiM interactions. Existing methods have focused on discovering SLiMs in the interacting proteins' sequence data. With the recent increase in protein structures, we have a new opportunity to detect SLiMs directly from the proteins' 3D structures instead of their linear sequences. In this chapter, we describe a computational method called SLiMDIet to directly detect SLiMs on domain interfaces extracted from 3D structures of PPIs. SLiMDIet comprises two steps: (1) interaction interfaces belonging to the same domain are extracted and grouped together using structural clustering and (2) the extracted interaction interfaces in each cluster are structurally aligned to extract the corresponding SLiM. Using SLiMDIet, de novo SLiMs interacting with protein domains can be computationally detected from structurally clustered domain-SLiM interactions for PFAM domains which have available 3D structures in the PDB database.

  11. FERM Domain Interaction Promotes FAK Signaling

    PubMed Central

    Dunty, Jill M.; Gabarra-Niecko, Veronica; King, Michelle L.; Ceccarelli, Derek F. J.; Eck, Michael J.; Schaller, Michael D.

    2004-01-01

    From the results of deletion analyses, the FERM domain of FAK has been proposed to inhibit enzymatic activity and repress FAK signaling. We have identified a sequence in the FERM domain that is important for FAK signaling in vivo. Point mutations in this sequence had little effect upon catalytic activity in vitro. However, the mutant exhibits reduced tyrosine phosphorylation and dramatically reduced Src family kinase binding. Further, the abilities of the mutant to transduce biochemical signals and to promote cell migration were severely impaired. The results implicate a FERM domain interaction in cell adhesion-dependent activation of FAK and downstream signaling. We also show that the purified FERM domain of FAK interacts with full-length FAK in vitro, and mutation of this sequence disrupts the interaction. These findings are discussed in the context of models of FAK regulation by its FERM domain. PMID:15169899

  12. The SH2 domain interaction landscape

    PubMed Central

    Tinti, Michele; Kiemer, Lars; Costa, Stefano; Miller, Martin; Sacco, Francesca; Olsen, Jesper V.; Carducci, Martina; Paoluzi, Serena; Langone, Francesca; Workman, Christopher T.; Blom, Nikolaj; Machida, Kazuya; Thompson, Christopher M.; Schutkowski, Mike; Brunak, Søren; Mann, Matthias; Mayer, Bruce J.; Castagnoli, Luisa; Cesareni, Gianni

    2014-01-01

    Summary Members of the SH2 domain family modulate signal transduction by binding to short peptides containing phosphorylated tyrosines. Each domain displays a distinct preference for the sequence context of the phosphorylated residue. We have developed a new high-density peptide chip technology that allows probing the affinity of most SH2 domains for a large fraction of the entire complement of tyrosine phosphopeptides in the human proteome. Using this technique we have experimentally identified thousands of putative SH2- peptide interactions for more than 70 different SH2 domains. By integrating this rich data set with orthogonal context-specific information, we have assembled an SH2 mediated probabilistic interaction network, which we make available as a community resource in the PepSpotDB database. A new predicted dynamic interaction between the SH2 domains of the tyrosine phosphatase SHP2 and the phosphorylated tyrosine in the ERK activation loop was validated by experiments in living cells. PMID:23545499

  13. Characterizing WW Domain Interactions of Tumor Suppressor WWOX Reveals Its Association with Multiprotein Networks*

    PubMed Central

    Abu-Odeh, Mohammad; Bar-Mag, Tomer; Huang, Haiming; Kim, TaeHyung; Salah, Zaidoun; Abdeen, Suhaib K.; Sudol, Marius; Reichmann, Dana; Sidhu, Sachdev; Kim, Philip M.; Aqeilan, Rami I.

    2014-01-01

    WW domains are small modules present in regulatory and signaling proteins that mediate specific protein-protein interactions. The WW domain-containing oxidoreductase (WWOX) encodes a 46-kDa tumor suppressor that contains two N-terminal WW domains and a central short-chain dehydrogenase/reductase domain. Based on its ligand recognition motifs, the WW domain family is classified into four groups. The largest one, to which WWOX belongs, recognizes ligands with a PPXY motif. To pursue the functional properties of the WW domains of WWOX, we employed mass spectrometry and phage display experiments to identify putative WWOX-interacting partners. Our analysis revealed that the first WW (WW1) domain of WWOX is the main functional interacting domain. Furthermore, our study uncovered well known and new PPXY-WW1-interacting partners and shed light on novel LPXY-WW1-interacting partners of WWOX. Many of these proteins are components of multiprotein complexes involved in molecular processes, including transcription, RNA processing, tight junction, and metabolism. By utilizing GST pull-down and immunoprecipitation assays, we validated that WWOX is a substrate of the E3 ubiquitin ligase ITCH, which contains two LPXY motifs. We found that ITCH mediates Lys-63-linked polyubiquitination of WWOX, leading to its nuclear localization and increased cell death. Our data suggest that the WW1 domain of WWOX provides a versatile platform that links WWOX with individual proteins associated with physiologically important networks. PMID:24550385

  14. Characterizing WW domain interactions of tumor suppressor WWOX reveals its association with multiprotein networks.

    PubMed

    Abu-Odeh, Mohammad; Bar-Mag, Tomer; Huang, Haiming; Kim, TaeHyung; Salah, Zaidoun; Abdeen, Suhaib K; Sudol, Marius; Reichmann, Dana; Sidhu, Sachdev; Kim, Philip M; Aqeilan, Rami I

    2014-03-28

    WW domains are small modules present in regulatory and signaling proteins that mediate specific protein-protein interactions. The WW domain-containing oxidoreductase (WWOX) encodes a 46-kDa tumor suppressor that contains two N-terminal WW domains and a central short-chain dehydrogenase/reductase domain. Based on its ligand recognition motifs, the WW domain family is classified into four groups. The largest one, to which WWOX belongs, recognizes ligands with a PPXY motif. To pursue the functional properties of the WW domains of WWOX, we employed mass spectrometry and phage display experiments to identify putative WWOX-interacting partners. Our analysis revealed that the first WW (WW1) domain of WWOX is the main functional interacting domain. Furthermore, our study uncovered well known and new PPXY-WW1-interacting partners and shed light on novel LPXY-WW1-interacting partners of WWOX. Many of these proteins are components of multiprotein complexes involved in molecular processes, including transcription, RNA processing, tight junction, and metabolism. By utilizing GST pull-down and immunoprecipitation assays, we validated that WWOX is a substrate of the E3 ubiquitin ligase ITCH, which contains two LPXY motifs. We found that ITCH mediates Lys-63-linked polyubiquitination of WWOX, leading to its nuclear localization and increased cell death. Our data suggest that the WW1 domain of WWOX provides a versatile platform that links WWOX with individual proteins associated with physiologically important networks.

  15. [HPV diagnosis: woman's process of interaction with her partner].

    PubMed

    Vargens, Octavio Muniz da Costa; Silva, Carla Marins; Azevedo E Silva, Gulnar; Girianelli, Vânia Reis

    2013-01-01

    This is a descriptive research, with qualitative approach, which aimed at analyze the interaction process between woman and her partner starting from the diagnosis of infection by the human papilomavirus (HPV). It was accomplished in 13 communities in the cities of Duque de Caxias and Nova Iguaçu, Rio de Janeiro state, Brazil, from October/2006 to September/2008. Twenty women, diagnosed with HPV infection related to oncogenic high risk, were interviewed. The Symbolic Interactionism and Grounded Theory perspectives guided data collection and analysis. The results revealed that the HPV diagnosis means serious challenges in the women's relationship with her partner mainly regarding to the adoption of preventive initiatives. It is concluded that these issues lead to the need of a humanized care in order to favor the women's empowerment.

  16. Partnering.

    DTIC Science & Technology

    1991-06-01

    AD-A240,6490 PARTNERING A Special Research Problem Presented to The Faculty of the School of Civil Engineering Georgia Institute of Technology by...OF GEORGIA SCHOOL OF CIVIL ENGINEERING .ATLANTA, GEORGIA 30332 9 1 D 17 116 PARTNERING Approved: Faculty Adv ,’*/Da e TABLE OF CONTENTS Page LIST OF...It appears so. The results from the Oliver Lock and Dam, and the Operational Control Center indicate a successful project. The investment in the

  17. Cooperative interactions between paired domain and homeodomain.

    PubMed

    Jun, S; Desplan, C

    1996-09-01

    The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs,the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire.

  18. Predicting domain-domain interaction based on domain profiles with feature selection and support vector machines

    PubMed Central

    2010-01-01

    Background Protein-protein interaction (PPI) plays essential roles in cellular functions. The cost, time and other limitations associated with the current experimental methods have motivated the development of computational methods for predicting PPIs. As protein interactions generally occur via domains instead of the whole molecules, predicting domain-domain interaction (DDI) is an important step toward PPI prediction. Computational methods developed so far have utilized information from various sources at different levels, from primary sequences, to molecular structures, to evolutionary profiles. Results In this paper, we propose a computational method to predict DDI using support vector machines (SVMs), based on domains represented as interaction profile hidden Markov models (ipHMM) where interacting residues in domains are explicitly modeled according to the three dimensional structural information available at the Protein Data Bank (PDB). Features about the domains are extracted first as the Fisher scores derived from the ipHMM and then selected using singular value decomposition (SVD). Domain pairs are represented by concatenating their selected feature vectors, and classified by a support vector machine trained on these feature vectors. The method is tested by leave-one-out cross validation experiments with a set of interacting protein pairs adopted from the 3DID database. The prediction accuracy has shown significant improvement as compared to InterPreTS (Interaction Prediction through Tertiary Structure), an existing method for PPI prediction that also uses the sequences and complexes of known 3D structure. Conclusions We show that domain-domain interaction prediction can be significantly enhanced by exploiting information inherent in the domain profiles via feature selection based on Fisher scores, singular value decomposition and supervised learning based on support vector machines. Datasets and source code are freely available on the web at http

  19. Predicting domain-domain interaction based on domain profiles with feature selection and support vector machines.

    PubMed

    González, Alvaro J; Liao, Li

    2010-10-29

    Protein-protein interaction (PPI) plays essential roles in cellular functions. The cost, time and other limitations associated with the current experimental methods have motivated the development of computational methods for predicting PPIs. As protein interactions generally occur via domains instead of the whole molecules, predicting domain-domain interaction (DDI) is an important step toward PPI prediction. Computational methods developed so far have utilized information from various sources at different levels, from primary sequences, to molecular structures, to evolutionary profiles. In this paper, we propose a computational method to predict DDI using support vector machines (SVMs), based on domains represented as interaction profile hidden Markov models (ipHMM) where interacting residues in domains are explicitly modeled according to the three dimensional structural information available at the Protein Data Bank (PDB). Features about the domains are extracted first as the Fisher scores derived from the ipHMM and then selected using singular value decomposition (SVD). Domain pairs are represented by concatenating their selected feature vectors, and classified by a support vector machine trained on these feature vectors. The method is tested by leave-one-out cross validation experiments with a set of interacting protein pairs adopted from the 3DID database. The prediction accuracy has shown significant improvement as compared to InterPreTS (Interaction Prediction through Tertiary Structure), an existing method for PPI prediction that also uses the sequences and complexes of known 3D structure. We show that domain-domain interaction prediction can be significantly enhanced by exploiting information inherent in the domain profiles via feature selection based on Fisher scores, singular value decomposition and supervised learning based on support vector machines. Datasets and source code are freely available on the web at http

  20. The interaction of transverse domain walls.

    PubMed

    Krüger, Benjamin

    2012-01-18

    The interaction between transverse domain walls is calculated analytically using a multipole expansion up to third order. Starting from an analytical expression for the magnetization in the wall, the monopole, dipole, and quadrupole moments are derived and their impact on the interaction is investigated using the surface and volume charges. The surface charges are important for the dipole moment while the volume charges constitute the monopole and quadrupole moments. For domain walls that are situated in different wires it is found that there is a strong deviation from the interaction of two monopoles. This deviation is caused by the interaction of the monopole of the wall in the first wire with the dipole of the wall in the second wire and vice versa. The dipole-dipole and the quadrupole-monopole interactions are found to be also of considerable size and non-negligible. A comparison with micromagnetic simulations shows a good agreement.

  1. THE PARITY PARTNER OF THE NUCLEON IN QUENCHED QCD WITH DOMAIN WALL FERMIONS

    SciTech Connect

    SASAKI,S.

    2000-07-12

    The authors present preliminary results for the mass spectrum of the nucleon and its low-lying excited states from quenched lattice QCD using the domain wall fermion method which preserves the chiral symmetry at finite lattice cutoff. Definite mass splitting is observed between the nucleon and its parity partner. This splitting grows with decreasing valence quark mass. They also present preliminary data regarding the first positive-parity excited state.

  2. Partnering

    DTIC Science & Technology

    1991-12-01

    to meet the de- successful despite the site challenges and sign intent, business changes during the project. (Groves was acquired by the Torno ...34 Complete the contract without need for America company during construction- litigation. Torno embraced Partnering and continued the process which was

  3. New Reactions and Products Resulting from Alternative Interactions between the P450 Enzyme and Redox Partners

    PubMed Central

    2015-01-01

    Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C–H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein–protein interactions in modulating the catalytic activity of P450 enzymes. PMID:24521145

  4. Conditional random field approach to prediction of protein-protein interactions using domain information.

    PubMed

    Hayashida, Morihiro; Kamada, Mayumi; Song, Jiangning; Akutsu, Tatsuya

    2011-06-20

    For understanding cellular systems and biological networks, it is important to analyze functions and interactions of proteins and domains. Many methods for predicting protein-protein interactions have been developed. It is known that mutual information between residues at interacting sites can be higher than that at non-interacting sites. It is based on the thought that amino acid residues at interacting sites have coevolved with those at the corresponding residues in the partner proteins. Several studies have shown that such mutual information is useful for identifying contact residues in interacting proteins. We propose novel methods using conditional random fields for predicting protein-protein interactions. We focus on the mutual information between residues, and combine it with conditional random fields. In the methods, protein-protein interactions are modeled using domain-domain interactions. We perform computational experiments using protein-protein interaction datasets for several organisms, and calculate AUC (Area Under ROC Curve) score. The results suggest that our proposed methods with and without mutual information outperform EM (Expectation Maximization) method proposed by Deng et al., which is one of the best predictors based on domain-domain interactions. We propose novel methods using conditional random fields with and without mutual information between domains. Our methods based on domain-domain interactions are useful for predicting protein-protein interactions.

  5. CONTESTED DOMAINS, VERBAL 'AMPLIFIERS,' AND INTIMATE PARTNER VIOLENCE IN YOUNG ADULTHOOD.

    PubMed

    Giordano, Peggy C; Copp, Jennifer E; Longmore, Monica A; Manning, Wendy D

    2015-12-01

    We draw on structured and qualitative data to examine relationship dynamics associated with intimate partner violence (IPV) that occurs during the young adult period. Relying on a symbolic interactionist perspective, we identify specific contested domains associated with what has been called 'situational couple violence,' and explore the degree to which certain forms of communication about contested areas ('verbal amplifiers') exacerbate the risk of violence. Consistent with this relational focus, measures index respondent as well as partner concerns and use of these negative forms of communication. Results of analyses of interview data from a large, diverse sample of young adults show that net of family background, history of antisocial behavior, and other controls, concerns about the partner's or individual's own economic viability, disagreements about time spent with friends, and issues of infidelity are significantly related to IPV perpetration. Yet the analyses indicate that infidelity is particularly central as a source of conflict associated with violence, and the use of verbal amplifiers explained additional variance. Further, while research has highlighted important differences in the meaning and consequences of male and female IPV, findings point to some areas of overlap in the relationship concerns and communication processes associated with variations in self-reports of the use of violence. In-depth "relationship history narratives" elicited from a subset of respondents and a sample of their partners support the quantitative results, but also highlight variations within the sample, the sequencing of these interrelated processes, and ways in which gender may have influenced respondents' perspectives and behavior.

  6. Mast cell: an emerging partner in immune interaction.

    PubMed

    Gri, Giorgia; Frossi, Barbara; D'Inca, Federica; Danelli, Luca; Betto, Elena; Mion, Francesca; Sibilano, Riccardo; Pucillo, Carlo

    2012-01-01

    Mast cells (MCs) are currently recognized as effector cells in many settings of the immune response, including host defense, immune regulation, allergy, chronic inflammation, and autoimmune diseases. MC pleiotropic functions reflect their ability to secrete a wide spectrum of preformed or newly synthesized biologically active products with pro-inflammatory, anti-inflammatory and/or immunosuppressive properties, in response to multiple signals. Moreover, the modulation of MC effector phenotypes relies on the interaction of a wide variety of membrane molecules involved in cell-cell or cell-extracellular-matrix interaction. The delivery of co-stimulatory signals allows MC to specifically communicate with immune cells belonging to both innate and acquired immunity, as well as with non-immune tissue-specific cell types. This article reviews and discusses the evidence that MC membrane-expressed molecules play a central role in regulating MC priming and activation and in the modulation of innate and adaptive immune response not only against host injury, but also in peripheral tolerance and tumor-surveillance or -escape. The complex expression of MC surface molecules may be regarded as a measure of connectivity, with altered patterns of cell-cell interaction representing functionally distinct MC states. We will focalize our attention on roles and functions of recently discovered molecules involved in the cross-talk of MCs with other immune partners.

  7. Mast Cell: An Emerging Partner in Immune Interaction

    PubMed Central

    Gri, Giorgia; Frossi, Barbara; D’Inca, Federica; Danelli, Luca; Betto, Elena; Mion, Francesca; Sibilano, Riccardo; Pucillo, Carlo

    2012-01-01

    Mast cells (MCs) are currently recognized as effector cells in many settings of the immune response, including host defense, immune regulation, allergy, chronic inflammation, and autoimmune diseases. MC pleiotropic functions reflect their ability to secrete a wide spectrum of preformed or newly synthesized biologically active products with pro-inflammatory, anti-inflammatory and/or immunosuppressive properties, in response to multiple signals. Moreover, the modulation of MC effector phenotypes relies on the interaction of a wide variety of membrane molecules involved in cell–cell or cell-extracellular-matrix interaction. The delivery of co-stimulatory signals allows MC to specifically communicate with immune cells belonging to both innate and acquired immunity, as well as with non-immune tissue-specific cell types. This article reviews and discusses the evidence that MC membrane-expressed molecules play a central role in regulating MC priming and activation and in the modulation of innate and adaptive immune response not only against host injury, but also in peripheral tolerance and tumor-surveillance or -escape. The complex expression of MC surface molecules may be regarded as a measure of connectivity, with altered patterns of cell–cell interaction representing functionally distinct MC states. We will focalize our attention on roles and functions of recently discovered molecules involved in the cross-talk of MCs with other immune partners. PMID:22654879

  8. The Different Roles of Aggrecan Interaction Domains

    PubMed Central

    2012-01-01

    The aggregating proteoglycans of the lectican family are important components of extracellular matrices. Aggrecan is the most well studied of these and is central to cartilage biomechanical properties and skeletal development. Key to its biological function is the fixed charge of the many glycosaminoglycan chains, that provide the basis for the viscoelastic properties necessary for load distribution over the articular surface. This review is focused on the globular domains of aggrecan and their role in anchoring the proteoglycans to other extracellular matrix components. The N-terminal G1 domain is vital in that it binds the proteoglycan to hyaluronan in ternary complex with link protein, retaining the proteoglycan in the tissue. The importance of the C-terminal G3 domain interactions has recently been emphasized by two different human hereditary disorders: autosomal recessive aggrecan-type spondyloepimetaphyseal dysplasia and autosomal dominant familial osteochondritis dissecans. In these two conditions, different missense mutations in the aggrecan C-type lectin repeat have been described. The resulting amino acid replacements affect the ligand interactions of the G3 domain, albeit with widely different phenotypic outcomes. PMID:23019016

  9. Evolution of a protein domain interaction network

    NASA Astrophysics Data System (ADS)

    Gao, Li-Feng; Shi, Jian-Jun; Guan, Shan

    2010-01-01

    In this paper, we attempt to understand complex network evolution from the underlying evolutionary relationship between biological organisms. Firstly, we construct a Pfam domain interaction network for each of the 470 completely sequenced organisms, and therefore each organism is correlated with a specific Pfam domain interaction network; secondly, we infer the evolutionary relationship of these organisms with the nearest neighbour joining method; thirdly, we use the evolutionary relationship between organisms constructed in the second step as the evolutionary course of the Pfam domain interaction network constructed in the first step. This analysis of the evolutionary course shows: (i) there is a conserved sub-network structure in network evolution; in this sub-network, nodes with lower degree prefer to maintain their connectivity invariant, and hubs tend to maintain their role as a hub is attached preferentially to new added nodes; (ii) few nodes are conserved as hubs; most of the other nodes are conserved as one with very low degree; (iii) in the course of network evolution, new nodes are added to the network either individually in most cases or as clusters with relative high clustering coefficients in a very few cases.

  10. In Situ Detection of Interactions Between Nuclear Envelope Proteins and Partners.

    PubMed

    Barateau, Alice; Buendia, Brigitte

    2016-01-01

    Proximity ligation assay (PLA) appears as a quick and easy technique to visualize within fixed cells the occurrence and in situ distribution of protein complexes. PLA has been validated to detect protein-protein interactions within the nuclear compartment. Here, we describe a protocol which allows the detection of interactions between A-type nuclear lamins and either LEM-domain proteins (such as emerin, integrated within the inner nuclear membrane, and LAP2α which accumulates within the nucleoplasm) or gene regulatory factors (e.g., the transcription factor SREBP1). The distinct amounts and patterns of PLA signals obtained for various complexes highlight the pertinence of using PLA to reveal in situ where and to which extent nuclear envelope proteins bind specific partners.

  11. Characterization of domain-peptide interaction interface: prediction of SH3 domain-mediated protein-protein interaction network in yeast by generic structure-based models.

    PubMed

    Hou, Tingjun; Li, Nan; Li, Youyong; Wang, Wei

    2012-05-04

    Determination of the binding specificity of SH3 domain, a peptide recognition module (PRM), is important to understand their biological functions and reconstruct the SH3-mediated protein-protein interaction network. In the present study, the SH3-peptide interactions for both class I and II SH3 domains were characterized by the intermolecular residue-residue interaction network. We developed generic MIEC-SVM models to infer SH3 domain-peptide recognition specificity that achieved satisfactory prediction accuracy. By investigating the domain-peptide recognition mechanisms at the residue level, we found that the class-I and class-II binding peptides have different binding modes even though they occupy the same binding site of SH3. Furthermore, we predicted the potential binding partners of SH3 domains in the yeast proteome and constructed the SH3-mediated protein-protein interaction network. Comparison with the experimentally determined interactions confirmed the effectiveness of our approach. This study showed that our sophisticated computational approach not only provides a powerful platform to decipher protein recognition code at the molecular level but also allows identification of peptide-mediated protein interactions at a proteomic scale. We believe that such an approach is general to be applicable to other domain-peptide interactions.

  12. Effects of administered alcohol on intimate partner interactions in a conflict resolution paradigm.

    PubMed

    Testa, Maria; Crane, Cory A; Quigley, Brian M; Levitt, Ash; Leonard, Kenneth E

    2014-03-01

    Although couples' alcohol use has been associated with intimate partner aggression and poorer marital functioning, few studies have examined the proximal effects of alcohol on couple interactions. The current experimental study examined the effects of alcohol, administered independently to male and female intimate partners, on positive and negative interaction behaviors within a naturalistic conflict resolution paradigm. Married and cohabiting couples (n = 152) were recruited from the community and each partner randomly assigned to receive either alcohol (target dose: .08 mg/kg) or no alcohol. They engaged in two 15-minute interactions regarding current disagreements in their relationship, one before and one after beverage administration. Videotaped interactions were coded by trained observers using the Rapid Marital Interaction Coding System, and positive and negative interaction behaviors were analyzed using the Actor-Partner Interdependence Model. Participants displayed decreased negativity and increased positivity following alcohol consumption when their partners were sober but no differences in negativity or positivity when their partners also consumed alcohol. There were no gender differences. Although participants with a history of perpetrating intimate partner aggression displayed more negativity, prior aggression did not interact with beverage condition. The immediate effects of alcohol consumption on couple interaction behaviors appeared more positive than negative. Contrary to hypotheses, congruent partner drinking had neither particularly positive nor particularly negative effects. These unique findings represent a rare glimpse into the immediate consequences of alcohol consumption on couple interaction and stand in contrast to its delayed or long-term effects.

  13. Institutional, individual, and socio-cultural domains of partnerships: a typology of USDA Forest Service recreation partners.

    PubMed

    Seekamp, Erin; Cerveny, Lee K; McCreary, Allie

    2011-09-01

    Federal land management agencies, such as the USDA Forest Service, have expanded the role of recreation partners reflecting constrained growth in appropriations and broader societal trends towards civic environmental governance. Partnerships with individual volunteers, service groups, commercial outfitters, and other government agencies provide the USDA Forest Service with the resources necessary to complete projects and meet goals under fiscal constraints. Existing partnership typologies typically focus on collaborative or strategic alliances and highlight organizational dimensions (e.g., structure and process) defined by researchers. This paper presents a partner typology constructed from USDA Forest Service partnership practitioners' conceptualizations of 35 common partner types. Multidimensional scaling of data from unconstrained pile sorts identified 3 distinct cultural dimensions of recreation partners--specifically, partnership character, partner impact, and partner motivations--that represent institutional, individual, and socio-cultural cognitive domains. A hierarchical agglomerative cluster analysis provides further insight into the various domains of agency personnel's conceptualizations. While three dimensions with high reliability (RSQ = 0.83) and corresponding hierarchical clusters illustrate commonality between agency personnel's partnership suppositions, this study also reveals variance in personnel's familiarity and affinity for specific partnership types. This real-world perspective on partner types highlights that agency practitioners not only make strategic choices when selecting and cultivating partnerships to accomplish critical task, but also elect to work with partners for the primary purpose of providing public service and fostering land stewardship.

  14. Coevolution between positive reciprocity, punishment, and partner switching in repeated interactions.

    PubMed

    Wubs, Matthias; Bshary, Redouan; Lehmann, Laurent

    2016-06-15

    Cooperation based on mutual investments can occur between unrelated individuals when they are engaged in repeated interactions. Individuals then need to use a conditional strategy to deter their interaction partners from defecting. Responding to defection such that the future payoff of a defector is reduced relative to cooperating with it is called a partner control mechanism. Three main partner control mechanisms are (i) to switch from cooperation to defection when being defected ('positive reciprocity'), (ii) to actively reduce the payoff of a defecting partner ('punishment'), or (iii) to stop interacting and switch partner ('partner switching'). However, such mechanisms to stabilize cooperation are often studied in isolation from each other. In order to better understand the conditions under which each partner control mechanism tends to be favoured by selection, we here analyse by way of individual-based simulations the coevolution between positive reciprocity, punishment, and partner switching. We show that random interactions in an unstructured population and a high number of rounds increase the likelihood that selection favours partner switching. In contrast, interactions localized in small groups (without genetic structure) increase the likelihood that selection favours punishment and/or positive reciprocity. This study thus highlights the importance of comparing different control mechanisms for cooperation under different conditions. © 2016 The Author(s).

  15. A Strategy for Interaction Site Prediction between Phospho-binding Modules and their Partners Identified from Proteomic Data*

    PubMed Central

    Aucher, Willy; Becker, Emmanuelle; Ma, Emilie; Miron, Simona; Martel, Arnaud; Ochsenbein, Françoise; Marsolier-Kergoat, Marie-Claude; Guerois, Raphaël

    2010-01-01

    Small and large scale proteomic technologies are providing a wealth of potential interactions between proteins bearing phospho-recognition modules and their substrates. Resulting interaction maps reveal such a dense network of interactions that the functional dissection and understanding of these networks often require to break specific interactions while keeping the rest intact. Here, we developed a computational strategy, called STRIP, to predict the precise interaction site involved in an interaction with a phospho-recognition module. The method was validated by a two-hybrid screen carried out using the ForkHead Associated (FHA)1 domain of Rad53, a key protein of Saccharomyces cerevisiae DNA checkpoint, as a bait. In this screen we detected 11 partners, including Cdc7 and Cdc45, essential components of the DNA replication machinery. FHA domains are phospho-threonine binding modules and the threonines involved in both interactions could be predicted using the STRIP strategy. The threonines T484 and T189 in Cdc7 and Cdc45, respectively, were mutated and loss of binding could be monitored experimentally with the full-length proteins. The method was further tested for the analysis of 63 known Rad53 binding partners and provided several key insights regarding the threonines likely involved in these interactions. The STRIP method relies on a combination of conservation, phosphorylation likelihood, and binding specificity criteria and can be accessed via a web interface at http://biodev.extra.cea.fr/strip/. PMID:20733106

  16. Ras GTPases’ interaction with effector domains

    PubMed Central

    Patel, Manishha; Côté, Jean-François

    2013-01-01

    The Ras superfamily of proteins consists of five branches: Ras, Rho, Arf, Rab and Ran subfamilies. These proteins are involved in a plethora of biological functions spanning cytoskeletal organization, cell proliferation, transcription and intracellular trafficking. Ras-Binding Domains (RBDs) have classically been identified as autonomous ubiquitin-like folded regions that bind certain activated Ras GTPases of the Ras subfamily. In general, RBDs in many proteins have been tagged with membrane-targeting functions as in the case of the well-characterized c-Raf-RBD/Ras interaction. However, it is becoming apparent that the definition and functions of RBDs need to be revamped in order to reflect the new discoveries associated with this domain. Here, we discuss in more detail the recent advances associated with these RBDs. We highlight research identifying RBDs in formins, ELMOs and the RhoGEF, Syx and discuss the emerging role for RBDs in controlling autoinhibition relief and the newly recognized versatility of RBDs to interact with Rho and Arf family GTPases. In addition, these recent findings raise the exciting hypothesis that functional RBDs remain hidden in the proteome and are ready to be uncovered. PMID:23986800

  17. CDD: a conserved domain database for interactive domain family analysis.

    PubMed

    Marchler-Bauer, Aron; Anderson, John B; Derbyshire, Myra K; DeWeese-Scott, Carol; Gonzales, Noreen R; Gwadz, Marc; Hao, Luning; He, Siqian; Hurwitz, David I; Jackson, John D; Ke, Zhaoxi; Krylov, Dmitri; Lanczycki, Christopher J; Liebert, Cynthia A; Liu, Chunlei; Lu, Fu; Lu, Shennan; Marchler, Gabriele H; Mullokandov, Mikhail; Song, James S; Thanki, Narmada; Yamashita, Roxanne A; Yin, Jodie J; Zhang, Dachuan; Bryant, Stephen H

    2007-01-01

    The conserved domain database (CDD) is part of NCBI's Entrez database system and serves as a primary resource for the annotation of conserved domain footprints on protein sequences in Entrez. Entrez's global query interface can be accessed at http://www.ncbi.nlm.nih.gov/Entrez and will search CDD and many other databases. Domain annotation for proteins in Entrez has been pre-computed and is readily available in the form of 'Conserved Domain' links. Novel protein sequences can be scanned against CDD using the CD-Search service; this service searches databases of CDD-derived profile models with protein sequence queries using BLAST heuristics, at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. Protein query sequences submitted to NCBI's protein BLAST search service are scanned for conserved domain signatures by default. The CDD collection contains models imported from Pfam, SMART and COG, as well as domain models curated at NCBI. NCBI curated models are organized into hierarchies of domains related by common descent. Here we report on the status of the curation effort and present a novel helper application, CDTree, which enables users of the CDD resource to examine curated hierarchies. More importantly, CDD and CDTree used in concert, serve as a powerful tool in protein classification, as they allow users to analyze protein sequences in the context of domain family hierarchies.

  18. CONTESTED DOMAINS, VERBAL ‘AMPLIFIERS,’ AND INTIMATE PARTNER VIOLENCE IN YOUNG ADULTHOOD

    PubMed Central

    Giordano, Peggy C.; Copp, Jennifer E.; Longmore, Monica A.; Manning, Wendy D.

    2015-01-01

    We draw on structured and qualitative data to examine relationship dynamics associated with intimate partner violence (IPV) that occurs during the young adult period. Relying on a symbolic interactionist perspective, we identify specific contested domains associated with what has been called ‘situational couple violence,’ and explore the degree to which certain forms of communication about contested areas (‘verbal amplifiers’) exacerbate the risk of violence. Consistent with this relational focus, measures index respondent as well as partner concerns and use of these negative forms of communication. Results of analyses of interview data from a large, diverse sample of young adults show that net of family background, history of antisocial behavior, and other controls, concerns about the partner’s or individual’s own economic viability, disagreements about time spent with friends, and issues of infidelity are significantly related to IPV perpetration. Yet the analyses indicate that infidelity is particularly central as a source of conflict associated with violence, and the use of verbal amplifiers explained additional variance. Further, while research has highlighted important differences in the meaning and consequences of male and female IPV, findings point to some areas of overlap in the relationship concerns and communication processes associated with variations in self-reports of the use of violence. In-depth “relationship history narratives” elicited from a subset of respondents and a sample of their partners support the quantitative results, but also highlight variations within the sample, the sequencing of these interrelated processes, and ways in which gender may have influenced respondents’ perspectives and behavior. PMID:26617420

  19. Institutional, Individual, and Socio-Cultural Domains of Partnerships: A Typology of USDA Forest Service Recreation Partners

    NASA Astrophysics Data System (ADS)

    Seekamp, Erin; Cerveny, Lee K.; McCreary, Allie

    2011-09-01

    Federal land management agencies, such as the USDA Forest Service, have expanded the role of recreation partners reflecting constrained growth in appropriations and broader societal trends towards civic environmental governance. Partnerships with individual volunteers, service groups, commercial outfitters, and other government agencies provide the USDA Forest Service with the resources necessary to complete projects and meet goals under fiscal constraints. Existing partnership typologies typically focus on collaborative or strategic alliances and highlight organizational dimensions (e.g., structure and process) defined by researchers. This paper presents a partner typology constructed from USDA Forest Service partnership practitioners' conceptualizations of 35 common partner types. Multidimensional scaling of data from unconstrained pile sorts identified 3 distinct cultural dimensions of recreation partners—specifically, partnership character, partner impact, and partner motivations—that represent institutional, individual, and socio-cultural cognitive domains. A hierarchical agglomerative cluster analysis provides further insight into the various domains of agency personnel's conceptualizations. While three dimensions with high reliability (RSQ = 0.83) and corresponding hierarchical clusters illustrate commonality between agency personnel's partnership suppositions, this study also reveals variance in personnel's familiarity and affinity for specific partnership types. This real-world perspective on partner types highlights that agency practitioners not only make strategic choices when selecting and cultivating partnerships to accomplish critical task, but also elect to work with partners for the primary purpose of providing public service and fostering land stewardship.

  20. Sequential unfolding of the hemolysin two-partner secretion domain from Proteus mirabilis

    PubMed Central

    Wimmer, Megan R; Woods, Christopher N; Adamczak, Kyle J; Glasgow, Evan M; Novak, Walter RP; Grilley, Daniel P; Weaver, Todd M

    2015-01-01

    Protein secretion is a major contributor to Gram-negative bacterial virulence. Type Vb or two-partner secretion (TPS) pathways utilize a membrane bound β-barrel B component (TpsB) to translocate large and predominantly virulent exoproteins (TpsA) through a nucleotide independent mechanism. We focused our studies on a truncated TpsA member termed hemolysin A (HpmA265), a structurally and functionally characterized TPS domain from Proteus mirabilis. Contrary to the expectation that the TPS domain of HpmA265 would denature in a single cooperative transition, we found that the unfolding follows a sequential model with three distinct transitions linking four states. The solvent inaccessible core of HpmA265 can be divided into two different regions. The C-proximal region contains nonpolar residues and forms a prototypical hydrophobic core as found in globular proteins. The N-proximal region of the solvent inaccessible core, however, contains polar residues. To understand the contributions of the hydrophobic and polar interiors to overall TPS domain stability, we conducted unfolding studies on HpmA265 and site-specific mutants of HpmA265. By correlating the effect of individual site-specific mutations with the sequential unfolding results we were able to divide the HpmA265 TPS domain into polar core, nonpolar core, and C-terminal subdomains. Moreover, the unfolding studies provide quantitative evidence that the folding free energy for the polar core subdomain is more favorable than for the nonpolar core and C-terminal subdomains. This study implicates the hydrogen bonds shared among these conserved internal residues as a primary means for stabilizing the N-proximal polar core subdomain. PMID:26350294

  1. Sequential unfolding of the hemolysin two-partner secretion domain from Proteus mirabilis.

    PubMed

    Wimmer, Megan R; Woods, Christopher N; Adamczak, Kyle J; Glasgow, Evan M; Novak, Walter R P; Grilley, Daniel P; Weaver, Todd M

    2015-11-01

    Protein secretion is a major contributor to Gram-negative bacterial virulence. Type Vb or two-partner secretion (TPS) pathways utilize a membrane bound β-barrel B component (TpsB) to translocate large and predominantly virulent exoproteins (TpsA) through a nucleotide independent mechanism. We focused our studies on a truncated TpsA member termed hemolysin A (HpmA265), a structurally and functionally characterized TPS domain from Proteus mirabilis. Contrary to the expectation that the TPS domain of HpmA265 would denature in a single cooperative transition, we found that the unfolding follows a sequential model with three distinct transitions linking four states. The solvent inaccessible core of HpmA265 can be divided into two different regions. The C-proximal region contains nonpolar residues and forms a prototypical hydrophobic core as found in globular proteins. The N-proximal region of the solvent inaccessible core, however, contains polar residues. To understand the contributions of the hydrophobic and polar interiors to overall TPS domain stability, we conducted unfolding studies on HpmA265 and site-specific mutants of HpmA265. By correlating the effect of individual site-specific mutations with the sequential unfolding results we were able to divide the HpmA265 TPS domain into polar core, nonpolar core, and C-terminal subdomains. Moreover, the unfolding studies provide quantitative evidence that the folding free energy for the polar core subdomain is more favorable than for the nonpolar core and C-terminal subdomains. This study implicates the hydrogen bonds shared among these conserved internal residues as a primary means for stabilizing the N-proximal polar core subdomain. © 2015 The Protein Society.

  2. A beta-sheet interaction interface directs the tetramerisation of the Miz-1 POZ domain.

    PubMed

    Stead, Mark A; Trinh, Chi H; Garnett, James A; Carr, Stephen B; Baron, Andrew J; Edwards, Thomas A; Wright, Stephanie C

    2007-11-02

    The POZ/BTB domain is an evolutionarily conserved motif found in approximately 40 zinc-finger transcription factors (POZ-ZF factors). Several POZ-ZF factors are implicated in human cancer, and POZ domain interaction interfaces represent an attractive target for therapeutic intervention. Miz-1 (Myc-interacting zinc-finger protein) is a POZ-ZF factor that regulates DNA-damage-induced cell cycle arrest and plays an important role in human cancer by virtue of its interaction with the c-Myc and BCL6 oncogene products. The Miz-1 POZ domain mediates both self-association and the recruitment of non-POZ partners. POZ-ZF factors generally function as homodimers, although higher-order associations and heteromeric interactions are known to be physiologically important; crucially, the interaction interfaces in such large complexes have not been characterised. We report here the crystal structure of the Miz-1 POZ domain up to 2.1 A resolution. The tetrameric organisation of Miz-1 POZ reveals two types of interaction interface between subunits; an interface of alpha-helices resembles the dimerisation interface of reported POZ domain structures, whereas a novel beta-sheet interface directs the association of two POZ domain dimers. We show that the beta-sheet interface directs the tetramerisation of the Miz-1 POZ domain in solution and therefore represents a newly described candidate interface for the higher-order homo- and hetero-oligomerisation of POZ-ZF proteins in vivo.

  3. Interacting protein partners of Arabidopsis RNA-binding protein AtRBP45b.

    PubMed

    Muthuramalingam, M; Wang, Y; Li, Y; Mahalingam, R

    2017-05-01

    RNA binding proteins, important players in post-transcriptional gene regulation, usually exist in ribonuclear complexes. However, even in model systems like Arabidopsis characterisation of RBP associated proteins is limited. In this study, we investigated the interacting proteins of the Arabidopsis AtRBP45b, which is involved in stress signalling. In vivo localisation of AtRBP45b was conducted using 35S-GFP. FLAG-tagged AtRBP45b under control of the 35S promoter in the Atrbp45b-1 mutant background was used to pull down AtRBP45b interacting proteins. Yeast two-hybrid analysis, fluorescence energy resonance transfer assays were used to confirm the veracity of the AtRBP45b interacting proteins. In planta GFP-tagging indicated AtRBP45b is localised to the nucleus and the cytosol. AtRBP45b protein has a N-terminal proline-rich region and a C-terminal glutamine-rich domain that are usually involved in protein-protein interactions. Co-immunoprecipitation followed by mass spectrometry-based protein sequencing led to identification of 30 proteins that interacted with AtRBP45b. Using information from interactome databases (BIOGRID, INTACT and STRING), pull-down assays and localisation data, 12 putative interacting proteins were selected for yeast two-hybrid analysis. Cap-binding protein (CBP20, At5g44200) and polyA-binding protein (PAB8, At1g49760) were shown to interact with AtRBP45b. Based on its interacting partners we speculate that AtRBP45b may play an important role in RNA metabolism, especially in aspects related to mRNA stability and translation initiation during stress conditions in plants.

  4. When an Intramolecular Disulfide Bridge Governs the Interaction of DUOX2 with Its Partner DUOXA2

    PubMed Central

    Carré, Aurore; Louzada, Ruy A.N.; Fortunato, Rodrigo S.; Ameziane-El-Hassani, Rabii; Morand, Stanislas; Ogryzko, Vasily; de Carvalho, Denise Pires; Grasberger, Helmut; Leto, Thomas L.

    2015-01-01

    Abstract Aims: The dual oxidase 2 (DUOX2) protein belongs to the NADPH oxidase (NOX) family. As H2O2 generator, it plays a key role in both thyroid hormone biosynthesis and innate immunity. DUOX2 forms with its maturation factor, DUOX activator 2 (DUOXA2), a stable complex at the cell surface that is crucial for the H2O2-generating activity, but the nature of their interaction is unknown. The contribution of some cysteine residues located in the N-terminal ectodomain of DUOX2 in a surface protein–protein interaction is suggested. We have investigated the involvement of different cysteine residues in the formation of covalent bonds that could be of critical importance for the function of the complex. Results: We report the identification and the characterization of an intramolecular disulfide bond between cys-124 of the N-terminal ectodomain and cys-1162 of an extracellular loop of DUOX2, which has important functional implications in both export and activity of DUOX2. This intramolecular bridge provides structural support for the formation of interdisulfide bridges between the N-terminal domain of DUOX2 and the two extracellular loops of its partner, DUOXA2. Innovation: Both stability and function of the maturation factor, DUOXA2, are dependent on the oxidative folding of DUOX2, indicating that DUOX2 displays a chaperone-like function with respect to its partner. Conclusions: The oxidative folding of DUOX2 that takes place in the endoplasmic reticulum (ER) appears to be a key event in the trafficking of the DUOX2/DUOXA2 complex as it promotes an appropriate conformation of the N-terminal region, which is propitious to subsequent covalent interactions with the maturation factor, DUOXA2. Antioxid. Redox Signal. 23, 724–733. PMID:25761904

  5. Protein Interacting with C Kinase 1 (PICK1) Reduces Reinsertion Rates of Interaction Partners Sorted to Rab11-dependent Slow Recycling Pathway*

    PubMed Central

    Madsen, Kenneth L.; Thorsen, Thor S.; Rahbek-Clemmensen, Troels; Eriksen, Jacob; Gether, Ulrik

    2012-01-01

    The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the β2-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway. PMID:22303009

  6. Protein interacting with C kinase 1 (PICK1) reduces reinsertion rates of interaction partners sorted to Rab11-dependent slow recycling pathway.

    PubMed

    Madsen, Kenneth L; Thorsen, Thor S; Rahbek-Clemmensen, Troels; Eriksen, Jacob; Gether, Ulrik

    2012-04-06

    The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the β(2)-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway.

  7. Conformational selectivity in cytochrome P450 redox partner interactions

    PubMed Central

    Hollingsworth, Scott A.; Batabyal, Dipanwita; Nguyen, Brian D.; Poulos, Thomas L.

    2016-01-01

    The heme iron of cytochromes P450 must be reduced to bind and activate molecular oxygen for substrate oxidation. Reducing equivalents are derived from a redox partner, which requires the formation of a protein–protein complex. A subject of increasing discussion is the role that redox partner binding plays, if any, in favoring significant structural changes in the P450s that are required for activity. Many P450s now have been shown to experience large open and closed motions. Several structural and spectral studies indicate that the well-studied P450cam adopts the open conformation when its redox partner, putidaredoxin (Pdx), binds, whereas recent NMR studies indicate that this view is incorrect. Given the relevance of this discrepancy to P450 chemistry, it is important to determine whether Pdx favors the open or closed form of P450cam. Here, we have used both computational and experimental isothermal titration calorimetry studies that unequivocally show Pdx favors binding to the open form of P450cam. Analyses of molecular-dynamic trajectories also provide insights into intermediate conformational states that could be relevant to catalysis. PMID:27439869

  8. The good, the bad, and the rare: memory for partners in social interactions.

    PubMed

    Volstorf, Jenny; Rieskamp, Jörg; Stevens, Jeffrey R

    2011-04-29

    For cooperation to evolve via direct reciprocity, individuals must track their partners' behavior to avoid exploitation. With increasing size of the interaction group, however, memory becomes error prone. To decrease memory effort, people could categorize partners into types, distinguishing cooperators and cheaters. We explored two ways in which people might preferentially track one partner type: remember cheaters or remember the rare type in the population. We assigned participants to one of three interaction groups which differed in the proportion of computer partners' types (defectors rare, equal proportion, or cooperators rare). We extended research on both hypotheses in two ways. First, participants experienced their partners repeatedly by interacting in Prisoner's Dilemma games. Second, we tested categorization of partners as cooperators or defectors in memory tests after a short and long retention interval (10 min and 1 week). Participants remembered rare partner types better than they remembered common ones at both retention intervals. We propose that the flexibility of responding to the environment suggests an ecologically rational memory strategy in social interactions.

  9. The Good, the Bad, and the Rare: Memory for Partners in Social Interactions

    PubMed Central

    Volstorf, Jenny; Rieskamp, Jörg; Stevens, Jeffrey R.

    2011-01-01

    For cooperation to evolve via direct reciprocity, individuals must track their partners' behavior to avoid exploitation. With increasing size of the interaction group, however, memory becomes error prone. To decrease memory effort, people could categorize partners into types, distinguishing cooperators and cheaters. We explored two ways in which people might preferentially track one partner type: remember cheaters or remember the rare type in the population. We assigned participants to one of three interaction groups which differed in the proportion of computer partners' types (defectors rare, equal proportion, or cooperators rare). We extended research on both hypotheses in two ways. First, participants experienced their partners repeatedly by interacting in Prisoner's Dilemma games. Second, we tested categorization of partners as cooperators or defectors in memory tests after a short and long retention interval (10 min and 1 week). Participants remembered rare partner types better than they remembered common ones at both retention intervals. We propose that the flexibility of responding to the environment suggests an ecologically rational memory strategy in social interactions. PMID:21559490

  10. Structural characterization of ANGPTL8 (betatrophin) with its interacting partner lipoprotein lipase.

    PubMed

    Siddiqa, Amnah; Ahmad, Jamil; Ali, Amjad; Paracha, Rehan Zafar; Bibi, Zurah; Aslam, Babar

    2016-04-01

    Angiopoietin-like protein 8 (ANGPTL8) (also known as betatrophin) is a newly identified secretory protein with a potential role in autophagy, lipid metabolism and pancreatic beta-cell proliferation. Its structural characterization is required to enhance our current understanding of its mechanism of action which could help in identifying its receptor and/or other binding partners. Based on the physiological significance and necessity of exploring structural features of ANGPTL8, the present study is conducted with a specific aim to model the structure of ANGPTL8 and study its possible interactions with Lipoprotein Lipase (LPL). To the best of our knowledge, this is the first attempt to predict 3-dimensional (3D) structure of ANGPTL8. Three different approaches were used for modeling of ANGPTL8 including homology modeling, de-novo structure prediction and their amalgam which is then proceeded by structure verification using ERRATT, PROSA, Qmean and Ramachandran plot scores. The selected models of ANGPTL8 were further evaluated for protein-protein interaction (PPI) analysis with LPL using CPORT and HADDOCK server. Our results have shown that the crystal structure of iSH2 domain of Phosphatidylinositol 3-kinase (PI3K) p85β subunit (PDB entry: 3mtt) is a good candidate for homology modeling of ANGPTL8. Analysis of inter-molecular interactions between the structure of ANGPTL8 and LPL revealed existence of several non-covalent interactions. The residues of LPL involved in these interactions belong from its lid region, thrombospondin (TSP) region and heparin binding site which is suggestive of a possible role of ANGPTL8 in regulating the proteolysis, motility and localization of LPL. Besides, the conserved residues of SE1 region of ANGPTL8 formed interactions with the residues around the hinge region of LPL. Overall, our results support a model of inhibition of LPL by ANGPTL8 through the steric block of its catalytic site which will be further explored using wet lab

  11. FTIR studies of the redox partner interaction in cytochrome P450: the Pdx-P450cam couple.

    PubMed

    Karyakin, Andrey; Motiejunas, Domantas; Wade, Rebecca C; Jung, Christiane

    2007-03-01

    Recently we have developed a new approach to study protein-protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam-Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP-FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in beta-sheets and alpha-helix content, a decrease in the population of random coil/3(10)-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam-Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx-P450cam complex.

  12. Contribution of DEAF1 structural domains to the interaction with the breast cancer oncogene LMO4.

    PubMed

    Cubeddu, Liza; Joseph, Soumya; Richard, Derek J; Matthews, Jacqueline M

    2012-01-01

    The proteins LMO4 and DEAF1 contribute to the proliferation of mammary epithelial cells. During breast cancer LMO4 is upregulated, affecting its interaction with other protein partners. This may set cells on a path to tumour formation. LMO4 and DEAF1 interact, but it is unknown how they cooperate to regulate cell proliferation. In this study, we identify a specific LMO4-binding domain in DEAF1. This domain contains an unstructured region that directly contacts LMO4, and a coiled coil that contains the DEAF1 nuclear export signal (NES). The coiled coil region can form tetramers and has the typical properties of a coiled coil domain. Using a simple cell-based assay, we show that LMO4 modulates the activity of the DEAF NES, causing nuclear accumulation of a construct containing the LMO4-interaction region of DEAF1.

  13. The SAM domain inhibits EphA2 interactions in the plasma membrane.

    PubMed

    Singh, Deo R; Ahmed, Fozia; Paul, Michael D; Gedam, Manasee; Pasquale, Elena B; Hristova, Kalina

    2017-01-01

    All members of the Eph receptor family of tyrosine kinases contain a SAM domain near the C terminus, which has been proposed to play a role in receptor homotypic interactions and/or interactions with binding partners. The SAM domain of EphA2 is known to be important for receptor function, but its contribution to EphA2 lateral interactions in the plasma membrane has not been determined. Here we use a FRET-based approach to directly measure the effect of the SAM domain on the stability of EphA2 dimers on the cell surface in the absence of ligand binding. We also investigate the functional consequences of EphA2 SAM domain deletion. Surprisingly, we find that the EphA2 SAM domain inhibits receptor dimerization and decreases EphA2 tyrosine phosphorylation. This role is dramatically different from the role of the SAM domain of the related EphA3 receptor, which we previously found to stabilize EphA3 dimers and increase EphA3 tyrosine phosphorylation in cells in the absence of ligand. Thus, the EphA2 SAM domain likely contributes to a unique mode of EphA2 interaction that leads to distinct signaling outputs. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. When Humanoid Robots Become Human-Like Interaction Partners: Corepresentation of Robotic Actions

    ERIC Educational Resources Information Center

    Stenzel, Anna; Chinellato, Eris; Bou, Maria A. Tirado; del Pobil, Angel P.; Lappe, Markus; Liepelt, Roman

    2012-01-01

    In human-human interactions, corepresenting a partner's actions is crucial to successfully adjust and coordinate actions with others. Current research suggests that action corepresentation is restricted to interactions between human agents facilitating social interaction with conspecifics. In this study, we investigated whether action…

  15. When Humanoid Robots Become Human-Like Interaction Partners: Corepresentation of Robotic Actions

    ERIC Educational Resources Information Center

    Stenzel, Anna; Chinellato, Eris; Bou, Maria A. Tirado; del Pobil, Angel P.; Lappe, Markus; Liepelt, Roman

    2012-01-01

    In human-human interactions, corepresenting a partner's actions is crucial to successfully adjust and coordinate actions with others. Current research suggests that action corepresentation is restricted to interactions between human agents facilitating social interaction with conspecifics. In this study, we investigated whether action…

  16. Adamantyl-Substituted Retinoid-Derived Molecules That Interact with the Orphan Nuclear Receptor Small Heterodimer Partner: Effects of Replacing the 1-Adamantyl or Hydroxyl Group on Inhibition of Cancer Cell Growth, Induction of Cancer Cell Apoptosis, and Inhibition of Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase-2 Activity

    PubMed Central

    Dawson, Marcia I.; Xia, Zebin; Jiang, Tao; Ye, Mao; Fontana, Joseph A.; Farhana, Lulu; Patel, Bhaumik; Xue, Li Ping; Bhuiyan, Mohammad; Pellicciari, Roberto; Macchiarulo, Antonio; Nuti, Roberto; Zhang, Xiao-Kun; Han, Young-Hoon; Tautz, Lutz; Hobbs, Peter D.; Jong, Ling; Waleh, Nahid; Chao, Wan-ru; Feng, Gen-Sheng; Pang, Yuhong; Su, Ying

    2014-01-01

    (E)-4-[3-(1-Adamantyl)-4′-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC) induces the cell-cycle arrest and apoptosis of leukemia and cancer cells. Studies demonstrated that 3-Cl-AHPC bound to the atypical orphan nuclear receptor small heterodimer partner (SHP). Although missing a DNA-binding domain, SHP heterodimerizes with the ligand-binding domains of other nuclear receptors to repress their abilities to induce or inhibit gene expression. 3-Cl-AHPC analogues having the 1-adamantyl and phenolic hydroxyl pharmacophoric elements replaced with isosteric groups were designed, synthesized, and evaluated for their inhibition of proliferation and induction of human cancer cell apoptosis. Structure–anticancer activity relationship studies indicated the importance of both groups to apoptotic activity. Docking of 3-Cl-AHPC and its analogues to an SHP computational model that was based on the crystal structure of ultraspiracle complexed with 1-stearoyl-2-palmitoylglycero-3-phosphoethanolamine suggested why these 3-Cl-AHPC groups could influence SHP activity. Inhibitory activity against Src homology 2 domain-containing protein tyrosine phosphatase 2 (Shp-2) was also assessed. The most active Shp-2 inhibitor was found to be the 3′-(3,3-dimethylbutynyl) analogue of 3-Cl-AHPC. PMID:18759424

  17. A Quantitative Fluorescence-Based Assay for Assessing LIM Domain-Peptide Interactions.

    PubMed

    Robertson, Neil O; Shah, Manan; Matthews, Jacqueline M

    2016-10-10

    We have developed Förster resonance energy transfer (FRET)-based experiments for measuring the binding affinity, off-rates, and inferred on-rates for interactions between a family of transcriptional regulators and their intrinsically disordered binding partners. It was difficult to evaluate these interactions previously, as the transcriptional regulators are obligate binding proteins that aggregate in the absence of a binding partner. The assays rely on fusion constructs where binding domains are linked by a flexible tether containing a specific protease site, with fluorescent proteins at either end that display FRET when the complex is formed. Loss of FRET is monitored after cutting the tether followed by dilution or competition with a non-fluorescent peptide. These methods allowed a wide range of binding affinities (10(-9) -10(-5)  m) to be determined. Our data indicate that interactions of closely related proteins can have surprisingly different binding properties.

  18. The HP1a Disordered C-terminus and Chromo Shadow Domain Cooperate to Select Target Peptide Partners

    PubMed Central

    Mendez, Deanna L.; Kim, Daesung; Chruszcz, Maksymilian; Stephens, Gena E.; Minor, Wladek

    2011-01-01

    Drosophila melanogaster Heterochromatin Protein 1a (HP1a) is essential for compacted heterochromatin structure and associated gene silencing. Its chromo shadow domain (CSD) is well-known for binding to peptides that contain a PXVXL motif. Heterochromatin protein 2 (HP2) is a nonhistone chromosomal protein that associates with HP1a in the pericentric heterochromatin, telomeres and the fourth chromosome. Using NMR spectroscopy, fluorescence polarization and site-directed mutagenesis, we identified an LCVKI motif in HP2 that binds to the HP1a CSD. The binding affinity of the HP2 fragment is approximately two orders of magnitude higher than that of peptides from PIWI (with a PRVKV motif), AF10 (with a PLVVL motif), or CG15356 (with LYPLL and LSIVA motifs). To delineate differential interactions of the HP1a CSD, we characterized its structure, backbone dynamics and dimerization constant. We find that the dimerization constant is bracketed by the affinities of HP2 and PIWI, which dock to the same HP1a homodimer surface. This suggests that HP2, but not PIWI, interaction can drive homodimerization of HP1a. Interestingly, the integrity of the disordered C-terminal extension (CTE) of HP1a is essential for discriminatory binding, whereas swapping the PXVXL motifs does not confer specificity. Serine phosphorylation at the peptide binding surface of the CSD is thought to regulate heterochromatin assembly. Glutamic acid substitution at these sites destabilizes HP1a dimers, but improves the interaction with both binding partners. Our studies underscore the importance of CSD dimerization and cooperation with the CTE in forming distinct complexes of HP1a. PMID:21472955

  19. A novel TPR-BEN domain interaction mediates PICH-BEND3 association.

    PubMed

    Pitchai, Ganesha P; Kaulich, Manuel; Bizard, Anna H; Mesa, Pablo; Yao, Qi; Sarlos, Kata; Streicher, Werner W; Nigg, Erich A; Montoya, Guillermo; Hickson, Ian D

    2017-09-05

    PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH-BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR-BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR-BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein-protein interaction mediated by a BEN domain. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Interaction between Functional Domains of Bacillus thuringiensis Insecticidal Crystal Proteins

    PubMed Central

    Rang, Cécile; Vachon, Vincent; de Maagd, Ruud A.; Villalon, Mario; Schwartz, Jean-Louis; Bosch, Dirk; Frutos, Roger; Laprade, Raynald

    1999-01-01

    Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C. PMID:10388684

  1. Specificity of broad protein interaction surfaces for proteins with multiple binding partners.

    PubMed

    Uchikoga, Nobuyuki; Matsuzaki, Yuri; Ohue, Masahito; Akiyama, Yutaka

    2016-01-01

    Analysis of protein-protein interaction networks has revealed the presence of proteins with multiple interaction ligand proteins, such as hub proteins. For such proteins, multiple ligands would be predicted as interacting partners when predicting all-to-all protein-protein interactions (PPIs). In this work, to obtain a better understanding of PPI mechanisms, we focused on protein interaction surfaces, which differ between protein pairs. We then performed rigid-body docking to obtain information of interfaces of a set of decoy structures, which include many possible interaction surfaces between a certain protein pair. Then, we investigated the specificity of sets of decoy interactions between true binding partners in each case of alpha-chymotrypsin, actin, and cyclin-dependent kinase 2 as test proteins having multiple true binding partners. To observe differences in interaction surfaces of docking decoys, we introduced broad interaction profiles (BIPs), generated by assembling interaction profiles of decoys for each protein pair. After cluster analysis, the specificity of BIPs of true binding partners was observed for each receptor. We used two types of BIPs: those involved in amino acid sequences (BIP-seqs) and those involved in the compositions of interacting amino acid residue pairs (BIP-AAs). The specificity of a BIP was defined as the number of group members including all true binding partners. We found that BIP-AA cases were more specific than BIP-seq cases. These results indicated that the composition of interacting amino acid residue pairs was sufficient for determining the properties of protein interaction surfaces.

  2. Domain Interaction Studies of Herpes Simplex Virus 1 Tegument Protein UL16 Reveal Its Interaction with Mitochondria.

    PubMed

    Chadha, Pooja; Sarfo, Akua; Zhang, Dan; Abraham, Thomas; Carmichael, Jillian; Han, Jun; Wills, John W

    2017-01-15

    The UL16 tegument protein of herpes simplex virus 1 (HSV-1) is conserved among all herpesviruses and plays many roles during replication. This protein has an N-terminal domain (NTD) that has been shown to bind to several viral proteins, including UL11, VP22, and glycoprotein E, and these interactions are negatively regulated by a C-terminal domain (CTD). Thus, in pairwise transfections, UL16 binding is enabled only when the CTD is absent or altered. Based on these results, we hypothesized that direct interactions occur between the NTD and the CTD. Here we report that the separated and coexpressed functional domains of UL16 are mutually responsive to each other in transfected cells and form complexes that are stable enough to be captured in coimmunoprecipitation assays. Moreover, we found that the CTD can associate with itself. To our surprise, the CTD was also found to contain a novel and intrinsic ability to localize to specific spots on mitochondria in transfected cells. Subsequent analyses of HSV-infected cells by immunogold electron microscopy and live-cell confocal imaging revealed a population of UL16 that does not merely accumulate on mitochondria but in fact makes dynamic contacts with these organelles in a time-dependent manner. These findings suggest that the domain interactions of UL16 serve to regulate not just the interaction of this tegument protein with its viral binding partners but also its interactions with mitochondria. The purpose of this novel interaction remains to be determined.

  3. The Intermediate Filament Protein Peripherin Is the Specific Interaction Partner of Mouse BPAG1-n (Dystonin) in Neurons

    PubMed Central

    Leung, Conrad L.; Sun, Dongming; Liem, Ronald K.H.

    1999-01-01

    The dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons. The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n). BPAG1-n contains an actin-binding domain at its NH2 terminus and a putative intermediate filament-binding domain at its COOH terminus. Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments. In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. Cell. 86:655–665), mouse BPAG1 fails to associate with full-length NFTPs. The tail domains interfered with the association of the NFTPs with BPAG1. In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology. Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo. PMID:9971739

  4. Interaction between persons with profound intellectual and multiple disabilities and their partners: a literature review.

    PubMed

    Hostyn, Ine; Maes, Bea

    2009-12-01

    High quality interactions are of crucial importance for quality of life of persons with profound intellectual and multiple disabilities (PIMD). This literature review describes and synthesises studies addressing the interaction between persons with PIMD and their partners. A computerised literature search using defined inclusion criteria yielded 15 articles. The literature analysis revealed four components important in interactions: sensitive responsiveness, joint attention, co-regulation, and an emotional component. The abilities and disabilities, interactive behaviours, and personality of persons with PIMD influence these interactions. Additional influences are the partners' interactive strategies, knowledge, and perceptions and the context of the interaction. An overview model integrates the results and forms a vehicle to facilitate our understanding of interactions with persons with high support needs. Methodological analyses of the studies show lacunae in current research. This review offers a starting point to guide future research and intervention.

  5. Expert-Novice Interactions: Influence of Partner Status.

    ERIC Educational Resources Information Center

    Verba, Mina; Winnykamen, Fajda

    1992-01-01

    Presents study results showing that a range of modes of interactive organization coexist in expert-novice problem-solving activity and vary with the asymmetry of the relationships. Reports that by combining high achieving status with task-related expertise, the resulting interactive dynamic is guidance/tutoring. Argues for multidimensional…

  6. Predicting PDZ domain mediated protein interactions from structure

    PubMed Central

    2013-01-01

    Background PDZ domains are structural protein domains that recognize simple linear amino acid motifs, often at protein C-termini, and mediate protein-protein interactions (PPIs) in important biological processes, such as ion channel regulation, cell polarity and neural development. PDZ domain-peptide interaction predictors have been developed based on domain and peptide sequence information. Since domain structure is known to influence binding specificity, we hypothesized that structural information could be used to predict new interactions compared to sequence-based predictors. Results We developed a novel computational predictor of PDZ domain and C-terminal peptide interactions using a support vector machine trained with PDZ domain structure and peptide sequence information. Performance was estimated using extensive cross validation testing. We used the structure-based predictor to scan the human proteome for ligands of 218 PDZ domains and show that the predictions correspond to known PDZ domain-peptide interactions and PPIs in curated databases. The structure-based predictor is complementary to the sequence-based predictor, finding unique known and novel PPIs, and is less dependent on training–testing domain sequence similarity. We used a functional enrichment analysis of our hits to create a predicted map of PDZ domain biology. This map highlights PDZ domain involvement in diverse biological processes, some only found by the structure-based predictor. Based on this analysis, we predict novel PDZ domain involvement in xenobiotic metabolism and suggest new interactions for other processes including wound healing and Wnt signalling. Conclusions We built a structure-based predictor of PDZ domain-peptide interactions, which can be used to scan C-terminal proteomes for PDZ interactions. We also show that the structure-based predictor finds many known PDZ mediated PPIs in human that were not found by our previous sequence-based predictor and is less dependent on

  7. Interaction between Persons with Profound Intellectual and Multiple Disabilities and Their Partners: A Literature Review

    ERIC Educational Resources Information Center

    Hostyn, Ine; Maes, Bea

    2009-01-01

    Background: High quality interactions are of crucial importance for quality of life of persons with profound intellectual and multiple disabilities (PIMD). This literature review describes and synthesises studies addressing the interaction between persons with PIMD and their partners. Method: A computerised literature search using defined…

  8. Interaction between Persons with Profound Intellectual and Multiple Disabilities and Their Partners: A Literature Review

    ERIC Educational Resources Information Center

    Hostyn, Ine; Maes, Bea

    2009-01-01

    Background: High quality interactions are of crucial importance for quality of life of persons with profound intellectual and multiple disabilities (PIMD). This literature review describes and synthesises studies addressing the interaction between persons with PIMD and their partners. Method: A computerised literature search using defined…

  9. Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3-kinases: binding partners of SH3 domains?

    PubMed Central

    Ponting, C. P.

    1996-01-01

    Two SH3 domain-containing cytosolic components of the NADPH oxidase, p47phox and p40phox, are shown by analyses of their sequences to contain single copies of a novel class of domain, the PX (phox) domain. Homologous domains are demonstrated to be present in the Cpk class of phosphatidylinositol 3-kinase, S. cerevisiae Bem1p, and S. pombe Scd2, and a large family of human sorting nexin 1 (SNX1) homologues. The majority of these domains contains a polyproline motif, typical of SH3 domain-binding proteins. Two further findings are reported. A third NADPH oxidase subunit, p67phox, is shown to contain four tetratricopeptide repeats (TPRs) within its N-terminal RaclGTP-binding region, and a 28 residue motif in p40phox is demonstrated to be present in protein kinase C isoforms iota/lambda and zeta, and in three ZZ domain-containing proteins. PMID:8931154

  10. Domain specificity in social interactions, social thought, and social development.

    PubMed

    Turiel, Elliot

    2010-01-01

    J. E. Grusec and M. Davidov (this issue) have taken good steps in formulating a domain-specific view of parent-child interactions. This commentary supports the introduction of domain specificity to analyses of parenting. Their formulation is an advance over formulations that characterized parental practices globally. This commentary calls for inclusion of definitions of the classification system of domain-specific interactions and criteria for each domain. It is also maintained that Grusec and Davidov's domains of social interaction imply that processes of development are involved, along with socialization; that bidirectionality in parent-child relations needs to be extended to include mutual influences and the construction of domains of social thought; and that conflicts and opposition within families coexist with compliance and social harmony.

  11. Understanding and predicting synthetic lethal genetic interactions in Saccharomyces cerevisiae using domain genetic interactions

    PubMed Central

    2011-01-01

    Background Synthetic lethal genetic interactions among proteins have been widely used to define functional relationships between proteins and pathways. However, the molecular mechanism of synthetic lethal genetic interactions is still unclear. Results In this study, we demonstrated that yeast synthetic lethal genetic interactions can be explained by the genetic interactions between domains of those proteins. The domain genetic interactions rarely overlap with the domain physical interactions from iPfam database and provide a complementary view about domain relationships. Moreover, we found that domains in multidomain yeast proteins contribute to their genetic interactions differently. The domain genetic interactions help more precisely define the function related to the synthetic lethal genetic interactions, and then help understand how domains contribute to different functionalities of multidomain proteins. Using the probabilities of domain genetic interactions, we were able to predict novel yeast synthetic lethal genetic interactions. Furthermore, we had also identified novel compensatory pathways from the predicted synthetic lethal genetic interactions. Conclusion The identification of domain genetic interactions helps the understanding of originality of functional relationship in SLGIs at domain level. Our study significantly improved the understanding of yeast mulitdomain proteins, the synthetic lethal genetic interactions and the functional relationships between proteins and pathways. PMID:21586150

  12. Thermotoga maritima NusG: domain interaction mediates autoinhibition and thermostability

    PubMed Central

    Drögemüller, Johanna; Schneider, Christin; Schweimer, Kristian; Strauß, Martin; Wöhrl, Birgitta M.; Rösch, Paul; Knauer, Stefan H.

    2017-01-01

    NusG, the only universally conserved transcription factor, comprises an N- and a C-terminal domain (NTD, CTD) that are flexibly connected and move independently in Escherichia coli and other organisms. In NusG from the hyperthermophilic bacterium Thermotoga maritima (tmNusG), however, NTD and CTD interact tightly. This closed state stabilizes the CTD, but masks the binding sites for the interaction partners Rho, NusE and RNA polymerase (RNAP), suggesting that tmNusG is autoinhibited. Furthermore, tmNusG and some other bacterial NusGs have an additional domain, DII, of unknown function. Here we demonstrate that tmNusG is indeed autoinhibited and that binding to RNAP may stabilize the open conformation. We identified two interdomain salt bridges as well as Phe336 as major determinants of the domain interaction. By successive weakening of this interaction we show that after domain dissociation tmNusG-CTD can bind to Rho and NusE, similar to the Escherichia coli NusG-CTD, indicating that these interactions are conserved in bacteria. Furthermore, we show that tmNusG-DII interacts with RNAP as well as nucleic acids with a clear preference for double stranded DNA. We suggest that tmNusG-DII supports tmNusG recruitment to the transcription elongation complex and stabilizes the tmNusG:RNAP complex, a necessary adaptation to high temperatures. PMID:27899597

  13. Thermodynamics of heme-induced conformational changes in hemopexin: role of domain-domain interactions.

    PubMed Central

    Wu, M. L.; Morgan, W. T.

    1995-01-01

    Hemopexin is a serum glycoprotein that binds heme with high affinity and delivers heme to the liver cells via receptor-mediated endocytosis. A hinge region connects the two non-disulfide-linked domains of hemopexin, a 35-kDa N-terminal domain (domain I) that binds heme, and a 25-kDa C-terminal domain (domain II). Although domain II does not bind heme, it assumes one structural state in apo-hemopexin and another in heme-hemopexin, and this change is important in facilitating the association of heme-hemopexin with its receptor. In order to elucidate the structure and function of hemopexin, it is important to understand how structural information is transmitted to domain II when domain I binds heme. Here we report a study of the protein-protein interactions between domain I and domain II using analytical ultracentrifugation and isothermal titration calorimetry. Sedimentation equilibrium analysis showed that domain I associates with domain II both in the presence and absence of heme with Kd values of 0.8 microM and 55 microM, respectively. The interaction between heme-domain I and domain II has a calorimetric enthalpy of +11 kcal/mol, a heat capacity (delta Cp) of -720 cal/mol.K, and a calculated entropy of +65 cal/mol.K. By varying the temperature of the centrifugation equilibrium runs, a van't Hoff plot with an apparent change in enthalpy (delta H) of -3.6 kcal/mol and change in entropy (delta S) of +8.1 cal/mol.K for the association of apo-domain I with domain II was obtained.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7773173

  14. Novel interaction partners of the TPR/MET tyrosine kinase.

    PubMed

    Schaaf, Christian P; Benzing, Jörg; Schmitt, Thomas; Erz, Dorothee H R; Tewes, Magdalena; Bartram, Claus R; Janssen, Johannes W G

    2005-02-01

    A large variety of biological processes is mediated by stimulation of the receptor tyrosine kinase MET. Screening a mouse embryo cDNA library, we were able to identify several novel, putative intracellular TPR/MET-substrates: SNAPIN, DCOHM, VAV-1, Sorting nexin 2, Death associated protein kinase 3, SMC-1, Centromeric protein C, and hTID-1. Interactions as identified by yeast two-hybrid analysis were validated in vitro and in vivo by mammalian two-hybrid studies, a far-western assay and coimmunoprecipitation. Participation in apoptosis-regulating mechanisms through interaction with DAPK-3 and cell cycle control via binding to nuclear proteins such as CENPC and SMC-1 are possible new aspects of intracellular MET signaling.

  15. The Curricular Domain of Educational Interactive Videodisc.

    ERIC Educational Resources Information Center

    Helsel, Sandra Kay

    1987-01-01

    Discusses curriculum theory in relation to educational interactive videodisc. Highlights include the influence of cultural values on curriculum, curriculum design components, and curriculum engineering, which includes planning, implementation, and evaluation. Instructional design for interactive videodisc is discussed, and 41 references are…

  16. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins.

    PubMed

    Enz, Ralf

    2012-01-01

    Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g., night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson's disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors' C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners.

  17. Fanconi Anemia Proteins and Their Interacting Partners: A Molecular Puzzle

    PubMed Central

    Kaddar, Tagrid; Carreau, Madeleine

    2012-01-01

    In recent years, Fanconi anemia (FA) has been the subject of intense investigations, primarily in the DNA repair research field. Many discoveries have led to the notion of a canonical pathway, termed the FA pathway, where all FA proteins function sequentially in different protein complexes to repair DNA cross-link damages. Although a detailed architecture of this DNA cross-link repair pathway is emerging, the question of how a defective DNA cross-link repair process translates into the disease phenotype is unresolved. Other areas of research including oxidative metabolism, cell cycle progression, apoptosis, and transcriptional regulation have been studied in the context of FA, and some of these areas were investigated before the fervent enthusiasm in the DNA repair field. These other molecular mechanisms may also play an important role in the pathogenesis of this disease. In addition, several FA-interacting proteins have been identified with roles in these “other” nonrepair molecular functions. Thus, the goal of this paper is to revisit old ideas and to discuss protein-protein interactions related to other FA-related molecular functions to try to give the reader a wider perspective of the FA molecular puzzle. PMID:22737580

  18. Domain Specificity in Social Interactions, Social Thought, and Social Development

    ERIC Educational Resources Information Center

    Turiel, Elliot

    2010-01-01

    J. E. Grusec and M. Davidov (this issue) have taken good steps in formulating a domain-specific view of parent-child interactions. This commentary supports the introduction of domain specificity to analyses of parenting. Their formulation is an advance over formulations that characterized parental practices globally. This commentary calls for…

  19. Membrane-Mediated Interactions Measured Using Membrane Domains

    PubMed Central

    Semrau, Stefan; Idema, Timon; Schmidt, Thomas; Storm, Cornelis

    2009-01-01

    Abstract Cell membrane organization is the result of the collective effect of many driving forces. Several of these, such as electrostatic and van der Waals forces, have been identified and studied in detail. In this article, we investigate and quantify another force, the interaction between inclusions via deformations of the membrane shape. For electrically neutral systems, this interaction is the dominant organizing force. As a model system to study membrane-mediated interactions, we use phase-separated biomimetic vesicles that exhibit coexistence of liquid-ordered and liquid-disordered lipid domains. The membrane-mediated interactions between these domains lead to a rich variety of effects, including the creation of long-range order and the setting of a preferred domain size. Our findings also apply to the interaction of membrane protein patches, which induce similar membrane shape deformations and hence experience similar interactions. PMID:19527649

  20. Quantum Oscillations of Interacting Nanoscale Structural Inhomogeneities in a Domain Wall of Magnetic Stripe Domain

    NASA Astrophysics Data System (ADS)

    Shevchenko, Andriy; Barabash, Maksym

    2016-10-01

    It was established that at low temperatures, quantum oscillations of a pair of interacting nanoscale structural inhomogeneities (vertical Bloch lines) occur in a domain wall of stripe domain in uniaxial ferromagnetic film. The effective mass of vertical Bloch line and conditions for this effect were determined. The effect can be used in the hybrid storage devices bit + q-bit.

  1. Human endothelial and platelet septin SEPT11: cloning of novel variants and characterisation of interaction partners.

    PubMed

    Bartsch, Ingrid; Bläser, Susanne; Röseler, Sabrina; Sandrock, Kirstin; Busse, Anja; Huber, Michael; Rempp, Hansjörg; Lieber, Mareike; Horn, Julia; Brendle, Cornelia; Zieger, Barbara

    2010-12-01

    Septins are cytoskeletal GTPases forming heteropolymeric complexes involved in processes characterised by active membrane movement such as cytokinesis, vesicle trafficking, and exocytosis. Septins are expressed in non-mitotic cells such as neurons and platelets. SEPT11 belongs to the SEPT6 group and was identified as interaction partner of SEPT5. We cloned and characterised novel SEPT11 variants and investigated interaction partners of SEPT11 in platelets and human umbilical vein endothelial cells. An endothelial cell library was used for cloning novel SEPT11 variants. Using Northern analysis the different SEPT11 transcripts were illustrated. Interaction studies were performed using yeast two-hybrid system, precipitation, FRET, and immunofluorescence microscopy. We demonstrate that SEPT11 partners with SEPT2, SEPT4 and SEPT7 using yeast two-hybrid system and precipitation. The interaction of SEPT11 with SEPT7 is also demonstrated by FRET. In addition to the known SEPT11 transcript (SEPT11_v1) we identified a novel SEPT11 variant (SEPT11_v2) as interaction partner of SEPT4 and SEPT7. Library screening of an endothelial cell library also revealed the presence of this novel SEPT11_v2 transcript. In addition, a third SEPT11 variant (SEPT11_v3) was identified. Expression of SEPT11_v1 and of SEPT11_v2 and SEPT11_v3 in human brain regions was investigated by Northern analysis. Further interaction partners of SEPT11 are characterised using immunofluorescence. Co-localisation of SEPT2, SEPT4, SEPT7 and SEPT11 with tubulin and transferrin receptor (endocytotic marker) is demonstrated. In addition, co-localisation of SEPT4 and SEPT11 with the vesicle-associated protein synaptobrevin 1 (VAMP1), but not clearly with actin, was shown. Only SEPT2 and SEPT7 definitely co-localised with actin, but not clearly with VAMP1.

  2. Effects of MyTeachingPartner-Math/Science on Teacher-Child Interactions in Prekindergarten Classrooms

    ERIC Educational Resources Information Center

    Whittaker, Jessica Vick; Kinzie, Mable B.; Williford, Amanda; DeCoster, Jamie

    2016-01-01

    Research Findings: This study examined the impact of MyTeachingPartner-Math/Science, a system of math and science curricula and professional development, on the quality of teachers' interactions with children in their classrooms. Schools were randomly assigned to 1 of 2 intervention conditions (Basic: curricula providing within-activity, embedded…

  3. Effects of MyTeachingPartner-Math/Science on Teacher-Child Interactions in Prekindergarten Classrooms

    ERIC Educational Resources Information Center

    Whittaker, Jessica Vick; Kinzie, Mable B.; Williford, Amanda; DeCoster, Jamie

    2016-01-01

    Research Findings: This study examined the impact of MyTeachingPartner-Math/Science, a system of math and science curricula and professional development, on the quality of teachers' interactions with children in their classrooms. Schools were randomly assigned to 1 of 2 intervention conditions (Basic: curricula providing within-activity, embedded…

  4. Interactions of Student Partners in a High School Astronomy Computer Lab.

    ERIC Educational Resources Information Center

    McLellan, Hilary

    1994-01-01

    Reports the results of a study that examined the patterns of interactions of high school student partners engaged in problem-solving activities while working through computer simulation activities in an astronomy lab. Observations, interviews with teachers and students, and a software program evaluation are discussed. (LRW)

  5. The (pro)renin receptor and its interaction partners.

    PubMed

    Peters, Jörg

    2017-06-15

    The prorenin receptor was originally discovered as a receptor that binds renin and prorenin, thereby inducing pro-fibrotic intracellular signal cascades. These effects are partially mediated in vitro by angiotensin (ANG) and partially independent of ANG. Consequently, inhibitors of the interaction between the prorenin and the (pro)renin receptor (PRR) were designed hoping that they may prevent fibrotic tissue damage, for instance, in the kidney. However, this concept was challenged by the fact that overexpression of the PRR was not harmful at all, whereas depletion of the PRR was lethal or markedly detrimental. Furthermore, the high levels of prorenin needed to activate the PRR may not be reached in vivo. As it turned out, the PRR instead exhibited a variety of important functions that has nothing to do with the name given to the protein. Thus, the PRR was identified as an accessory subunit of vesicular (v)-ATPases, representing an essential chaperon for the assembly of v-ATPase subunits. In this respect, the gene encoding the PRR was also named ATP6AP2. Finally, the PRR is an essential component of the canonical and non-canonical PCP Wnt pathways. Thus, the PRR is essential for lysosomal functions, such as endocytosis, secretion, and autophagy as well as for cell division and differentiation, embryonic development, organogenesis, and stem cell biology.

  6. Appraisal distortions and intimate partner violence: gender, power, and interaction.

    PubMed

    Whiting, Jason B; Oka, Megan; Fife, Stephen T

    2012-06-01

    In relationships characterized by control, abuse, or violence, many appraisal distortions occur including denial and minimization. However, the nature of the distortion varies depending on the individual's role in the relationship (i.e., abuser or victim). Reducing these distortions is an important component in treatment success and involves accepting responsibility for actions and attributions. This study used constructivist grounded theory methods to explore the following questions: (1) What are the types of distortions that are used by individuals who have been in violent or abusive relationships? (2) What are the gender and power differences in the appraisal distortions used? (3) What are the functions and interactions of the distortions in the relationship dynamics? Qualitative analysis of interviews with 29 individuals who had been in abusive relationships found that there were several types of distortions used by participants, but there were differences in the function of the distortion, depending on the individual's role in the abuse. These generally corresponded to power and gender, where the male as perpetrator used different distortions (or used similar distortions for different reasons) than did the female as victim. Suggestions for research as well as treatment implications for both offenders and survivors of abuse are given.

  7. Putative Domain-Domain Interactions in the Vesicular Stomatitis Virus L Polymerase Protein Appendage Region

    PubMed Central

    Ruedas, John B.

    2014-01-01

    ABSTRACT The multidomain polymerase protein (L) of nonsegmented negative-strand (NNS) RNA viruses catalyzes transcription and replication of the virus genome. The N-terminal half of the protein forms a ring-like polymerase structure, while the C-terminal half encoding viral mRNA transcript modifications consists of a flexible appendage with three distinct globular domains. To gain insight into putative transient interactions between L domains during viral RNA synthesis, we exchanged each of the four distinct regions encompassing the appendage region of vesicular stomatitis virus (VSV) Indiana serotype L protein with their counterparts from VSV New Jersey and analyzed effects on virus polymerase activity in a minigenome system. The methyltransferase domain exchange yielded a fully active polymerase protein, which functioned as well as wild-type L in the context of a recombinant virus. Exchange of the downstream C-terminal nonconserved region abolished activity, but coexchanging it with the methyltransferase domain generated a polymerase favoring replicase over transcriptase activity, providing strong evidence of interaction between these two regions. Exchange of the capping enzyme domain or the adjacent nonconserved region thought to function as an “unstructured” linker also abrogated polymerase activity even when either domain was coexchanged with other appendage domains. Further probing of the putative linker segment using in-frame enhanced green fluorescent protein (EGFP) insertions similarly abrogated activity. We discuss the implications of these findings with regard to L protein appendage domain structure and putative domain-domain interactions required for polymerase function. IMPORTANCE NNS viruses include many well-known human pathogens (e.g., rabies, measles, and Ebola viruses), as well as emerging viral threats (e.g., Nipah and Hendra viruses). These viruses all encode a large L polymerase protein similarly organized into multiple domains that work in

  8. Characterization of the SAM domain of the PKD-related protein ANKS6 and its interaction with ANKS3.

    PubMed

    Leettola, Catherine N; Knight, Mary Jane; Cascio, Duilio; Hoffman, Sigrid; Bowie, James U

    2014-07-07

    Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder leading to end-stage renal failure in humans. In the PKD/Mhm(cy/+) rat model of ADPKD, the point mutation R823W in the sterile alpha motif (SAM) domain of the protein ANKS6 is responsible for disease. SAM domains are known protein-protein interaction domains, capable of binding each other to form polymers and heterodimers. Despite its physiological importance, little is known about the function of ANKS6 and how the R823W point mutation leads to PKD. Recent work has revealed that ANKS6 interacts with a related protein called ANKS3. Both ANKS6 and ANKS3 have a similar domain structure, with ankyrin repeats at the N-terminus and a SAM domain at the C-terminus. The SAM domain of ANKS3 is identified as a direct binding partner of the ANKS6 SAM domain. We find that ANKS3-SAM polymerizes and ANKS6-SAM can bind to one end of the polymer. We present crystal structures of both the ANKS3-SAM polymer and the ANKS3-SAM/ANKS6-SAM complex, revealing the molecular details of their association. We also learn how the R823W mutation disrupts ANKS6 function by dramatically destabilizing the SAM domain such that the interaction with ANKS3-SAM is lost. ANKS3 is a direct interacting partner of ANKS6. By structurally and biochemically characterizing the interaction between the ANKS3 and ANKS6 SAM domains, our work provides a basis for future investigation of how the interaction between these proteins mediates kidney function.

  9. Characterization of the SAM domain of the PKD-related protein ANKS6 and its interaction with ANKS3

    PubMed Central

    2014-01-01

    Background Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder leading to end-stage renal failure in humans. In the PKD/Mhm(cy/+) rat model of ADPKD, the point mutation R823W in the sterile alpha motif (SAM) domain of the protein ANKS6 is responsible for disease. SAM domains are known protein-protein interaction domains, capable of binding each other to form polymers and heterodimers. Despite its physiological importance, little is known about the function of ANKS6 and how the R823W point mutation leads to PKD. Recent work has revealed that ANKS6 interacts with a related protein called ANKS3. Both ANKS6 and ANKS3 have a similar domain structure, with ankyrin repeats at the N-terminus and a SAM domain at the C-terminus. Results The SAM domain of ANKS3 is identified as a direct binding partner of the ANKS6 SAM domain. We find that ANKS3-SAM polymerizes and ANKS6-SAM can bind to one end of the polymer. We present crystal structures of both the ANKS3-SAM polymer and the ANKS3-SAM/ANKS6-SAM complex, revealing the molecular details of their association. We also learn how the R823W mutation disrupts ANKS6 function by dramatically destabilizing the SAM domain such that the interaction with ANKS3-SAM is lost. Conclusions ANKS3 is a direct interacting partner of ANKS6. By structurally and biochemically characterizing the interaction between the ANKS3 and ANKS6 SAM domains, our work provides a basis for future investigation of how the interaction between these proteins mediates kidney function. PMID:24998259

  10. PTEN-PDZ domain interactions: binding of PTEN to PDZ domains of PTPN13.

    PubMed

    Sotelo, Natalia S; Schepens, Jan T G; Valiente, Miguel; Hendriks, Wiljan J A J; Pulido, Rafael

    2015-05-01

    Protein modular interactions mediated by PDZ domains are essential for the establishment of functional protein networks controlling diverse cellular functions. The tumor suppressor PTEN possesses a C-terminal PDZ-binding motif (PDZ-BM) that is recognized by a specific set of PDZ domains from scaffolding and regulatory proteins. Here, we review the current knowledge on PTEN-PDZ domain interactions and tumor suppressor networks, describe methodology suitable to analyze these interactions, and report the binding of PTEN and the PDZ domain-containing protein tyrosine phosphatase PTPN13. Yeast two-hybrid and GST pull-down analyses showed that PTEN binds to PDZ2/PTPN13 domain in a manner that depends on the specific PTPN13 PDZ domain arrangement involving the interdomain region between PDZ1 and PDZ2. Furthermore, a specific binding profile of PTEN to PDZ2/PTPN13 domain was observed by mutational analysis of the PTEN PDZ-BM. Our results disclose a PDZ-mediated physical interaction of PTEN and PTPN13 with potential relevance in tumor suppression and cell homeostasis. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Differential effects of intranasal oxytocin on sexual experiences and partner interactions in couples.

    PubMed

    Behnia, Behnoush; Heinrichs, Markus; Bergmann, Wiebke; Jung, Stefanie; Germann, Janine; Schedlowski, Manfred; Hartmann, Uwe; Kruger, Tillmann H C

    2014-03-01

    Knowledge about the effects of the neuropeptide oxytocin (OXT) on human sexual behaviors and partner interactions remains limited. Based on our previous studies, we hypothesize that OXT should be able to positively influence parameters of sexual function and couple interactions. Employing a naturalistic setting involving 29 healthy heterosexual couples (n=58 participants), we analyzed the acute effects of intranasally administered OXT (24IU) on sexual drive, arousal, orgasm and refractory aspects of sexual behavior together with partner interactions. Data were assessed by psychometric instruments (Acute Sexual Experiences Scale, Arizona Sexual Experience Scale) as well as biomarkers, such as cortisol, α-amylase and heart rate. Intranasal OXT administration did not alter "classical" parameters of sexual function, such as sexual drive, arousal or penile erection and lubrication. However, analysis of variance and a hierarchical linear model (HLM) revealed specific effects related to the orgasmic/post-orgasmic interval as well as parameters of partner interactions. According to HLM analysis, OXT increased the intensity of orgasm, contentment after sexual intercourse and the effect of study participation. According to ANOVA analysis, these effects were more pronounced in men. Men additionally indicated higher levels of sexual satiety after sexual intercourse with OXT administration. Women felt more relaxed and subgroups indicated better abilities to share sexual desires or to empathize with their partners. The effect sizes were small to moderate. Biomarkers indicated moderate psychophysiological activation but were not affected by OXT, gender or method of contraception. Using a naturalistic setting, intranasal OXT administration in couples exerted differential effects on parameters of sexual function and partner interactions. These results warrant further investigations, including subjects with sexual and relationship problems. Copyright © 2014 Elsevier Inc. All

  12. Assembly of cell regulatory systems through protein interaction domains.

    PubMed

    Pawson, Tony; Nash, Piers

    2003-04-18

    The sequencing of complete genomes provides a list that includes the proteins responsible for cellular regulation. However, this does not immediately reveal what these proteins do, nor how they are assembled into the molecular machines and functional networks that control cellular behavior. The regulation of many different cellular processes requires the use of protein interaction domains to direct the association of polypeptides with one another and with phospholipids, small molecules, or nucleic acids. The modular nature of these domains, and the flexibility of their binding properties, have likely facilitated the evolution of cellular pathways. Conversely, aberrant interactions can induce abnormal cellular behavior and disease. The fundamental properties of protein interaction domains are discussed in this review and in detailed reviews on individual domains at Science's STKE at http://www.sciencemag.org/cgi/content/full/300/5618/445/DC1.

  13. Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II.

    PubMed

    Chang, A; Cheang, S; Espanel, X; Sudol, M

    2000-07-07

    RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation.

  14. Solution structure and intermolecular interactions of the third metal-binding domain of ATP7A, the Menkes disease protein.

    PubMed

    Banci, Lucia; Bertini, Ivano; Cantini, Francesca; DellaMalva, Nunzia; Herrmann, Torsten; Rosato, Antonio; Wüthrich, Kurt

    2006-09-29

    The third metal-binding domain of the human Menkes protein (MNK3), a copper(I)-transporting ATPase, has been expressed in Escherichia coli and characterized in solution. The solution structure of MNK3, its copper(I)-binding properties, and its interaction with the physiological partner, HAH1, have been studied. MNK3 is the domain most dissimilar in structure from the other domains of the Menkes protein. This is reflected in a significant rearrangement of the last strand of the four-stranded beta-sheet when compared with the other known homologous proteins or protein domains. MNK3 is also peculiar with respect to its interaction with the copper(I) ion, as it was found to be a comparatively weak binder. Copper(I) transfer from metal-loaded HAH1 was observed experimentally, but the metal distribution was shifted toward binding by HAH1. This is at variance with what is observed for the other Menkes domains.

  15. Candidate Cell and Matrix Interaction Domains on the Collagen Fibril, the Predominant Protein of Vertebrates

    SciTech Connect

    Sweeney, Shawn M.; Orgel, Joseph P.; Fertala, Andrzej; McAuliffe, Jon D.; Turner, Kevin R.; Di Lullo, Gloria A.; Chen, Steven; Antipova, Olga; Perumal, Shiamalee; Ala-Kokko, Leena; Forlinoi, Antonella; Cabral, Wayne A.; Barnes, Aileen M.; Marini, Joan C.; San Antonio, James D.

    2008-07-18

    Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The 'cell interaction domain' is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The 'matrix interaction domain' may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.

  16. In vivo analysis of human nucleoporin repeat domain interactions

    PubMed Central

    Xu, Songli; Powers, Maureen A.

    2013-01-01

    The nuclear pore complex (NPC), assembled from ∼30 proteins termed nucleoporins (Nups), mediates selective nucleocytoplasmic trafficking. A subset of nucleoporins bear a domain with multiple phenylalanine–glycine (FG) motifs. As binding sites for transport receptors, FG Nups are critical in translocation through the NPC. Certain FG Nups are believed to associate via low-affinity, cohesive interactions to form the permeability barrier of the pore, although the form and composition of this functional barrier are debated. We used green fluorescent protein–Nup98/HoxA9 constructs with various numbers of repeats and also substituted FG domains from other nucleoporins for the Nup98 domain to directly compare cohesive interactions in live cells by fluorescence recovery after photobleaching (FRAP). We find that cohesion is a function of both number and type of FG repeats. Glycine–leucine–FG (GLFG) repeat domains are the most cohesive. FG domains from several human nucleoporins showed no interactions in this assay; however, Nup214, with numerous VFG motifs, displayed measurable cohesion by FRAP. The cohesive nature of a human nucleoporin did not necessarily correlate with that of its yeast orthologue. The Nup98 GLFG domain also functions in pore targeting through binding to Nup93, positioning the GLFG domain in the center of the NPC and supporting a role for this nucleoporin in the permeability barrier. PMID:23427268

  17. The role of cytochrome b5 structural domains in interaction with cytochromes P450.

    PubMed

    Sergeev, G V; Gilep, A A; Usanov, S A

    2014-05-01

    To understand the role of the structural elements of cytochrome b5 in its interaction with cytochrome P450 and the catalysis performed by this heme protein, we carried out comparative structural and functional analysis of the two major mammalian forms of membrane-bound cytochrome b5 - microsomal and mitochondrial, designed chimeric forms of the heme proteins in which the hydrophilic domain of one heme protein is replaced by the hydrophilic domain of another one, and investigated the effect of the highly purified native and chimeric heme proteins on the enzymatic activity of recombinant cytochromes P4503A4 and P45017A1 (CYP3A4 and CYP17A1). We show that the presence of a hydrophobic domain in the structure of cytochrome b5 is necessary for its effective interaction with its redox partners, while the nature of the hydrophobic domain has no significant effect on the ability of cytochrome b5 to stimulate the activity of cytochrome P450-catalyzed reactions. Thus, the functional properties of cytochrome b5 are mainly determined by the structure of the heme-binding domain.

  18. Computer modeling of the membrane interaction of FYVE domains.

    PubMed

    Diraviyam, Karthikeyan; Stahelin, Robert V; Cho, Wonhwa; Murray, Diana

    2003-05-02

    FYVE domains are membrane targeting domains that are found in proteins involved in endosomal trafficking and signal transduction pathways. Most FYVE domains bind specifically to phosphatidylinositol 3-phosphate (PI(3)P), a lipid that resides mainly in endosomal membranes. Though the specific interactions between FYVE domains and the headgroup of PI(3)P have been well characterized, principally through structural studies, the available experimental structures suggest several different models for FYVE/membrane association. Thus, the manner in which FYVE domains adsorb to the membrane surface remains to be elucidated. Towards this end, recent experiments have shown that FYVE domains bind PI(3)P in the context of phospholipid bilayers and that hydrophobic residues on a conserved loop are able to penetrate the membrane interface in a PI(3)P-dependent manner.Here, the finite difference Poisson-Boltzmann (FDPB) method has been used to calculate the energetic interactions of FYVE domains with phospholipid membranes. Based on the computational analysis, it is found that (1) recruitment to membranes is facilitated by non-specific electrostatic interactions that occur between basic residues on the domains and acidic phospholipids in the membrane, (2) the energetic analysis can quantitatively differentiate among the modes of membrane association proposed by the experimentally determined structures, (3) FDPB calculations predict energetically feasible models for the membrane-associated states of FYVE domains, (4) these models are consistent with the observation that conserved hydrophobic residues insert into the membrane interface, and (5) the calculations provide a molecular model for the hydrophobic partitioning: binding of PI(3)P significantly neutralizes positive potential in the region of the hydrophobic residues, which acts as an "electrostatic switch" by reducing the energetic barrier for membrane penetration. Finally, the computational results are extended to FYVE

  19. Co-evolutionary Analysis of Domains in Interacting Proteins Reveals Insights into Domain–Domain Interactions Mediating Protein–Protein Interactions

    PubMed Central

    Jothi, Raja; Cherukuri, Praveen F.; Tasneem, Asba; Przytycka, Teresa M.

    2006-01-01

    Recent advances in functional genomics have helped generate large-scale high-throughput protein interaction data. Such networks, though extremely valuable towards molecular level understanding of cells, do not provide any direct information about the regions (domains) in the proteins that mediate the interaction. Here, we performed co-evolutionary analysis of domains in interacting proteins in order to understand the degree of co-evolution of interacting and non-interacting domains. Using a combination of sequence and structural analysis, we analyzed protein–protein interactions in F1-ATPase, Sec23p/Sec24p, DNA-directed RNA polymerase and nuclear pore complexes, and found that interacting domain pair(s) for a given interaction exhibits higher level of co-evolution than the noninteracting domain pairs. Motivated by this finding, we developed a computational method to test the generality of the observed trend, and to predict large-scale domain–domain interactions. Given a protein–protein interaction, the proposed method predicts the domain pair(s) that is most likely to mediate the protein interaction. We applied this method on the yeast interactome to predict domain–domain interactions, and used known domain–domain interactions found in PDB crystal structures to validate our predictions. Our results show that the prediction accuracy of the proposed method is statistically significant. Comparison of our prediction results with those from two other methods reveals that only a fraction of predictions are shared by all the three methods, indicating that the proposed method can detect known interactions missed by other methods. We believe that the proposed method can be used with other methods to help identify previously unrecognized domain–domain interactions on a genome scale, and could potentially help reduce the search space for identifying interaction sites. PMID:16949097

  20. The nature of protein domain evolution: shaping the interaction network.

    PubMed

    Bagowski, Christoph P; Bruins, Wouter; Te Velthuis, Aartjan J W

    2010-08-01

    The proteomes that make up the collection of proteins in contemporary organisms evolved through recombination and duplication of a limited set of domains. These protein domains are essentially the main components of globular proteins and are the most principal level at which protein function and protein interactions can be understood. An important aspect of domain evolution is their atomic structure and biochemical function, which are both specified by the information in the amino acid sequence. Changes in this information may bring about new folds, functions and protein architectures. With the present and still increasing wealth of sequences and annotation data brought about by genomics, new evolutionary relationships are constantly being revealed, unknown structures modeled and phylogenies inferred. Such investigations not only help predict the function of newly discovered proteins, but also assist in mapping unforeseen pathways of evolution and reveal crucial, co-evolving inter- and intra-molecular interactions. In turn this will help us describe how protein domains shaped cellular interaction networks and the dynamics with which they are regulated in the cell. Additionally, these studies can be used for the design of new and optimized protein domains for therapy. In this review, we aim to describe the basic concepts of protein domain evolution and illustrate recent developments in molecular evolution that have provided valuable new insights in the field of comparative genomics and protein interaction networks.

  1. The Nature of Protein Domain Evolution: Shaping the Interaction Network

    PubMed Central

    Bagowski, Christoph P; Bruins, Wouter; te Velthuis, Aartjan J.W

    2010-01-01

    The proteomes that make up the collection of proteins in contemporary organisms evolved through recombination and duplication of a limited set of domains. These protein domains are essentially the main components of globular proteins and are the most principal level at which protein function and protein interactions can be understood. An important aspect of domain evolution is their atomic structure and biochemical function, which are both specified by the information in the amino acid sequence. Changes in this information may bring about new folds, functions and protein architectures. With the present and still increasing wealth of sequences and annotation data brought about by genomics, new evolutionary relationships are constantly being revealed, unknown structures modeled and phylogenies inferred. Such investigations not only help predict the function of newly discovered proteins, but also assist in mapping unforeseen pathways of evolution and reveal crucial, co-evolving inter- and intra-molecular interactions. In turn this will help us describe how protein domains shaped cellular interaction networks and the dynamics with which they are regulated in the cell. Additionally, these studies can be used for the design of new and optimized protein domains for therapy. In this review, we aim to describe the basic concepts of protein domain evolution and illustrate recent developments in molecular evolution that have provided valuable new insights in the field of comparative genomics and protein interaction networks. PMID:21286315

  2. Cholesterol and the interaction of proteins with membrane domains.

    PubMed

    Epand, Richard M

    2006-07-01

    Cholesterol is not uniformly distributed in biological membranes. One of the factors influencing the formation of cholesterol-rich domains in membranes is the unequal lateral distribution of proteins in membranes. Certain proteins are found in cholesterol-rich domains. In some of these cases, it is as a consequence of the proteins interacting directly with cholesterol. There are several structural features of a protein that result in the protein preferentially associating with cholesterol-rich domains. One of the best documented of these is certain types of lipidations. In addition, however, there are segments of a protein that can preferentially sequester cholesterol. We discuss two examples of these cholesterol-recognition elements: the cholesterol recognition/interaction amino acid consensus (CRAC) domain and the sterol-sensing domain (SSD). The requirements for a CRAC motif are quite flexible and predict that a large number of sequences could recognize cholesterol. There are, however, certain proteins that are known to interact with cholesterol-rich domains of cell membranes that have CRAC motifs, and synthetic peptides corresponding to these segments also promote the formation of cholesterol-rich domains. Modeling studies have provided a rationale for certain requirements of the CRAC motif. The SSD is a larger protein segment comprising five transmembrane domains. The amino acid sequence YIYF is found in several SSD and in certain other proteins for which there is evidence that they interact with cholesterol-rich domains. The CRAC sequences as well as YIYF are generally found adjacent to a transmembrane helical segment. These regions appear to have a strong influence of the localization of certain proteins into domains in biological membranes. In addition to the SSD, there is also a domain found in soluble proteins, the START domain, that binds lipids. Certain proteins with START domains specifically bind cholesterol and are believed to function in

  3. Electroweak interactions in the nuclear domain

    SciTech Connect

    Pollock, S.J.

    1988-01-01

    A variety of electroweak interactions with nucleons and nuclei is considered as a means to yield tests of the Standard Model, to provide measurements of hadronic structure, and to serve as a guide to experimental efforts. In Part I, single nucleon elastic electroweak processes are studied. The general formalism is outlined, and formulae are presented for cross section for e{sup {minus}} and {upsilon} neutral current processes, {epsilon} charge-changing events, and parity violation. Means have been found to extract both vector and axial form factors from experiment, at arbitrary q{sup 2}. Numerical predictions are presented for these processes, assuming a set of phenomenological form factors. Low every structure in charge-changing reactions would provide tests of CVC and a measurement of the pseudoscalar form factor. Relations between the processes which yield tests of the Standard Model and provide an experimental means to determine the effects of intrinsic parity violation, isospin breaking, and heavy quark content are presented. Parity violation is discussed as a means to measure sin{sup 2}{theta}{sub 2} in the low energy quark-lepton sector, and to measure the weak form factors of the nucleon. Sources of uncertainity are considered, including poorly known electromagnetic neutron form factors, and axial weak form factors. A means to detect anomalous effective axial isoscalar current is provided and the bounds on extra heavy neutral Z bosons a CEBAF parity experiment would provide are discussed. In Part II, coincidence cross sections are studied. The formalism for electroweak single-particle coincidence experiments is outlined. The general angular distribution for single-nucleon coincidence measurements on a deuterium (spin 1) target is derived.

  4. The phosphoCTD-interacting domain of Topoisomerase I

    SciTech Connect

    Wu, Jianhong; Phatnani, Hemali P.; Hsieh, Tao-Shih; Greenleaf, Arno L.

    2010-06-18

    The N-terminal domain (NTD) of Drosophila melanogaster (Dm) Topoisomerase I has been shown to bind to RNA polymerase II, but the domain of RNAPII with which it interacts is not known. Using bacterially-expressed fusion proteins carrying all or half of the NTDs of Dm and human (Homo sapiens, Hs) Topo I, we demonstrate that the N-terminal half of each NTD binds directly to the hyperphosphorylated C-terminal repeat domain (phosphoCTD) of the largest RNAPII subunit, Rpb1. Thus, the amino terminal segment of metazoan Topo I (1-157 for Dm and 1-114 for Hs) contains a novel phosphoCTD-interacting domain that we designate the Topo I-Rpb1 interacting (TRI) domain. The long-known in vivo association of Topo I with active genes presumably can be attributed, wholly or in part, to the TRI domain-mediated binding of Topo I to the phosphoCTD of transcribing RNAPII.

  5. Using interactive theater to create socioculturally relevant community-based intimate partner violence prevention.

    PubMed

    Yoshihama, Mieko; Tolman, Richard M

    2015-03-01

    This article describes the use of interactive theater, audience response assessment, and peer educators to create community-generated approaches for bystander interventions (i.e., actions taken by people who become aware of controlling, abusive and violent behavior of others) to prevent intimate partner violence (IPV) and to foster change in community norms. We include a case example of an ongoing university-community partnership, which mobilizes community members to develop and implement socioculturally relevant IPV prevention programs in multiple Asian communities. We used interactive theater at a community event--a walk to raise awareness about IPV in South Asian communities--and examined how the enacted bystander interventions reflect specific community contexts. We detail the challenges and limitations we have encountered in our attempts to implement this approach in collaboration with our community partners.

  6. Positive Interactions and Avoidant and Anxious Representations in Relationships with Parents, Friends, and Romantic Partners.

    PubMed

    Furman, Wyndol; Stephenson, J Claire; Rhoades, Galena K

    2014-12-01

    We examined associations between positive interactions and avoidant and anxious representations in relationships with parents, friends, and romantic partners. Two hundred adolescents completed questionnaires, observations, and attachment interviews. From a between-person perspective, those adolescents with more positive interactions overall had less avoidant representations. Within persons, more positive interactions were relative to one's own average level in relationships, the less avoidant representations were for that type of relationship. Adolescents were less anxious about a particular type of relationship if they have positive interactions in their other types of relationships. Finally, representations were primarily predicted by interactions in the same type of relationship; interactions in other relationships contributed little. The findings underscore the importance of examining representations of particular types of relationships.

  7. Reconstituting Protein Interaction Networks Using Parameter-Dependent Domain-Domain Interactions

    DTIC Science & Technology

    2013-05-07

    for yeast ( Saccharomyces cerevisiae ) using sequences of 5,884 proteins,a downloaded from the Saccharomyces Genome Database (SGD) [31] and yeast...Riles L, Mortimer RK, et al: Genetic and physical maps of Saccharomyces cerevisiae . Nature 1997, 387(6632 Suppl):67–73. 32. Finn RD, Mistry J, Tate J...downloaded from the Saccharomyces Genome Database [31]. NP, proteins with at least one domain annotation. NS, protein-domain amino acid sequence

  8. Interacting Regions of CD81 and Two of Its Partners, EWI-2 and EWI-2wint, and Their Effect on Hepatitis C Virus Infection*

    PubMed Central

    Montpellier, Claire; Tews, Birke Andrea; Poitrimole, Julien; Rocha-Perugini, Vera; D'Arienzo, Valentina; Potel, Julie; Zhang, Xin A.; Rubinstein, Eric; Dubuisson, Jean; Cocquerel, Laurence

    2011-01-01

    CD81 is a tetraspanin protein that is involved in several essential cellular functions, as well as in the hepatitis C virus (HCV) infection. CD81 interacts with a high stoichiometry with its partner proteins EWI-2, EWI-2wint, and EWI-F. These latter proteins modify the functions of CD81 and can thereby potentially inhibit infection or modulate cell migration. Here, we characterized the cleavage of EWI-2 leading to the production of EWI-2wint, which has been shown to inhibit HCV infection. We determined the regions of EWI-2/EWI-2wint and CD81 that are important for their interaction and their functionality. More precisely, we identified a glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint that is essential for the interaction with CD81. In addition, we found that palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for their interaction with CD81 as well as with CD9, another tetraspanin. Thus, we have shown that palmitoylation of a tetraspanin partner protein can influence the interaction with a tetraspanin. We therefore propose that palmitoylation not only of tetraspanins, but also of their partner proteins is important in regulating the composition of complexes in tetraspanin networks. Finally, we identified the regions in CD81 that are necessary for its functionality in HCV entry and we demonstrated that EWI-2wint needs to interact with CD81 to exert its inhibitory effect on HCV infection. PMID:21343309

  9. TolA central domain interacts with Escherichia coli porins.

    PubMed Central

    Derouiche, R; Gavioli, M; Bénédetti, H; Prilipov, A; Lazdunski, C; Lloubès, R

    1996-01-01

    TolA is an inner membrane protein with three domains: a transmembrane N-terminus and periplasmic central and C-terminal domains. The interaction of TolA with outer membrane porins of Escherichia coli was investigated. Western blot analyses of cell extracts with anti-TolA antibodies indicated that TolA forms high molecular weight complexes specifically with trimeric OmpF, OmpC, PhoE and LamB, but not with OmpA. The interaction of purified TolA domains with purified porins was also studied. TolA interacted with OmpF, PhoE and LamB porins via its central domain, but not with either their denatured monomeric forms or OmpA. Moreover, the presence or absence of lipopolysaccharides associated with trimeric porins did not modify the interactions. These results suggest that the specific interaction of TolA with outer membrane porins might be relevant to the function of Tol proteins. Images PMID:8978668

  10. Does Enhancing Partner Support and Interaction Improve Smoking Cessation? A Meta-Analysis

    PubMed Central

    Park, Eal-Whan; Tudiver, Fred; Schultz, Jennifer K.; Campbell, Thomas

    2004-01-01

    BACKGROUND We wanted to determine whether an intervention to enhance partner support helps as an adjunct to a smoking cessation program. METHODS We undertook a meta-analysis of English-language, randomized controlled trials of smoking cessation interventions through July 2002 using the following data sources: Cochrane Tobacco Addiction Group specialized register, Cochrane controlled trials register, CDC Tobacco Information and Prevention Database, MEDLINE, Cancer Lit, EMBASE, CINAHL, PsycINFO, ERIC, PsycLIT, Dissertation Abstracts, SSCI and HealthSTAR, with reviews of bibliographies of included articles. Included were trials that assessed a partner support component with a minimum follow-up of 6 months. The outcomes measured were abstinence and biochemical assessment at 6 to 9 months and more than 12 months after treatment. Partner Interaction Questionnaire scores were primary and secondary outcomes. RESULTS Nine studies (31 articles) met inclusion criteria. Partner definition varied among studies. All studies included self-reported smoking cessation rates, but there was limited biochemical validation of abstinence. For self-reported abstinence at 6 to 9 months after treatment, the Peto odds ratio (OR) = 1.08 (95% confidence interval [CI], 0.81–1.44) and at 12 months Peto OR = 1.0 (95% CI, 0.75–1.34). Sensitivity analysis of studies using live-in, married, and equivalent-to-married partners found a higher odds ratio at 6 to 9 months after treatment, Peto OR = 1.64 (95% CI, 0.5–4.64). Sensitivity analysis of studies reporting significant increases in partner support found at 6 to 9 months after treatment Peto OR = 1.83 (95% CI, 0.9–3.47); and at 12 months Peto OR =1.22 (95% CI, 0.67–2.23). CONCLUSIONS Interventions to enhance partner support showed the most promise for clinical practice when implemented with live-in, married, and equivalent-to-married partners. Such interventions should focus on enhancing supportive behaviors, while minimizing behaviors

  11. Structure and Regulatory Interactions of the Cytoplasmic Terminal Domains of Serotonin Transporter

    PubMed Central

    2014-01-01

    Uptake of neurotransmitters by sodium-coupled monoamine transporters of the NSS family is required for termination of synaptic transmission. Transport is tightly regulated by protein–protein interactions involving the small cytoplasmic segments at the amino- and carboxy-terminal ends of the transporter. Although structures of homologues provide information about the transmembrane regions of these transporters, the structural arrangement of the terminal domains remains largely unknown. Here, we combined molecular modeling, biochemical, and biophysical approaches in an iterative manner to investigate the structure of the 82-residue N-terminal and 30-residue C-terminal domains of human serotonin transporter (SERT). Several secondary structures were predicted in these domains, and structural models were built using the Rosetta fragment-based methodology. One-dimensional 1H nuclear magnetic resonance and circular dichroism spectroscopy supported the presence of helical elements in the isolated SERT N-terminal domain. Moreover, introducing helix-breaking residues within those elements altered the fluorescence resonance energy transfer signal between terminal cyan fluorescent protein and yellow fluorescent protein tags attached to full-length SERT, consistent with the notion that the fold of the terminal domains is relatively well-defined. Full-length models of SERT that are consistent with these and published experimental data were generated. The resultant models predict confined loci for the terminal domains and predict that they move apart during the transport-related conformational cycle, as predicted by structures of homologues and by the “rocking bundle” hypothesis, which is consistent with spectroscopic measurements. The models also suggest the nature of binding to regulatory interaction partners. This study provides a structural context for functional and regulatory mechanisms involving SERT terminal domains. PMID:25093911

  12. Lipids Regulate Lck Protein Activity through Their Interactions with the Lck Src Homology 2 Domain.

    PubMed

    Sheng, Ren; Jung, Da-Jung; Silkov, Antonina; Kim, Hyunjin; Singaram, Indira; Wang, Zhi-Gang; Xin, Yao; Kim, Eui; Park, Mi-Jeong; Thiagarajan-Rosenkranz, Pallavi; Smrt, Sean; Honig, Barry; Baek, Kwanghee; Ryu, Sungho; Lorieau, Justin; Kim, You-Me; Cho, Wonhwa

    2016-08-19

    Lymphocyte-specific protein-tyrosine kinase (Lck) plays an essential role in T cell receptor (TCR) signaling and T cell development, but its activation mechanism is not fully understood. To explore the possibility that plasma membrane (PM) lipids control TCR signaling activities of Lck, we measured the membrane binding properties of its regulatory Src homology 2 (SH2) and Src homology 3 domains. The Lck SH2 domain binds anionic PM lipids with high affinity but with low specificity. Electrostatic potential calculation, NMR analysis, and mutational studies identified the lipid-binding site of the Lck SH2 domain that includes surface-exposed basic, aromatic, and hydrophobic residues but not the phospho-Tyr binding pocket. Mutation of lipid binding residues greatly reduced the interaction of Lck with the ζ chain in the activated TCR signaling complex and its overall TCR signaling activities. These results suggest that PM lipids, including phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, modulate interaction of Lck with its binding partners in the TCR signaling complex and its TCR signaling activities in a spatiotemporally specific manner via its SH2 domain. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. CFTR chloride channel in the apical compartments: spatiotemporal coupling to its interacting partners.

    PubMed

    Li, Chunying; Naren, Anjaparavanda P

    2010-04-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel located primarily at the apical or luminal surfaces of epithelial cells in the airway, intestine, pancreas, kidney, sweat gland, as well as male reproductive tract, where it plays a crucial role in transepithelial fluid homeostasis. CFTR dysfunction can be detrimental and may result in life-threatening disorders. CFTR hypofunctioning because of genetic defects leads to cystic fibrosis, the most common lethal genetic disease in Caucasians, whereas CFTR hyperfunctioning resulting from various infections evokes secretory diarrhea, the leading cause of mortality in early childhood. Therefore, maintaining a dynamic balance between CFTR up-regulating processes and CFTR down-regulating processes is essential for maintaining fluid and body homeostasis. Accumulating evidence suggests that protein-protein interactions play a critical role in the fine-tuned regulation of CFTR function. A growing number of proteins have been reported to interact directly or indirectly with CFTR chloride channel, suggesting that CFTR might be coupled spatially and temporally to a wide variety of interacting partners including ion channels, receptors, transporters, scaffolding proteins, enzyme molecules, signaling molecules, and effectors. Most interactions occur primarily between the opposing terminal tails (amino or carboxyl) of CFTR protein and its binding partners, either directly or mediated through various PDZ scaffolding proteins. These dynamic interactions impact the channel function, as well as localization and processing of CFTR protein within cells. This article reviews the most recent progress and findings about the interactions between CFTR and its binding partners through PDZ scaffolding proteins, as well as the spatiotemporal regulation of CFTR-containing macromolecular signaling complexes in the apical compartments of polarized cells lining the secretory epithelia.

  14. Interacting partners of FEN1 and its role in the development of anticancer therapeutics.

    PubMed

    Kathera, Chandrasekhar; Zhang, Jing; Janardhan, Avilala; Sun, Hongfang; Ali, Wajid; Zhou, Xiaolong; He, Lingfeng; Guo, Zhigang

    2017-02-07

    Protein-protein interaction (PPI) plays a key role in cellular communication, Protein-protein interaction connected with each other with hubs and nods involved in signaling pathways. These interactions used to develop network based biomarkers for early diagnosis of cancer. FEN1(Flap endonuclease 1) is a central component in cellular metabolism, over expression and decrease of FEN1 levels may cause cancer, these regulation changes of Flap endonuclease 1reported in many cancer cells, to consider this data may needs to develop a network based biomarker. The current review focused on types of PPI, based on nature, detection methods and its role in cancer. Interacting partners of Flap endonuclease 1 role in DNA replication repair and development of anticancer therapeutics based on Protein-protein interaction data.

  15. CD6-ligand interactions: a paradigm for SRCR domain function?

    PubMed

    Aruffo, A; Bowen, M A; Patel, D D; Haynes, B F; Starling, G C; Gebe, J A; Bajorath, J

    1997-10-01

    The scavenger receptor cysteine-rich (SRCR) superfamily, which includes proteins expressed by leukocytes, can be subdivided into groups A and B. Group B contains the lymphocyte cell-surface receptor CD6. This article reviews recent progress in understanding the interaction between CD6 and its ligand, activated leukocyte cell adhesion molecule (ALCAM). Analysis of the CD6-ALCAM interaction may help to understand how other SRCR domains bind to their ligands.

  16. Time-domain Ramsey interferometry with interacting Rydberg atoms

    NASA Astrophysics Data System (ADS)

    Sommer, Christian; Pupillo, Guido; Takei, Nobuyuki; Takeda, Shuntaro; Tanaka, Akira; Ohmori, Kenji; Genes, Claudiu

    2016-11-01

    We theoretically investigate the dynamics of a gas of strongly interacting Rydberg atoms subject to a time-domain Ramsey interferometry protocol. The many-body dynamics is governed by an Ising-type Hamiltonian with long-range interactions of tunable strength. We analyze and model the contrast degradation and phase accumulation of the Ramsey signal and identify scaling laws for varying interrogation times, ensemble densities, and ensemble dimensionalities.

  17. Evaluation by Expert Dancers of a Robot That Performs Partnered Stepping via Haptic Interaction

    PubMed Central

    Chen, Tiffany L.; Bhattacharjee, Tapomayukh; McKay, J. Lucas; Borinski, Jacquelyn E.; Hackney, Madeleine E.; Ting, Lena H.; Kemp, Charles C.

    2015-01-01

    Our long-term goal is to enable a robot to engage in partner dance for use in rehabilitation therapy, assessment, diagnosis, and scientific investigations of two-person whole-body motor coordination. Partner dance has been shown to improve balance and gait in people with Parkinson's disease and in older adults, which motivates our work. During partner dance, dance couples rely heavily on haptic interaction to convey motor intent such as speed and direction. In this paper, we investigate the potential for a wheeled mobile robot with a human-like upper-body to perform partnered stepping with people based on the forces applied to its end effectors. Blindfolded expert dancers (N=10) performed a forward/backward walking step to a recorded drum beat while holding the robot's end effectors. We varied the admittance gain of the robot's mobile base controller and the stiffness of the robot's arms. The robot followed the participants with low lag (M=224, SD=194 ms) across all trials. High admittance gain and high arm stiffness conditions resulted in significantly improved performance with respect to subjective and objective measures. Biomechanical measures such as the human hand to human sternum distance, center-of-mass of leader to center-of-mass of follower (CoM-CoM) distance, and interaction forces correlated with the expert dancers' subjective ratings of their interactions with the robot, which were internally consistent (Cronbach's α=0.92). In response to a final questionnaire, 1/10 expert dancers strongly agreed, 5/10 agreed, and 1/10 disagreed with the statement "The robot was a good follower." 2/10 strongly agreed, 3/10 agreed, and 2/10 disagreed with the statement "The robot was fun to dance with." The remaining participants were neutral with respect to these two questions. PMID:25993099

  18. Evaluation by Expert Dancers of a Robot That Performs Partnered Stepping via Haptic Interaction.

    PubMed

    Chen, Tiffany L; Bhattacharjee, Tapomayukh; McKay, J Lucas; Borinski, Jacquelyn E; Hackney, Madeleine E; Ting, Lena H; Kemp, Charles C

    2015-01-01

    Our long-term goal is to enable a robot to engage in partner dance for use in rehabilitation therapy, assessment, diagnosis, and scientific investigations of two-person whole-body motor coordination. Partner dance has been shown to improve balance and gait in people with Parkinson's disease and in older adults, which motivates our work. During partner dance, dance couples rely heavily on haptic interaction to convey motor intent such as speed and direction. In this paper, we investigate the potential for a wheeled mobile robot with a human-like upper-body to perform partnered stepping with people based on the forces applied to its end effectors. Blindfolded expert dancers (N=10) performed a forward/backward walking step to a recorded drum beat while holding the robot's end effectors. We varied the admittance gain of the robot's mobile base controller and the stiffness of the robot's arms. The robot followed the participants with low lag (M=224, SD=194 ms) across all trials. High admittance gain and high arm stiffness conditions resulted in significantly improved performance with respect to subjective and objective measures. Biomechanical measures such as the human hand to human sternum distance, center-of-mass of leader to center-of-mass of follower (CoM-CoM) distance, and interaction forces correlated with the expert dancers' subjective ratings of their interactions with the robot, which were internally consistent (Cronbach's α=0.92). In response to a final questionnaire, 1/10 expert dancers strongly agreed, 5/10 agreed, and 1/10 disagreed with the statement "The robot was a good follower." 2/10 strongly agreed, 3/10 agreed, and 2/10 disagreed with the statement "The robot was fun to dance with." The remaining participants were neutral with respect to these two questions.

  19. Beyond the chalkboard: the interacting domains in undergraduate psychiatric teaching.

    PubMed

    Claassen, Johann; Kruger, Estie

    2005-06-01

    To identify and describe interacting domains that are present during undergraduate psychiatric teaching and impress on educators the roles of these domains. Undergraduate psychiatric teaching is an essential part of general family physicians' training and practice. Undergraduate students' exposure to psychiatry might be inadequate. Curricular goals, teaching and evaluation of students need to be aligned. Research and information technology can play a bigger role in teaching. Validation of patients, students and teachers during the teaching process is important, but not always emphasized. Educators should be aware of society's unwritten expectations, judgements and opinions of mental illness sufferers. Identifying and facilitating the various interacting domains during psychiatric teaching may contribute to better education in psychiatry, better retention of knowledge for students and an improved recruitment of students to a psychiatric career.

  20. The toxofilin-actin-PP2C complex of Toxoplasma: identification of interacting domains.

    PubMed

    Jan, Gaelle; Delorme, Violaine; David, Violaine; Revenu, Celine; Rebollo, Angelita; Cayla, Xavier; Tardieux, Isabelle

    2007-02-01

    Toxofilin is a 27 kDa protein isolated from the human protozoan parasite Toxoplasma gondii, which causes toxoplasmosis. Toxofilin binds to G-actin, and in vitro studies have shown that it controls elongation of actin filaments by sequestering actin monomers. Toxofilin affinity for G-actin is controlled by the phosphorylation status of its Ser53, which depends on the activities of a casein kinase II and a type 2C serine/threonine phosphatase (PP2C). To get insights into the functional properties of toxofilin, we undertook a structure-function analysis of the protein using a combination of biochemical techniques. We identified a domain that was sufficient to sequester G-actin and that contains three peptide sequences selectively binding to G-actin. Two of these sequences are similar to sequences present in several G- and F-actin-binding proteins, while the third appears to be specific to toxofilin. Additionally, we identified two toxofilin domains that interact with PP2C, one of which contains the Ser53 substrate. In addition to characterizing the interacting domains of toxofilin with its partners, the present study also provides information on an in vivo-based approach to selectively and competitively disrupt the protein-protein interactions that are important to parasite motility.

  1. Numerical evaluation of the interaction domain for gypsum pillars

    NASA Astrophysics Data System (ADS)

    Grisi, Stefano; Castellanza, Riccardo; di Prisco, Claudio; Battista Crosta, Giovanni; Agliardi, Federico

    2014-05-01

    The object of this work has been the evaluation of an interaction domain M-N-T for pillars located in an abandoned gypsum mine. The interaction domain is extremely useful for evaluating the limit failure when a pillar is simultaneously subjected to axial force N (from overburden loads) and to shear force T and momentum M (for instance seismic loads). The existing joints across the pillar suggest to overpass a typical continuum approach. In order to consider the presence of cracks during numerical simulations, a hybrid method FEM/DEM, which allows the transition from continuum to discontinum, was assumed. By means of a specific numerical code (ELFEN), this approach has been calibrated involving both physical quantities introduced by fracture mechanics and numerical aspects in order to support this hybrid method. Furthermore, the approaches FEM and FEM/DEM have been compared, showing advantages and disadvantages through experimental tests carried out to characterize geomechanical response of the pillar. The interaction domain has been calculated thanks to the implementation of both methods. The meaning of determining this domain is related to the evaluation of failure limit when a coupled system of loads (normal and tangential force and momentum) is acting on pillars. An application to a case study of an abandoned gypsum mine interacting with building in San Lazzaro di Savena (Bologna) will be shown.

  2. Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions.

    PubMed

    Pedersen, Søren W; Albertsen, Louise; Moran, Griffin E; Levesque, Brié; Pedersen, Stine B; Bartels, Lina; Wapenaar, Hannah; Ye, Fei; Zhang, Mingjie; Bowen, Mark E; Strømgaard, Kristian

    2017-09-15

    The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effect of phosphorylation on PSD-95, we used semisynthetic strategies to introduce phosphorylated amino acids at four positions within the PDZ domains and examined the effects on interactions with a large set of binding partners. We observed complex effects on affinity. Most notably, phosphorylation at Y397 induced a significant increase in affinity for stargazin, as confirmed by NMR and single molecule FRET. Additionally, we compared the effects of phosphorylation to phosphomimetic mutations, which revealed that phosphomimetics are ineffective substitutes for tyrosine phosphorylation. Our strategy to generate site-specifically phosphorylated PDZ domains provides a detailed understanding of the role of phosphorylation in the regulation of PSD-95 interactions.

  3. Asymmetric interaction and indeterminate fitness correlation between cooperative partners in the fig-fig wasp mutualism.

    PubMed

    Wang, Rui-Wu; Sun, Bao-Fa; Zheng, Qi; Shi, Lei; Zhu, Lixing

    2011-10-07

    Empirical observations have shown that cooperative partners can compete for common resources, but what factors determine whether partners cooperate or compete remain unclear. Using the reciprocal fig-fig wasp mutualism, we show that nonlinear amplification of interference competition between fig wasps-which limits the fig wasps' ability to use a common resource (i.e. female flowers)-keeps the common resource unsaturated, making cooperation locally stable. When interference competition was manually prevented, the fitness correlation between figs and fig wasps went from positive to negative. This indicates that genetic relatedness or reciprocal exchange between cooperative players, which could create spatial heterogeneity or self-restraint, was not sufficient to maintain stable cooperation. Moreover, our analysis of field-collected data shows that the fitness correlation between cooperative partners varies stochastically, and that the mainly positive fitness correlation observed during the warm season shifts to a negative correlation during the cold season owing to an increase in the initial oviposition efficiency of each fig wasp. This implies that the discriminative sanction of less-cooperative wasps (i.e. by decreasing the egg deposition efficiency per fig wasp) but reward to cooperative wasps by fig, a control of the initial value, will facilitate a stable mutualism. Our finding that asymmetric interaction leading to an indeterminate fitness interaction between symbiont (i.e. cooperative actors) and host (i.e. recipient) has the potential to explain why conflict has been empirically observed in both well-documented intraspecific and interspecific cooperation systems.

  4. Asymmetric interaction and indeterminate fitness correlation between cooperative partners in the fig–fig wasp mutualism

    PubMed Central

    Wang, Rui-Wu; Sun, Bao-Fa; Zheng, Qi; Shi, Lei; Zhu, Lixing

    2011-01-01

    Empirical observations have shown that cooperative partners can compete for common resources, but what factors determine whether partners cooperate or compete remain unclear. Using the reciprocal fig–fig wasp mutualism, we show that nonlinear amplification of interference competition between fig wasps—which limits the fig wasps' ability to use a common resource (i.e. female flowers)—keeps the common resource unsaturated, making cooperation locally stable. When interference competition was manually prevented, the fitness correlation between figs and fig wasps went from positive to negative. This indicates that genetic relatedness or reciprocal exchange between cooperative players, which could create spatial heterogeneity or self-restraint, was not sufficient to maintain stable cooperation. Moreover, our analysis of field-collected data shows that the fitness correlation between cooperative partners varies stochastically, and that the mainly positive fitness correlation observed during the warm season shifts to a negative correlation during the cold season owing to an increase in the initial oviposition efficiency of each fig wasp. This implies that the discriminative sanction of less-cooperative wasps (i.e. by decreasing the egg deposition efficiency per fig wasp) but reward to cooperative wasps by fig, a control of the initial value, will facilitate a stable mutualism. Our finding that asymmetric interaction leading to an indeterminate fitness interaction between symbiont (i.e. cooperative actors) and host (i.e. recipient) has the potential to explain why conflict has been empirically observed in both well-documented intraspecific and interspecific cooperation systems. PMID:21490005

  5. Analysing discourse in the traumatic brain injury population: telephone interactions with different communication partners.

    PubMed

    Togher, L; Hand, L; Code, C

    1997-03-01

    A range of discourse analyses are effective in identifying features which are aberrant following traumatic brain injury (TBI). We examined the exchanges of five traumatically brain-injured subjects and five matched controls across four speaking situations which included speaking to a therapist, to the bus timetable information service, to the police, and to their mothers on the telephone. Transcripts were analysed using the exchange structure analysis of systemic functional grammar. This analysis provided an indication of information giving (K1 moves per minute); information requesting and receiving (K2 moves per minute) and the amount of negotiation that was needed for the messages to be conveyed (dynamic moves per minute). Results indicated that the TBI subjects performed differently across the four conditions, and were differentiated from the matched controls on a number of measures. The role of different communication partners is also addressed. Communication partners were noted to interact differently with TBI subjects when compared with controls. This included increased information-giving to control subjects; more requests for information by police from TBI subjects and a greater use of dynamic moves by therapists with controls. The potential of exchange structure analysis is discussed as a useful way of examining the discourse of TBI subjects and their communication partners. Exchange structure analysis highlighted the dynamic nature of information exchange and the subtle ways speakers responded to familiarity and power imbalance in social interaction. This study has implications for family and community education regarding communication with people with TBI.

  6. PP2A binds to the LIM domains of lipoma-preferred partner through its PR130/B″ subunit to regulate cell adhesion and migration

    PubMed Central

    Janssens, Veerle; Zwaenepoel, Karen; Rossé, Carine; Petit, Marleen M. R.; Goris, Jozef; Parker, Peter J.

    2017-01-01

    Here, we identify the LIM protein lipoma-preferred partner (LPP) as a binding partner of a specific protein phosphatase 2A (PP2A) heterotrimer that is characterised by the regulatory PR130/B″α1 subunit (encoded by PPP2R3A). The PR130 subunit interacts with the LIM domains of LPP through a conserved Zn2+-finger-like motif in the differentially spliced N-terminus of PR130. Isolated LPP-associated PP2A complexes are catalytically active. PR130 colocalises with LPP at multiple locations within cells, including focal contacts, but is specifically excluded from mature focal adhesions, where LPP is still present. An LPP–PR130 fusion protein only localises to focal adhesions upon deletion of the domain of PR130 that binds to the PP2A catalytic subunit (PP2A/C), suggesting that PR130–LPP complex formation is dynamic and that permanent recruitment of PP2A activity might be unfavourable for focal adhesion maturation. Accordingly, siRNA-mediated knockdown of PR130 increases adhesion of HT1080 fibrosarcoma cells onto collagen I and decreases their migration in scratch wound and Transwell assays. Complex formation with LPP is mandatory for these PR130-PP2A functions, as neither phenotype can be rescued by re-expression of a PR130 mutant that no longer binds to LPP. Our data highlight the importance of specific, locally recruited PP2A complexes in cell adhesion and migration dynamics. PMID:26945059

  7. Identification of protein interaction partners in mammalian cells using SILAC-immunoprecipitation quantitative proteomics.

    PubMed

    Emmott, Edward; Goodfellow, Ian

    2014-07-06

    Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

  8. Domain-domain interactions in filamin A (16-23) impose a hierarchy of unfolding forces.

    PubMed

    Xu, Tianyou; Lannon, Herbert; Wolf, Sébastein; Nakamura, Fumihiko; Brujic, Jasna

    2013-05-07

    The quaternary structure of Filamin A (FLNa) 16-23 was recently shown to exhibit multiple domain-domain interactions that lead to a propeller-like construction. Here we present single-molecule force spectroscopy experiments to show a wide variety of mechanical responses of this molecule and compare it with its linear counterpart FLNa 1-8. The compact structure of FLNa 16-23 leads to a broad distribution of rupture forces and end-to-end lengths in the force-extension mode and multiple unraveling timescales in the force-clamp mode. Moreover, a subset of force-extension trajectories reveals a mechanical hierarchy in which the rupture of domain-domain interactions at high forces (>200 pN) liberates the unfolding of individual domains at low forces (∼100 pN). This mechanism may also explain the order-of-magnitude difference in the rates of the biexponential fits to the distribution of unfolding dwell times under force-clamp. Overall, FLNa 16-23 under a force of 100 pN is more compliant than the linear FLNa 1-8. Because a physiological role of FLNa is to crosslink actin filaments, this range of responses allows it to accommodate a broad spectrum of forces exerted by the cell and its environment. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  9. Identification of a novel putative interaction partner of the nucleoporin ALADIN

    PubMed Central

    Landgraf, Dana; Huebner, Angela; Koehler, Katrin

    2016-01-01

    ABSTRACT It has been shown that the nucleoporin ALADIN plays a significant role in the redox homeostasis of the cell, but its function in steroidogenesis contributing to adrenal atrophy in triple A syndrome remains largely unknown. In an attempt to identify new interaction partners of ALADIN, co-immunoprecipitation followed by proteome analysis was conducted in different expression models using the human adrenocortical tumour cell line NCI-H295R. Our results suggest an interaction of ALADIN with the microsomal protein PGRMC2. PGRMC2 is shown to be activity regulator of CYP P450 enzymes and, therefore, to be a possible target for adrenal dysregulation in triple A syndrome. We show that there is a sexual dimorphism regarding the expression of Pgrmc2 in adrenals and gonads of wild-type (WT) and Aaas knock-out (KO) mice. Female Aaas KO mice are sterile due to delayed oocyte maturation and meiotic spindle assembly. A participation in meiotic spindle assembly confirms the recently investigated involvement of ALADIN in mitosis and emphasises an interaction with PGRMC2 which is a regulator of the cell cycle. By identification of a novel interaction partner of ALADIN, we provide novel aspects for future research of the function of ALADIN during cell cycle and for new insights into the pathogenesis of triple A syndrome. PMID:27754849

  10. New insights into the activation, interaction partners and possible functions of MK5/PRAK.

    PubMed

    Perander, Maria; Keyse, Stephen M; Seternes, Ole-Morten

    2016-01-01

    MAP kinase-activated protein kinase 5 (MK5) was first described as a downstream target of the p38 MAP kinase pathway leading to its alternative acronym of p38-regulated/activated protein kinase (PRAK). However, since the discovery that MK5 is a bona fide interaction partner of the atypical MAP kinases ERK3 and ERK4 and that this interaction leads to both the activation and subcellular relocalisation of MK5, there has been considerable debate as to the relative roles of these MAPK pathways in mediating the activation and biological functions of MK5. Here we discuss recent progress in defining novel upstream components of the ERK3/ERK4 signalling pathway, our increased understanding of the mechanism by which MK5 interacts with and is activated by ERK3 and ERK4, and the discovery of novel interaction partners for MK5. Finally, we review recent literature that suggests novel biological functions for MK5 in a range of physiological and pathophysiological conditions including neuronal function and cancer.

  11. Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins.

    PubMed

    Anang, Saumya; Subramani, Chandru; Nair, Vidya P; Kaul, Sheetal; Kaushik, Nidhi; Sharma, Chandresh; Tiwari, Ashutosh; Ranjith-Kumar, C T; Surjit, Milan

    2016-04-26

    Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66(th),67(th) isoleucine and 101(st),102(nd) leucine, conserved across HEV genotypes. Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV.

  12. Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins

    PubMed Central

    Anang, Saumya; Subramani, Chandru; Nair, Vidya P.; Kaul, Sheetal; Kaushik, Nidhi; Sharma, Chandresh; Tiwari, Ashutosh; Ranjith-Kumar, CT; Surjit, Milan

    2016-01-01

    Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66th,67th isoleucine and 101st,102nd leucine, conserved across HEV genotypes. Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV. PMID:27113483

  13. Nuclear Localization of the Autism Candidate Gene Neurobeachin and Functional Interaction with the NOTCH1 Intracellular Domain Indicate a Role in Regulating Transcription

    PubMed Central

    Tuand, Krizia; Stijnen, Pieter; Volders, Karolien; Declercq, Jeroen; Nuytens, Kim; Meulemans, Sandra; Creemers, John

    2016-01-01

    Background Neurobeachin (NBEA) is an autism spectrum disorders (ASD) candidate gene. NBEA deficiency affects regulated secretion, receptor trafficking, synaptic architecture and protein kinase A (PKA)-mediated phosphorylation. NBEA is a large multidomain scaffolding protein. From N- to C-terminus, NBEA has a concanavalin A-like lectin domain flanked by armadillo repeats (ACA), an A-kinase anchoring protein domain that can bind to PKA, a domain of unknown function (DUF1088) and a BEACH domain, preceded by a pleckstrin homology-like domain and followed by WD40 repeats (PBW). Although most of these domains mediate protein-protein interactions, no interaction screen has yet been performed. Methods Yeast two-hybrid screens with the ACA and PBW domain modules of NBEA gave a list of interaction partners, which were analyzed for Gene Ontology (GO) enrichment. Neuro-2a cells were used for confocal microscopy and nuclear extraction analysis. NOTCH-mediated transcription was studied with luciferase reporter assays and qRT-PCR, combined with NBEA knockdown or overexpression. Results Both domain modules showed a GO enrichment for the nucleus. PBW almost exclusively interacted with transcription regulators, while ACA interacted with a number of PKA substrates. NBEA was partially localized in the nucleus of Neuro-2a cells, albeit much less than in the cytoplasm. A nuclear localization signal was found in the DUF1088 domain, which was shown to contribute to the nuclear localization of an EGFP-DPBW fusion protein. Yeast two-hybrid identified the Notch1 intracellular domain as a physical interactor of the PBW domain and a role for NBEA as a negative regulator in Notch-mediated transcription was demonstrated. Conclusion Defining novel interaction partners of conserved NBEA domain modules identified a role for NBEA as transcriptional regulator in the nucleus. The physical interaction of NBEA with NOTCH1 is most relevant for ASD pathogenesis because NOTCH signaling is essential for

  14. Kelch Domain of Gigaxonin Interacts with Intermediate Filament Proteins Affected in Giant Axonal Neuropathy

    PubMed Central

    Johnson-Kerner, Bethany L.; Garcia Diaz, Alejandro; Ekins, Sean; Wichterle, Hynek

    2015-01-01

    Patients with giant axonal neuropathy (GAN) show progressive loss of motor and sensory function starting in childhood and typically live for less than 30 years. GAN is caused by autosomal recessive mutations leading to low levels of gigaxonin (GIG), a ubiquitously-expressed BTB/Kelch cytoplasmic protein believed to be an E3 ligase substrate adaptor. GAN pathology is characterized by aggregates of intermediate filaments (IFs) in multiple tissues. To delineate the molecular pathway between GIG deficiency and IF pathology, we undertook a proteomic screen to identify the normal binding partners of GIG. Prominent among them were several classes of IFs, including the neurofilament subunits whose accumulation leads to the axonal swellings for which GAN is named. We showed these interactions were dependent on the Kelch domain of GIG. Furthermore, we identified the E3 ligase MYCBP2 and the heat shock proteins HSP90AA1/AB1 as interactors with the BTB domain that may result in the ubiquitination and subsequent degradation of intermediate filaments. Our open-ended proteomic screen provides support to GIG’s role as an adaptor protein, linking IF proteins through its Kelch domain to the ubiquitin pathway proteins via its BTB domain, and points to future approaches for reversing the phenotype in human patients. PMID:26460568

  15. Interaction matters: A perceived social partner alters the neural processing of human speech.

    PubMed

    Rice, Katherine; Redcay, Elizabeth

    2016-04-01

    Mounting evidence suggests that social interaction changes how communicative behaviors (e.g., spoken language, gaze) are processed, but the precise neural bases by which social-interactive context may alter communication remain unknown. Various perspectives suggest that live interactions are more rewarding, more attention-grabbing, or require increased mentalizing-thinking about the thoughts of others. Dissociating between these possibilities is difficult because most extant neuroimaging paradigms examining social interaction have not directly compared live paradigms to conventional "offline" (or recorded) paradigms. We developed a novel fMRI paradigm to assess whether and how an interactive context changes the processing of speech matched in content and vocal characteristics. Participants listened to short vignettes--which contained no reference to people or mental states--believing that some vignettes were prerecorded and that others were presented over a real-time audio-feed by a live social partner. In actuality, all speech was prerecorded. Simply believing that speech was live increased activation in each participant's own mentalizing regions, defined using a functional localizer. Contrasting live to recorded speech did not reveal significant differences in attention or reward regions. Further, higher levels of autistic-like traits were associated with altered neural specialization for live interaction. These results suggest that humans engage in ongoing mentalizing about social partners, even when such mentalizing is not explicitly required, illustrating how social context shapes social cognition. Understanding communication in social context has important implications for typical and atypical social processing, especially for disorders like autism where social difficulties are more acute in live interaction. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Structural Basis for the Interaction between Pyk2-FAT Domain and Leupaxin LD Repeats

    DOE PAGES

    Vanarotti, Murugendra S.; Finkelstein, David B.; Guibao, Cristina D.; ...

    2016-02-11

    Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase and belongs to the focal adhesion kinase (FAK) family. Like FAK, the C-terminal focal adhesion-targeting (FAT) domain of Pyk2 binds to paxillin, a scaffold protein in focal adhesions; however, the interaction between the FAT domain of Pyk2 and paxillin is dynamic and unstable. Leupaxin is another member in the paxillin family and was suggested to be the native binding partner of Pyk2; Pyk2 gene expression is strongly correlated with that of leupaxin in many tissues including primary breast cancer. Here, we report that leupaxin interacts with Pyk2-FAT. Leupaxin has fourmore » leucine–aspartate (LD) motifs. The first and third LD motifs of leupaxin preferably target the two LD-binding sites on the Pyk2-FAT domain, respectively. Moreover, the full-length leupaxin binds to Pyk2-FAT as a stable one-to-one complex. Together, we propose that there is an underlying selectivity between leupaxin and paxillin for Pyk2, which may influence the differing behavior of the two proteins at focal adhesion sites.« less

  17. Structural Basis for the Interaction between Pyk2-FAT Domain and Leupaxin LD Repeats

    PubMed Central

    2016-01-01

    Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase and belongs to the focal adhesion kinase (FAK) family. Like FAK, the C-terminal focal adhesion-targeting (FAT) domain of Pyk2 binds to paxillin, a scaffold protein in focal adhesions; however, the interaction between the FAT domain of Pyk2 and paxillin is dynamic and unstable. Leupaxin is another member in the paxillin family and was suggested to be the native binding partner of Pyk2; Pyk2 gene expression is strongly correlated with that of leupaxin in many tissues including primary breast cancer. Here, we report that leupaxin interacts with Pyk2-FAT. Leupaxin has four leucine–aspartate (LD) motifs. The first and third LD motifs of leupaxin preferably target the two LD-binding sites on the Pyk2-FAT domain, respectively. Moreover, the full-length leupaxin binds to Pyk2-FAT as a stable one-to-one complex. Together, we propose that there is an underlying selectivity between leupaxin and paxillin for Pyk2, which may influence the differing behavior of the two proteins at focal adhesion sites. PMID:26866573

  18. The Myb-domain protein ULTRAPETALA1 INTERACTING FACTOR 1 controls floral meristem activities in Arabidopsis.

    PubMed

    Moreau, Fanny; Thévenon, Emmanuel; Blanvillain, Robert; Lopez-Vidriero, Irene; Franco-Zorrilla, Jose Manuel; Dumas, Renaud; Parcy, François; Morel, Patrice; Trehin, Christophe; Carles, Cristel C

    2016-04-01

    Higher plants continuously and iteratively produce new above-ground organs in the form of leaves, stems and flowers. These organs arise from shoot apical meristems whose homeostasis depends on coordination between self-renewal of stem cells and their differentiation into organ founder cells. This coordination is stringently controlled by the central transcription factor WUSCHEL (WUS), which is both necessary and sufficient for stem cell specification in Arabidopsis thaliana ULTRAPETALA1 (ULT1) was previously identified as a plant-specific, negative regulator of WUS expression. However, molecular mechanisms underlying this regulation remain unknown. ULT1 protein contains a SAND putative DNA-binding domain and a B-box, previously proposed as a protein interaction domain in eukaryotes. Here, we characterise a novel partner of ULT1, named ULT1 INTERACTING FACTOR 1 (UIF1), which contains a Myb domain and an EAR motif. UIF1 and ULT1 function in the same pathway for regulation of organ number in the flower. Moreover, UIF1 displays DNA-binding activity and specifically binds to WUS regulatory elements. We thus provide genetic and molecular evidence that UIF1 and ULT1 work together in floral meristem homeostasis, probably by direct repression of WUS expression. © 2016. Published by The Company of Biologists Ltd.

  19. Interactions between the HIV TAT domain and cell membranes

    NASA Astrophysics Data System (ADS)

    Mishra, Abhijit; Wong, Gerard

    2005-03-01

    Biologically active molecules such as proteins and oligonucleotides can be transduced into cells with high efficiency when covalently linked to a Protein Transduction Domain (PTD), such as the TAT domain in the HIV virus. All PTDs have a high content of basic amino acids resulting in a net positive charge. Electrostatic interactions between cationic PTDs and the negatively charged phospholipids that constitute the plasma membrane seem to be responsible for peptide uptake, but no detailed structural studies exist. We present recent results on the structures of self-assembled complexes of the cationic TAT domain and anionic lipid bilayers using synchrotron x-ray scattering and electron microscopy, and examine possible mechanisms of the anomalous transduction.

  20. Copy Number Variations in DISC1 and DISC1-Interacting Partners in Major Mental Illness

    PubMed Central

    Johnstone, Mandy; MacLean, Alan; Heyrman, Lien; Lenaerts, An-Sofie; Nordin, Annelie; Nilsson, Lars-Göran; De Rijk, Peter; Goossens, Dirk; Adolfsson, Rolf; Clair, David M. St.; Hall, Jeremy; Lawrie, Stephen M.; McIntosh, Andrew M.; Del-Favero, Jurgen; Blackwood, Douglas H.R.; Pickard, Benjamin S.

    2015-01-01

    Robust statistical, genetic and functional evidence supports a role for DISC1 in the aetiology of major mental illness. Furthermore, many of its protein-binding partners show evidence for involvement in the pathophysiology of a range of neurodevelopmental and psychiatric disorders. Copy number variants (CNVs) are suspected to play an important causal role in these disorders. In this study, CNV analysis of DISC1 and its binding partners PAFAH1B1, NDE1, NDEL1, FEZ1, MAP1A, CIT and PDE4B in Scottish and Northern Swedish population-based samples was carried out using multiplex amplicon quantification. Here, we report the finding of rare CNVs in DISC1, NDE1 (together with adjacent genes within the 16p13.11 duplication), NDEL1 (including the overlapping MYH10 gene) and CIT. Our findings provide further evidence for involvement of DISC1 and its interaction partners in neuropsychiatric disorders and also for a role of structural variants in the aetiology of these devastating diseases. PMID:27239468

  1. Soluble protein expression in E. coli cells using IgG-binding domain of protein A as a solubilizing partner in the cold induced system.

    PubMed

    Inouye, Satoshi; Sahara, Yuiko

    2008-11-21

    We constructed a cold induced expression vector in Escherichia coli cells that consists of a histidine tag sequence for nickel chelate affinity purification, IgG-binding domain of protein A (ZZ-domain) and the multiple cloning sites. The role of ZZ-domain as a solubilizing partner at 15 degrees C was demonstrated by expressing the imidazopyrazinone-type luciferases of Renilla, Oplophorus, Gaussia, and Vargula (Cypridina) as well as the calcium-binding photoproteins and firefly luciferase. The fused protein with ZZ-domain was expressed efficiently as a soluble form in the cytoplasm of E. coli cells at low temperature.

  2. The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site

    PubMed Central

    Claveria-Gimeno, Rafael; Lanuza, Pilar M.; Morales-Chueca, Ignacio; Jorge-Torres, Olga C.; Vega, Sonia; Abian, Olga; Esteller, Manel; Velazquez-Campoy, Adrian

    2017-01-01

    Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities. PMID:28139759

  3. WW domain interactions regulate the Hippo tumor suppressor pathway

    PubMed Central

    Salah, Z; Aqeilan, R I

    2011-01-01

    The Hippo kinase pathway is emerging as a conserved signaling pathway that is essential for organ growth and tumorigenesis in Drosophila and mammalians. Although the signaling of the core kinases is relatively well understood, less is known about the upstream inputs, downstream outputs and regulation of the whole cascade. Enrichment of the Hippo pathway components with WW domains and their cognate proline-rich interacting motifs provides a versatile platform for further understanding the mechanisms that regulate organ growth and tumorigenesis. Here, we review recently discovered mechanisms of WW domain-mediated interactions that contribute to the regulation of the Hippo signaling pathway in tumorigenesis. We further discuss new insights and future directions on the emerging role of such regulation. PMID:21677687

  4. WW domain interactions regulate the Hippo tumor suppressor pathway.

    PubMed

    Salah, Z; Aqeilan, R I

    2011-06-16

    The Hippo kinase pathway is emerging as a conserved signaling pathway that is essential for organ growth and tumorigenesis in Drosophila and mammalians. Although the signaling of the core kinases is relatively well understood, less is known about the upstream inputs, downstream outputs and regulation of the whole cascade. Enrichment of the Hippo pathway components with WW domains and their cognate proline-rich interacting motifs provides a versatile platform for further understanding the mechanisms that regulate organ growth and tumorigenesis. Here, we review recently discovered mechanisms of WW domain-mediated interactions that contribute to the regulation of the Hippo signaling pathway in tumorigenesis. We further discuss new insights and future directions on the emerging role of such regulation.

  5. "Why can't you control this?" How women's interactions with intimate partners define menopause and family.

    PubMed

    Dillaway, Heather E

    2008-01-01

    In this article I explore women's discussions of the interactions that families have about menopause and, thus, attempt to broaden feminist knowledge of women's experiences of menopause within families. Data on which this article is based were collected in 61 in-depth interviews with menopausal women in a midwest state in 2001. Findings suggest that biomedical definitions of menopause are often reaffirmed within interactions between intimate partners. Thus, women reported negative familial interactions about menopause, as they were encouraged to define symptoms as problematic and seek medical treatment. Alternatively, some interviewees reported positive interactions about menopause, as a few partners helped them soothe symptoms and follow health regimens. Women interpreted these latter interactions as support or care, rather than surveillance or monitoring. The author concludes that familial interactions bolster dominant constructions of both menopause and family because, as menopause is discussed between intimate partners, definitions of gendered familial roles and responsibilities are cemented.

  6. Elucidating nitric oxide synthase domain interactions by molecular dynamics.

    PubMed

    Hollingsworth, Scott A; Holden, Jeffrey K; Li, Huiying; Poulos, Thomas L

    2016-02-01

    Nitric oxide synthase (NOS) is a multidomain enzyme that catalyzes the production of nitric oxide (NO) by oxidizing L-Arg to NO and L-citrulline. NO production requires multiple interdomain electron transfer steps between the flavin mononucleotide (FMN) and heme domain. Specifically, NADPH-derived electrons are transferred to the heme-containing oxygenase domain via the flavin adenine dinucleotide (FAD) and FMN containing reductase domains. While crystal structures are available for both the reductase and oxygenase domains of NOS, to date there is no atomic level structural information on domain interactions required for the final FMN-to-heme electron transfer step. Here, we evaluate a model of this final electron transfer step for the heme-FMN-calmodulin NOS complex based on the recent biophysical studies using a 105-ns molecular dynamics trajectory. The resulting equilibrated complex structure is very stable and provides a detailed prediction of interdomain contacts required for stabilizing the NOS output state. The resulting equilibrated complex model agrees well with previous experimental work and provides a detailed working model of the final NOS electron transfer step required for NO biosynthesis.

  7. Characterization of Domain–Peptide Interaction Interface: Prediction of SH3 Domain-Mediated Protein–Protein Interaction Network in Yeast by Generic Structure-Based Models

    PubMed Central

    Hou, Tingjun; Li, Nan; Li, Youyong; Wang, Wei

    2012-01-01

    Determination of the binding specificity of SH3 domain, a peptide recognition module (PRM), is important to understand their biological functions and reconstruct the SH3-mediated protein–protein interaction network. In the present study, the SH3-peptide interactions for both class I and II SH3 domains were characterized by the intermolecular residue–residue interaction network. We developed generic MIEC-SVM models to infer SH3 domain-peptide recognition specificity that achieved satisfactory prediction accuracy. By investigating the domain–peptide recognition mechanisms at the residue level, we found that the class-I and class-II binding peptides have different binding modes even though they occupy the same binding site of SH3. Furthermore, we predicted the potential binding partners of SH3 domains in the yeast proteome and constructed the SH3-mediated protein–protein interaction network. Comparison with the experimentally determined interactions confirmed the effectiveness of our approach. This study showed that our sophisticated computational approach not only provides a powerful platform to decipher protein recognition code at the molecular level but also allows identification of peptide-mediated protein interactions at a proteomic scale. We believe that such an approach is general to be applicable to other domain–peptide interactions. PMID:22468754

  8. SARAH Domain-Mediated MST2-RASSF Dimeric Interactions

    PubMed Central

    Matallanas, David; Romano, David; Nguyen, Lan K.; Kholodenko, Boris N.; Rosta, Edina; Kolch, Walter

    2016-01-01

    RASSF enzymes act as key apoptosis activators and tumor suppressors, being downregulated in many human cancers, although their exact regulatory roles remain unknown. A key downstream event in the RASSF pathway is the regulation of MST kinases, which are main effectors of RASSF-induced apoptosis. The regulation of MST1/2 includes both homo- and heterodimerization, mediated by helical SARAH domains, though the underlying molecular interaction mechanism is unclear. Here, we study the interactions between RASSF1A, RASSF5, and MST2 SARAH domains by using both atomistic molecular simulation techniques and experiments. We construct and study models of MST2 homodimers and MST2-RASSF SARAH heterodimers, and we identify the factors that control their high molecular stability. In addition, we also analyze both computationally and experimentally the interactions of MST2 SARAH domains with a series of synthetic peptides particularly designed to bind to it, and hope that our approach can be used to address some of the challenging problems in designing new anti-cancer drugs. PMID:27716844

  9. Assessing the co-occurrence of intimate partner violence domains across the life-course: relating typologies to mental health

    PubMed Central

    Armour, Cherie; Sleath, Emma

    2014-01-01

    Background The inter-generational transmission of violence (ITV) hypothesis and polyvictimisation have been studied extensively. The extant evidence suggests that individuals from violent families are at increased risk of subsequent intimate partner violence (IPV) and that a proportion of individuals experience victimisation across multiple rather than single IPV domains. Both ITV and polyvictimisation are shown to increase the risk of psychiatric morbidity, alcohol use, and anger expression. Objective The current study aimed to 1) ascertain if underlying typologies of victimisation across the life-course and over multiple victimisation domains were present and 2) ascertain if groupings differed on mean scores of posttraumatic stress disorder (PTSD), depression, alcohol use, and anger expression. Method University students (N=318) were queried in relation to victimisation experiences and psychological well-being. Responses across multiple domains of IPV spanning the life-course were used in a latent profile analysis. ANOVA was subsequently used to determine if profiles differed in their mean scores on PTSD, depression, alcohol use, and anger expression. Results Three distinct profiles were identified; one of which comprised individuals who experienced “life-course polyvictimisation,” another showing individuals who experienced “witnessing parental victimisation,” and one which experienced “psychological victimisation only.” Life-course polyvictims scored the highest across most assessed measures. Conclusion Witnessing severe physical aggression and injury in parental relationships as a child has an interesting impact on the ITV into adolescence and adulthood. Life-course polyvictims are shown to experience increased levels of psychiatric morbidity and issues with alcohol misuse and anger expression. PMID:25279106

  10. Cannibalism as an interacting phenotype: precannibalistic aggression is influenced by social partners in the endangered Socorro Isopod (Thermosphaeroma thermophilum).

    PubMed

    Bleakley, B H; Welter, S M; McCauley-Cole, K; Shuster, S M; Moore, A J

    2013-04-01

    Models for the evolution of cannibalism highlight the importance of asymmetries between individuals in initiating cannibalistic attacks. Studies may include measures of body size but typically group individuals into size/age classes or compare populations. Such broad comparisons may obscure the details of interactions that ultimately determine how socially contingent characteristics evolve. We propose that understanding cannibalism is facilitated by using an interacting phenotypes perspective that includes the influences of the phenotype of a social partner on the behaviour of a focal individual and focuses on variation in individual pairwise interactions. We investigated how relative body size, a composite trait between a focal individual and its social partner, and the sex of the partners influenced precannibalistic aggression in the endangered Socorro isopod, Thermosphaeroma thermophilum. We also investigated whether differences in mating interest among males and females influenced cannibalism in mixed sex pairs. We studied these questions in three populations that differ markedly in range of body size and opportunities for interactions among individuals. We found that relative body size influences the probability of and latency to attack. We observed differences in the likelihood of and latency to attack based on both an individual's sex and the sex of its partner but found no evidence of sexual conflict. The instigation of precannibalistic aggression in these isopods is therefore a property of both an individual and its social partner. Our results suggest that interacting phenotype models would be improved by incorporating a new conditional ψ, which describes the strength of a social partner's influence on focal behaviour.

  11. NMR assignments of the FKBP-type PPIase domain of the human aryl-hydrocarbon receptor-interacting protein (AIP).

    PubMed

    Linnert, Miriam; Haupt, Katja; Lin, Yi-Jan; Kissing, Sandra; Paschke, Anne-Katrin; Fischer, Gunter; Weiwad, Matthias; Lücke, Christian

    2012-10-01

    The aryl-hydrocarbon receptor-interacting protein (AIP) interacts with several protein binding partners and has been associated with pituitary tumor development. Here, we report nearly complete (1)H, (13)C and (15)N chemical shift assignments for the N-terminal AIP(2-166) segment, which has been predicted to represent a FKBP-type PPIase domain. Sequence alignment with the prototypic FKBP12, however, reveals disagreements between the AIP chemical shift index consensus and the corresponding FKBP12 secondary structure elements.

  12. Using support vector machine for improving protein-protein interaction prediction utilizing domain interactions

    SciTech Connect

    Singhal, Mudita; Shah, Anuj R.; Brown, Roslyn N.; Adkins, Joshua N.

    2010-10-02

    Understanding protein interactions is essential to gain insights into the biological processes at the whole cell level. The high-throughput experimental techniques for determining protein-protein interactions (PPI) are error prone and expensive with low overlap amongst them. Although several computational methods have been proposed for predicting protein interactions there is definite room for improvement. Here we present DomainSVM, a predictive method for PPI that uses computationally inferred domain-domain interaction values in a Support Vector Machine framework to predict protein interactions. DomainSVM method utilizes evidence of multiple interacting domains to predict a protein interaction. It outperforms existing methods of PPI prediction by achieving very high explanation ratios, precision, specificity, sensitivity and F-measure values in a 10 fold cross-validation study conducted on the positive and negative PPIs in yeast. A Functional comparison study using GO annotations on the positive and the negative test sets is presented in addition to discussing novel PPI predictions in Salmonella Typhimurium.

  13. Gender differences in partner interactions during an after-school science peer tutoring program

    NASA Astrophysics Data System (ADS)

    Brei-Crawley, M. Jo

    This teacher research study examined an after-school science program called SSTAR (Science Students Teaching as Resources) to determine if this program encourages early scientific involvement for girls, specifically the investigation of simple machines. SSTAR's overall goal was to develop scientific skills in fourth grade tutors who were partnered with second grade tutees. This study was conducted during two different SSTAR study sessions, identified as the pilot study (year one) and the expanded study (year two). The SSTAR program and the data collection instruments were refined and modified during this two-year process. Four data collection instruments were used to gather data and insights into this program; video-taped interactions between tutor and tutee, a writing assessment, a performance assessment and focus group discussions. The video taped partnership interactions found that tutors used similar instructional strategies and tutees gave similar response strategies. However, these strategies varied according to the gender of the partner. A written assessment, in the form of an open ended question was given to just the tutors at the beginning and end of their session. Additionally, a performance assessment was given. This assessment asked the tutors to construct a machine from the Legos(c) that were provided. This assessment was also done in a pretest/post-test format. Scores from the writing and performance assessment were then compared and the performance assessment showed more tutor growth in knowledge of simple machines than the writing assessment. Overall students made comments stating they enjoyed the SSTAR program and would sign up again. They had no preference for a same gender or opposite gender partner among either tutor or tutee discussions. All the data examined shows evidence that SSTAR was an effective program for tutor growth in the scientific area of simple machines. While the original study focus was specifically on girls, both genders

  14. Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins

    PubMed Central

    Smith, Everett Clinton; Smith, Stacy E.; Carter, James R.; Webb, Stacy R.; Gibson, Kathleen M.; Hellman, Lance M.; Fried, Michael G.; Dutch, Rebecca Ellis

    2013-01-01

    Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion. PMID:24178297

  15. The Interactive Effects of Emotion Regulation and Alcohol Intoxication on Lab-Based Intimate Partner Aggression

    PubMed Central

    Watkins, Laura E.; DiLillo, David; Maldonado, Rosalita C.

    2015-01-01

    This study draws on Finkel and Eckhardt’s (2013) I3 framework to examine the interactive effects of two emotion regulation strategies, anger rumination (an impellance factor) and reappraisal (an inhibition factor), and alcohol intoxication (a disinhibition factor), on intimate partner aggression (IPA) perpetration as measured with an analogue aggression task. Participants were 69 couples recruited from a large Midwestern university (total N = 138). Participants’ trait rumination and reappraisal were measured by self-report. Participants were randomized individually to an alcohol or placebo condition, then recalled an anger event while employing one of three randomly assigned emotion regulation conditions (rumination, reappraisal, or uninstructed). Following this, participants completed an analogue aggression task involving ostensibly assigning white noise blasts to their partner. Participants in the alcohol condition displayed greater IPA than participants in the placebo condition for provoked IPA, but not unprovoked IPA. Results also revealed interactions such that for those in the alcohol and rumination group, higher trait reappraisal was related to lower unprovoked IPA. For provoked IPA, higher trait rumination was related to greater IPA among those in the alcohol and rumination condition and those in the placebo and uninstructed condition. In general, results were consistent with I3 theory, suggesting that alcohol disinhibits, rumination impels, and trait reappraisal inhibits IPA. The theoretical and clinical implications of these findings are discussed in the context of current knowledge about the influence of alcohol intoxication and emotion regulation strategies on IPA perpetration. PMID:25844831

  16. The interactive effects of emotion regulation and alcohol intoxication on lab-based intimate partner aggression.

    PubMed

    Watkins, Laura E; DiLillo, David; Maldonado, Rosalita C

    2015-09-01

    This study draws on Finkel and Eckhardt's (2013) I³ framework to examine the interactive effects of 2 emotion regulation strategies-anger rumination (an impellance factor) and reappraisal (an inhibition factor), and alcohol intoxication (a disinhibition factor)-on intimate partner aggression (IPA) perpetration as measured with an analogue aggression task. Participants were 69 couples recruited from a large Midwestern university (total N = 138). Participants' trait rumination and reappraisal were measured by self-report. Participants were randomized individually to an alcohol or placebo condition, then recalled an anger event while using 1 of 3 randomly assigned emotion regulation conditions (rumination, reappraisal, or uninstructed). Following this, participants completed an analogue aggression task involving ostensibly assigning white noise blasts to their partner. Participants in the alcohol condition displayed greater IPA than participants in the placebo condition for provoked IPA, but not unprovoked IPA. Results also revealed interactions such that for those in the alcohol and rumination group, higher trait reappraisal was related to lower unprovoked IPA. For provoked IPA, higher trait rumination was related to greater IPA among those in the alcohol and rumination condition and those in the placebo and uninstructed condition. In general, results were consistent with I³ theory, suggesting that alcohol disinhibits, rumination impels, and trait reappraisal inhibits IPA. The theoretical and clinical implications of these findings are discussed in the context of current knowledge about the influence of alcohol intoxication and emotion regulation strategies on IPA perpetration.

  17. Proteome analysis of microtubule-associated proteins and their interacting partners from mammalian brain.

    PubMed

    Kozielski, Frank; Riaz, Tahira; DeBonis, Salvatore; Koehler, Christian J; Kroening, Mario; Panse, Isabel; Strozynski, Margarita; Donaldson, Ian M; Thiede, Bernd

    2011-07-01

    The microtubule (MT) cytoskeleton is essential for a variety of cellular processes. MTs are finely regulated by distinct classes of MT-associated proteins (MAPs), which themselves bind to and are regulated by a large number of additional proteins. We have carried out proteome analyses of tubulin-rich and tubulin-depleted MAPs and their interacting partners isolated from bovine brain. In total, 573 proteins were identified giving us unprecedented access to brain-specific MT-associated proteins from mammalian brain. Most of the standard MAPs were identified and at least 500 proteins have been reported as being associated with MTs. We identified protein complexes with a large number of subunits such as brain-specific motor/adaptor/cargo complexes for kinesins, dynein, and dynactin, and proteins of an RNA-transporting granule. About 25% of the identified proteins were also found in the synaptic vesicle proteome. Analysis of the MS/MS data revealed many posttranslational modifications, amino acid changes, and alternative splice variants, particularly in tau, a key protein implicated in Alzheimer's disease. Bioinformatic analysis of known protein-protein interactions of the identified proteins indicated that the number of MAPs and their associated proteins is larger than previously anticipated and that our database will be a useful resource to identify novel binding partners.

  18. Two-Partner Secretion: Combining Efficiency and Simplicity in the Secretion of Large Proteins for Bacteria-Host and Bacteria-Bacteria Interactions.

    PubMed

    Guérin, Jeremy; Bigot, Sarah; Schneider, Robert; Buchanan, Susan K; Jacob-Dubuisson, Françoise

    2017-01-01

    Initially identified in pathogenic Gram-negative bacteria, the two-partner secretion (TPS) pathway, also known as Type Vb secretion, mediates the translocation across the outer membrane of large effector proteins involved in interactions between these pathogens and their hosts. More recently, distinct TPS systems have been shown to secrete toxic effector domains that participate in inter-bacterial competition or cooperation. The effects of these systems are based on kin vs. non-kin molecular recognition mediated by specific immunity proteins. With these new toxin-antitoxin systems, the range of TPS effector functions has thus been extended from cytolysis, adhesion, and iron acquisition, to genome maintenance, inter-bacterial killing and inter-bacterial signaling. Basically, a TPS system is made up of two proteins, the secreted TpsA effector protein and its TpsB partner transporter, with possible additional factors such as immunity proteins for protection against cognate toxic effectors. Structural studies have indicated that TpsA proteins mainly form elongated β helices that may be followed by specific functional domains. TpsB proteins belong to the Omp85 superfamily. Open questions remain on the mechanism of protein secretion in the absence of ATP or an electrochemical gradient across the outer membrane. The remarkable dynamics of the TpsB transporters and the progressive folding of their TpsA partners at the bacterial surface in the course of translocation are thought to be key elements driving the secretion process.

  19. Identification through Combinatorial Random and Rational Mutagenesis of a Substrate-interacting Exosite in the γ Domain of Streptokinase*

    PubMed Central

    Yadav, Suman; Aneja, Rachna; Kumar, Prakash; Datt, Manish; Sinha, Sonali; Sahni, Girish

    2011-01-01

    To identify new structure-function correlations in the γ domain of streptokinase, mutants were generated by error-prone random mutagenesis of the γ domain and its adjoining region in the β domain followed by functional screening specifically for substrate plasminogen activation. Single-site mutants derived from various multipoint mutation clusters identified the importance of discrete residues in the γ domain that are important for substrate processing. Among the various residues, aspartate at position 328 was identified as critical for substrate human plasminogen activation through extensive mutagenesis of its side chain, namely D328R, D328H, D328N, and D328A. Other mutants found to be important in substrate plasminogen activation were, namely, R319H, N339S, K334A, K334E, and L335Q. When examined for their 1:1 interaction with human plasmin, these mutants were found to retain the native-like high affinity for plasmin and also to generate amidolytic activity with partner plasminogen in a manner similar to wild type streptokinase. Moreover, cofactor activities of the mutants precomplexed with plasmin against microplasminogen as the substrate as well as in silico modeling studies suggested that the region 315–340 of the γ domain interacts with the serine protease domain of the macromolecular substrate. Overall, our results identify the presence of a substrate specific exosite in the γ domain of streptokinase. PMID:21169351

  20. The neuronal RhoA GEF, Tech, interacts with the synaptic multi-PDZ-domain-containing protein, MUPP1.

    PubMed

    Estévez, Marcel A; Henderson, Jennifer A; Ahn, David; Zhu, Xin-Ran; Poschmann, Gereon; Lübbert, Hermann; Marx, Ruth; Baraban, Jay M

    2008-08-01

    Tech is a RhoA guanine nucleotide exchange factor (GEF) that is highly enriched in hippocampal and cortical neurons. To help define its function, we have conducted studies aimed at identifying partner proteins that bind to its C-terminal PDZ ligand motif. Yeast two hybrid studies using the Tech C-terminal segment as bait identified MUPP1, a protein that contains 13 PDZ domains and has been localized to the post-synaptic compartment, as a candidate partner protein for Tech. Co-transfection of Tech and MUPP1 in human embryonic kidney 293 cells confirmed that these full-length proteins interact in a PDZ-dependent fashion. Furthermore, we confirmed that endogenous Tech co-precipitates with MUPP1, but not PSD-95, from hippocampal and cortical extracts prepared from rat brain. In addition, immunostaining of primary cortical cultures revealed co-localization of MUPP1 and Tech puncta in the vicinity of synapses. In assessing which PDZ domains of MUPP1 mediate binding to Tech, we found that Tech can bind to either PDZ domain 10 or 13 of MUPP1 as mutation of both these domains is needed to disrupt their interaction. Taken together, these findings demonstrate that Tech binds to MUPP1 and suggest that it regulates RhoA signaling pathways in the vicinity of synapses.

  1. Great interactions: How binding incorrect partners can teach us about protein recognition and function

    PubMed Central

    Vamparys, Lydie; Laurent, Benoist; Carbone, Alessandra

    2016-01-01

    ABSTRACT Protein–protein interactions play a key part in most biological processes and understanding their mechanism is a fundamental problem leading to numerous practical applications. The prediction of protein binding sites in particular is of paramount importance since proteins now represent a major class of therapeutic targets. Amongst others methods, docking simulations between two proteins known to interact can be a useful tool for the prediction of likely binding patches on a protein surface. From the analysis of the protein interfaces generated by a massive cross‐docking experiment using the 168 proteins of the Docking Benchmark 2.0, where all possible protein pairs, and not only experimental ones, have been docked together, we show that it is also possible to predict a protein's binding residues without having any prior knowledge regarding its potential interaction partners. Evaluating the performance of cross‐docking predictions using the area under the specificity‐sensitivity ROC curve (AUC) leads to an AUC value of 0.77 for the complete benchmark (compared to the 0.5 AUC value obtained for random predictions). Furthermore, a new clustering analysis performed on the binding patches that are scattered on the protein surface show that their distribution and growth will depend on the protein's functional group. Finally, in several cases, the binding‐site predictions resulting from the cross‐docking simulations will lead to the identification of an alternate interface, which corresponds to the interaction with a biomolecular partner that is not included in the original benchmark. Proteins 2016; 84:1408–1421. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:27287388

  2. Great interactions: How binding incorrect partners can teach us about protein recognition and function.

    PubMed

    Vamparys, Lydie; Laurent, Benoist; Carbone, Alessandra; Sacquin-Mora, Sophie

    2016-10-01

    Protein-protein interactions play a key part in most biological processes and understanding their mechanism is a fundamental problem leading to numerous practical applications. The prediction of protein binding sites in particular is of paramount importance since proteins now represent a major class of therapeutic targets. Amongst others methods, docking simulations between two proteins known to interact can be a useful tool for the prediction of likely binding patches on a protein surface. From the analysis of the protein interfaces generated by a massive cross-docking experiment using the 168 proteins of the Docking Benchmark 2.0, where all possible protein pairs, and not only experimental ones, have been docked together, we show that it is also possible to predict a protein's binding residues without having any prior knowledge regarding its potential interaction partners. Evaluating the performance of cross-docking predictions using the area under the specificity-sensitivity ROC curve (AUC) leads to an AUC value of 0.77 for the complete benchmark (compared to the 0.5 AUC value obtained for random predictions). Furthermore, a new clustering analysis performed on the binding patches that are scattered on the protein surface show that their distribution and growth will depend on the protein's functional group. Finally, in several cases, the binding-site predictions resulting from the cross-docking simulations will lead to the identification of an alternate interface, which corresponds to the interaction with a biomolecular partner that is not included in the original benchmark. Proteins 2016; 84:1408-1421. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

  3. The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia

    PubMed Central

    Fuchs, Uta; Rehkamp, Gönna; Haas, Oskar A.; Slany, Robert; König, Margit; Bojesen, Stig; Bohle, Rainer M.; Damm-Welk, Christine; Ludwig, Wolf-Dieter; Harbott, Jochen; Borkhardt, Arndt

    2001-01-01

    We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to ABL. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins(11;9)(q23;q34)inv(11)(q13q23). The rearrangement resulted in a 5′-MLL/FBP17-3′ fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability. In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein. PMID:11438682

  4. A proteomic analysis of LRRK2 binding partners reveals interactions with multiple signaling components of the WNT/PCP pathway.

    PubMed

    Salašová, Alena; Yokota, Chika; Potěšil, David; Zdráhal, Zbyněk; Bryja, Vítězslav; Arenas, Ernest

    2017-07-11

    Autosomal-dominant mutations in the Park8 gene encoding Leucine-rich repeat kinase 2 (LRRK2) have been identified to cause up to 40% of the genetic forms of Parkinson's disease. However, the function and molecular pathways regulated by LRRK2 are largely unknown. It has been shown that LRRK2 serves as a scaffold during activation of WNT/β-catenin signaling via its interaction with the β-catenin destruction complex, DVL1-3 and LRP6. In this study, we examine whether LRRK2 also interacts with signaling components of the WNT/Planar Cell Polarity (WNT/PCP) pathway, which controls the maturation of substantia nigra dopaminergic neurons, the main cell type lost in Parkinson's disease patients. Co-immunoprecipitation and tandem mass spectrometry was performed in a mouse substantia nigra cell line (SN4741) and human HEK293T cell line in order to identify novel LRRK2 binding partners. Inhibition of the WNT/β-catenin reporter, TOPFlash, was used as a read-out of WNT/PCP pathway activation. The capacity of LRRK2 to regulate WNT/PCP signaling in vivo was tested in Xenopus laevis' early development. Our proteomic analysis identified that LRRK2 interacts with proteins involved in WNT/PCP signaling such as the PDZ domain-containing protein GIPC1 and Integrin-linked kinase (ILK) in dopaminergic cells in vitro and in the mouse ventral midbrain in vivo. Moreover, co-immunoprecipitation analysis revealed that LRRK2 binds to two core components of the WNT/PCP signaling pathway, PRICKLE1 and CELSR1, as well as to FLOTILLIN-2 and CULLIN-3, which regulate WNT secretion and inhibit WNT/β-catenin signaling, respectively. We also found that PRICKLE1 and LRRK2 localize in signalosomes and act as dual regulators of WNT/PCP and β-catenin signaling. Accordingly, analysis of the function of LRRK2 in vivo, in X. laevis revelaed that LRKK2 not only inhibits WNT/β-catenin pathway, but induces a classical WNT/PCP phenotype in vivo. Our study shows for the first time that LRRK2 activates the WNT

  5. Spin torque and interactions in ferromagnetic semiconductor domain walls

    NASA Astrophysics Data System (ADS)

    Golovatski, Elizabeth Ann

    transmission spectrum with many of the similar features from the 2pi wall. Looking at the individual walls, we find an interesting interaction that has three distinct regimes: (1) repulsion, where the left wall moves to the left and the right wall to the right; (2) motion together, where the two walls both move to the right, even at the same velocity for one special value of separation; and (3) attraction, where the left wall moves to the right and the right wall moves to the left. This speaks to a kind of natural equilibrium distance between the domain walls. This is of major interest for device purposes as it means that stacks of domain walls could be self-correcting in their motions along a track. Much experimental work needs to be done to make this a reality, however.

  6. Ubiquitin-Activated Interaction Traps (UBAITs) identify E3 ligase binding partners.

    PubMed

    O'Connor, Hazel F; Lyon, Nancy; Leung, Justin W; Agarwal, Poonam; Swaim, Caleb D; Miller, Kyle M; Huibregtse, Jon M

    2015-12-01

    We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ubiquitin-Activated Interaction Traps) are E3-ubiquitin fusion proteins and, in an E1- and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester-linked lariat intermediate or through an E2 thioester intermediate, and both WT and active-site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double-strand break repair. Using the RNF168 UBAIT, we identify H2AZ--a histone protein involved in DNA repair--as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.

  7. Evaluation of Nod-Like Receptor (NLR) Effector Domain Interactions

    PubMed Central

    Kufer, Thomas A.; Schwarzenbacher, Robert

    2009-01-01

    Members of the Nod-like receptor (NLR) family recognize intracellular pathogens and recruit a variety of effector molecules, including pro-caspases and kinases, which in turn are implicated in cytokine processing and NF-κB activation. In order to elucidate the intricate network of NLR signaling, which is still fragmentary in molecular terms, we applied comprehensive yeast two-hybrid analysis for unbiased evaluation of physical interactions between NLRs and their adaptors (ASC, CARD8) as well as kinase RIPK2 and inflammatory caspases (C1, C2, C4, C5) under identical conditions. Our results confirmed the interaction of NOD1 and NOD2 with RIPK2, and between NLRP3 and ASC, but most importantly, our studies revealed hitherto unrecognized interactions of NOD2 with members of the NLRP subfamily. We found that NOD2 specifically and directly interacts with NLRP1, NLRP3 and NLRP12. Furthermore, we observed homodimerization of the RIPK2 CARD domains and identified residues in NOD2 critical for interaction with RIPK2. In conclusion, our work provides further evidence for the complex network of protein-protein interactions underlying NLR function. PMID:19337385

  8. Secretory production of antimicrobial peptides in Escherichia coli using the catalytic domain of a cellulase as fusion partner.

    PubMed

    Yu, Huili; Li, Haoran; Gao, Dongfang; Gao, Cuijuan; Qi, Qingsheng

    2015-11-20

    Antimicrobial peptides (AMPs) are small molecules which serve as essential components of the innate immune system in various organisms. AMPs possess a broad spectrum of antimicrobial activities. However, the scaled production of such peptides in Escherichia coli faces many difficulties because of their small size and toxicity to the host. Here, we described a new fusion strategy to extracellularly produce significant amounts of these antimicrobial peptides in recombinant E. coli at significant amount. Employing the catalytic domain of a cellulase (Cel-CD) from Bacillus subtilis KSM-64 as the fusion partner, five recombinant antimicrobial peptides were confirmed to accumulate in the culture medium at concentrations ranging from 184 mg/L to 297 mg/L. The radical diffusion experiment demonstrated that the released model antimicrobial peptide, bombinin, had antibacterial activities against both E. coli and Staphylococcus aureus. This strategy will be suitable for the production of antimicrobial peptides and other toxicity proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Interactions of adolescent social experiences and dopamine genes to predict physical intimate partner violence perpetration

    PubMed Central

    Parker, Edith A.; Peek-Asa, Corinne

    2017-01-01

    Objectives We examined the interactions between three dopamine gene alleles (DAT1, DRD2, DRD4) previously associated with violent behavior and two components of the adolescent environment (exposure to violence, school social environment) to predict adulthood physical intimate partner violence (IPV) perpetration among white men and women. Methods We used data from Wave IV of the National Longitudinal Study of Adolescent to Adult Health, a cohort study following individuals from adolescence to adulthood. Based on the prior literature, we categorized participants as at risk for each of the three dopamine genes using this coding scheme: two 10-R alleles for DAT1; at least one A-1 allele for DRD2; at least one 7-R or 8-R allele for DRD4. Adolescent exposure to violence and school social environment was measured in 1994 and 1995 when participants were in high school or middle school. Intimate partner violence perpetration was measured in 2008 when participants were 24 to 32 years old. We used simple and multivariable logistic regression models, including interactions of genes and the adolescent environments for the analysis. Results Presence of risk alleles was not independently associated with IPV perpetration but increasing exposure to violence and disconnection from the school social environment was associated with physical IPV perpetration. The effects of these adolescent experiences on physical IPV perpetration varied by dopamine risk allele status. Among individuals with non-risk dopamine alleles, increased exposure to violence during adolescence and perception of disconnection from the school environment were significantly associated with increased odds of physical IPV perpetration, but individuals with high risk alleles, overall, did not experience the same increase. Conclusion Our results suggested the effects of adolescent environment on adulthood physical IPV perpetration varied by genetic factors. This analysis did not find a direct link between risk alleles

  10. BAR Domain-Containing FAM92 Proteins Interact with Chibby1 To Facilitate Ciliogenesis

    PubMed Central

    Li, Feng-Qian; Chen, Xingwang; Fisher, Cody; Siller, Saul S.; Zelikman, Klara; Kuriyama, Ryoko

    2016-01-01

    Chibby1 (Cby1) is a small, conserved coiled-coil protein that localizes to centrioles/basal bodies and plays a crucial role in the formation and function of cilia. During early stages of ciliogenesis, Cby1 is required for the efficient recruitment of small vesicles at the distal end of centrioles to facilitate basal body docking to the plasma membrane. Here, we identified family with sequence similarity 92, member A (FAM92A) and FAM92B, which harbor predicted lipid-binding BAR domains, as novel Cby1-interacting partners using tandem affinity purification and mass spectrometry. We found that in cultured cell lines, FAM92A colocalizes with Cby1 at the centrioles/basal bodies of primary cilia, while FAM92B is undetectable. In airway multiciliated cells, both FAM92A and -92B colocalize with Cby1 at the base of cilia. Notably, the centriolar localization of FAM92A and -92B depends largely on Cby1. Knockdown of FAM92A in RPE1 cells impairs ciliogenesis. Consistent with the membrane-remodeling properties of BAR domains, FAM92A and -92B in cooperation with Cby1 induce deformed membrane-like structures containing the small GTPase Rab8 in cultured cells. Our results therefore suggest that FAM92 proteins interact with Cby1 to promote ciliogenesis via regulation of membrane-remodeling processes. PMID:27528616

  11. Structural Analyses of Zinc Finger Domains for Specific Interactions with DNA.

    PubMed

    Eom, Ki Seong; Cheong, Jin Sung; Lee, Seung Jae

    2016-12-28

    Zinc finger proteins are among the most extensively applied metalloproteins in the field of biotechnology owing to their unique structural and functional aspects as transcriptional and translational regulators. The classical zinc fingers are the largest family of zinc proteins and they provide critical roles in physiological systems from prokaryotes to eukaryotes. Two cysteine and two histidine residues (Cys₂His₂) coordinate to the zinc ion for the structural functions to generate a ββα fold, and this secondary structure supports specific interactions with their binding partners, including DNA, RNA, lipids, proteins, and small molecules. In this account, the structural similarity and differences of well-known Cys₂His₂-type zinc fingers such as zinc interaction factor 268 (ZIF268), transcription factor IIIA (TFIIIA), GAGA, and Ros will be explained. These proteins perform their specific roles in species from archaea to eukaryotes and they show significant structural similarity; however, their aligned amino acids present low sequence homology. These zinc finger proteins have different numbers of domains for their structural roles to maintain biological progress through transcriptional regulations from exogenous stresses. The superimposed structures of these finger domains provide interesting details when these fingers are applied to specific gene binding and editing. The structural information in this study will aid in the selection of unique types of zinc finger applications in vivo and in vitro approaches, because biophysical backgrounds including complex structures and binding affinities aid in the protein design area.

  12. The SH3 domain of UNC-89 (obscurin) interacts with paramyosin, a coiled-coil protein, in Caenorhabditis elegans muscle

    PubMed Central

    Qadota, Hiroshi; Mayans, Olga; Matsunaga, Yohei; McMurry, Jonathan L.; Wilson, Kristy J.; Kwon, Grace E.; Stanford, Rachel; Deehan, Kevin; Tinley, Tina L.; Ngwa, Verra M.; Benian, Guy M.

    2016-01-01

    UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89’s SH3 domain and residues 294–376 of paramyosin and has a KD of ∼1.1 μM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89’s SH3 is α-helical and lacks prolines. Homology modeling of UNC-89’s SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a “skip residue,” which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity. PMID:27009202

  13. Learning in Interactive Work Situations: It Takes Two to Tango; Why Not Invite Both Partners to Dance?

    ERIC Educational Resources Information Center

    Koopmans, Hanneke; Doombos, Anja J.; van Eekelen, Ilse M.

    2006-01-01

    Learning that arises from interactions at work is the focus of this study. More specifically, the concrete activities of adult learners and their interaction partners were of interest because such learning activities largely determine the quality of learning outcomes. The results of the study are summarized in the form of a typology of interactive…

  14. Prediction of Cancer Proteins by Integrating Protein Interaction, Domain Frequency, and Domain Interaction Data Using Machine Learning Algorithms

    PubMed Central

    2015-01-01

    Many proteins are known to be associated with cancer diseases. It is quite often that their precise functional role in disease pathogenesis remains unclear. A strategy to gain a better understanding of the function of these proteins is to make use of a combination of different aspects of proteomics data types. In this study, we extended Aragues's method by employing the protein-protein interaction (PPI) data, domain-domain interaction (DDI) data, weighted domain frequency score (DFS), and cancer linker degree (CLD) data to predict cancer proteins. Performances were benchmarked based on three kinds of experiments as follows: (I) using individual algorithm, (II) combining algorithms, and (III) combining the same classification types of algorithms. When compared with Aragues's method, our proposed methods, that is, machine learning algorithm and voting with the majority, are significantly superior in all seven performance measures. We demonstrated the accuracy of the proposed method on two independent datasets. The best algorithm can achieve a hit ratio of 89.4% and 72.8% for lung cancer dataset and lung cancer microarray study, respectively. It is anticipated that the current research could help understand disease mechanisms and diagnosis. PMID:25866773

  15. Interaction partners of PSD-93 studied by X-ray crystallography and fluorescence polarization spectroscopy.

    PubMed

    Fiorentini, Monica; Bach, Anders; Strømgaard, Kristian; Kastrup, Jette S; Gajhede, Michael

    2013-04-01

    PSD-93 (chapsyn-110, DLG2) is a member of the family of membrane-associated guanylate kinase (MAGUK) proteins. The MAGUK proteins are involved in receptor localization and signalling pathways. The best characterized MAGUK protein, PSD-95, is known to be involved in NMDA receptor signalling via its PDZ domains. The PDZ domains of PSD-95 and PSD-93 are structurally very similar, but relatively little is known about the function of PSD-93. PSD-93 has been suggested to interact with GluD2 from the family of ionotropic glutamate receptors. Here, the interactions of four residues (GTSI) representing the extreme C-terminus of GluD2 with PSD-93 PDZ1 have been investigated in the crystalline phase. Two different binding modes of these residues were observed, suggesting that the peptide is not tightly bound to PSD-93 PDZ1. In accordance, the two N-terminal PSD-93 PDZ domains show no appreciable binding affinity for a GluD2-derived C-terminal octapeptide, whereas micromolar affinity was observed for a GluN2B-derived C-terminal octapeptide. This indicates that if present, the interactions between GluD2 and PSD-93 involve more than the extreme terminus of the receptor. In contrast, the tumour-suppressor protein SCRIB PDZ3 shows low micromolar affinity towards the GluD2-derived octapeptide, which is in agreement with previous findings using high-throughput assays.

  16. Quantification of interaction strengths between chaperones and tetratricopeptide repeat domain-containing membrane proteins.

    PubMed

    Schweiger, Regina; Soll, Jürgen; Jung, Kirsten; Heermann, Ralf; Schwenkert, Serena

    2013-10-18

    The three tetratricopeptide repeat domain-containing docking proteins Toc64, OM64, and AtTPR7 reside in the chloroplast, mitochondrion, and endoplasmic reticulum of Arabidopsis thaliana, respectively. They are suggested to act during post-translational protein import by association with chaperone-bound preprotein complexes. Here, we performed a detailed biochemical, biophysical, and computational analysis of the interaction between Toc64, OM64, and AtTPR7 and the five cytosolic chaperones HSP70.1, HSP90.1, HSP90.2, HSP90.3, and HSP90.4. We used surface plasmon resonance spectroscopy in combination with Interaction Map® analysis to distinguish between chaperone oligomerization and docking protein-chaperone interactions and to calculate binding affinities for all tested interactions. Complementary to this, we applied pulldown assays as well as microscale thermophoresis as surface immobilization independent techniques. The data revealed that OM64 prefers HSP70 over HSP90, whereas Toc64 binds all chaperones with comparable affinities. We could further show that AtTPR7 is able to bind HSP90 in addition to HSP70. Moreover, differences between the HSP90 isoforms were detected and revealed a weaker binding for HSP90.1 to AtTPR7 and OM64, showing that slight differences in the amino acid composition or structure of the chaperones influence binding to the tetratricopeptide repeat domain. The combinatory approach of several methods provided a powerful toolkit to determine binding affinities of similar interaction partners in a highly quantitative manner.

  17. Differential binding partners of the Mis18α/β YIPPEE domains regulates the Mis18 complex recruitment to centromeres

    PubMed Central

    Knippler, Christina M.; Foltz, Daniel R.

    2016-01-01

    The Mis18 complex specifies the site of new CENP-A nucleosome assembly by recruiting the CENP-A specific assembly factor HJURP (Holliday junction recognition protein). The human Mis18 complex consists of Mis18α, Mis18β and Mis18 binding protein 1 (Mis18BP1/hsKNL2). Although Mis18α and Mis18β are highly homologous proteins, we find that their conserved YIPPEE domains mediate distinct interactions that are essential to link new CENP-A deposition to existing centromeres. We find that Mis18α directly interacts with the N-terminus of Mis18BP1; whereas, Mis18β directly interacts with CENP-C during G1 phase, revealing that these proteins have evolved to serve distinct functions in centromeres of higher eukaryotes. The N-terminus of Mis18BP1, containing both the Mis18α and CENP-C binding domains, is necessary and sufficient for centromeric localization. Therefore, the Mis18 complex contains dual CENP-C recognition motifs that are combinatorially required to generate robust centromeric localization that leads to CENP-A deposition. PMID:27239045

  18. Interaction of FUN14 domain containing 1, a mitochondrial outer membrane protein, with kinesin light chain 1 via the tetratricopeptide repeat domain

    PubMed Central

    Jang, Won Hee; Jeong, Young Joo; Choi, Sun Hee; Urm, Sang-Hwa; Seog, Dae-Hyun

    2017-01-01

    Kinesin 1 is a member of the kinesin superfamily proteins (KIFs) of microtubule-dependent molecular motor proteins that transport organelles and protein complexes in cells. Kinesin 1 consists of a homo- or hetero-dimer of kinesin heavy chains (KHCs), often, although not always, associated with two kinesin light chains (KLCs). KLCs are non-motor proteins that associate with many different binding proteins and cargoes, but their binding partners have not yet been fully identified. In the present study, a yeast two-hybrid system was used to identify proteins that interact with the tetratricopeptide repeat (TPR) domain of KLC1. The results of the current study revealed an interaction between the TPR domain of KLC1 and FUN14 domain-containing protein 1 (FUNDC1), which is a mitochondrial outer membrane protein mediating hypoxia-induced mitophagy. FUNDC1 bound to the six TPR motif-containing regions of KLC1 and did not interact with KIF5B (a motor subunit of kinesin 1) and KIF3A (a motor subunit of kinesin 2) in the yeast two-hybrid assay. The cytoplasmic amino N-terminal domain of FUNDC1 is essential for interaction with KLC1. When co-expressed in HEK-293T cells, FUNDC1 co-localized with KLC1 and co-immunoprecipitated with KLC1, but not KIF5B. Collectively, these results indicate that KLC1 may potentially compete with LC3, a key component for autophagosome formation, to interact with FUNDC1. PMID:28123706

  19. Characterization of HelD, an interacting partner of RNA polymerase from Bacillus subtilis.

    PubMed

    Wiedermannová, Jana; Sudzinová, Petra; Kovaľ, Tomaš; Rabatinová, Alžbeta; Šanderova, Hana; Ramaniuk, Olga; Rittich, Šimon; Dohnálek, Jan; Fu, Zhihui; Halada, Petr; Lewis, Peter; Krásny, Libor

    2014-04-01

    Bacterial RNA polymerase (RNAP) is an essential multisubunit protein complex required for gene expression. Here, we characterize YvgS (HelD) from Bacillus subtilis, a novel binding partner of RNAP. We show that HelD interacts with RNAP-core between the secondary channel of RNAP and the alpha subunits. Importantly, we demonstrate that HelD stimulates transcription in an ATP-dependent manner by enhancing transcriptional cycling and elongation. We demonstrate that the stimulatory effect of HelD can be amplified by a small subunit of RNAP, delta. In vivo, HelD is not essential but it is required for timely adaptations of the cell to changing environment. In summary, this study establishes HelD as a valid component of the bacterial transcription machinery.

  20. Attuning: A Communication Process between People with Severe and Profound Intellectual Disability and Their Interaction Partners.

    PubMed

    Griffiths, Colin; Smith, Martine

    2016-03-01

    People with severe and profound intellectual disability typically demonstrate a limited ability to communicate effectively. Most of their communications are non-verbal, often idiosyncratic and ambiguous. This article aims to identify the process that regulates communications of this group of people with others and to describe the methodological approach that was used to achieve this. In this qualitative study, two dyads consisting of a person with severe or profound intellectual and multiple disability and a teacher or carer were filmed as they engaged in school-based activities. Two 1-hour videotapes were transcribed and analysed using grounded theory. Attuning was identified within the theory proposed here as a central process that calibrates and regulates communication. Attuning is conceptualized as a bidirectional, dyadic communication process. Understanding this process may support more effective communication between people with severe or profound intellectual and multiple disability and their interaction partners. © 2015 John Wiley & Sons Ltd.

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

    PubMed

    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.

  2. Structure and functional interactions of the Tsg101 UEV domain.

    PubMed

    Pornillos, Owen; Alam, Steven L; Rich, Rebecca L; Myszka, David G; Davis, Darrell R; Sundquist, Wesley I

    2002-05-15

    Human Tsg101 plays key roles in HIV budding and in cellular vacuolar protein sorting (VPS). In performing these functions, Tsg101 binds both ubiquitin (Ub) and the PTAP tetrapeptide 'late domain' motif located within the viral Gag protein. These interactions are mediated by the N-terminal domain of Tsg101, which belongs to the catalytically inactive ubiquitin E2 variant (UEV) family. We now report the structure of Tsg101 UEV and chemical shift mapping of the Ub and PTAP binding sites. Tsg101 UEV resembles canonical E2 ubiquitin conjugating enzymes, but has an additional N-terminal helix, an extended beta-hairpin that links strands 1 and 2, and lacks the two C-terminal helices normally found in E2 enzymes. PTAP-containing peptides bind in a hydrophobic cleft exposed by the absence of the C-terminal helices, whereas ubiquitin binds in a novel site surrounding the beta-hairpin. These studies provide a structural framework for understanding how Tsg101 mediates the protein-protein interactions required for HIV budding and VPS.

  3. A Skeletal Muscle Ryanodine Receptor Interaction Domain in Triadin

    PubMed Central

    Wium, Elize; Dulhunty, Angela F.; Beard, Nicole A.

    2012-01-01

    Excitation-contraction coupling in skeletal muscle depends, in part, on a functional interaction between the ligand-gated ryanodine receptor (RyR1) and integral membrane protein Trisk 95, localized to the sarcoplasmic reticulum membrane. Various domains on Trisk 95 can associate with RyR1, yet the domain responsible for regulating RyR1 activity has remained elusive. We explored the hypothesis that a luminal Trisk 95 KEKE motif (residues 200–232), known to promote RyR1 binding, may also form the RyR1 activation domain. Peptides corresponding to Trisk 95 residues 200–232 or 200–231 bound to RyR1 and increased the single channel activity of RyR1 by 1.49±0.11-fold and 1.8±0.15-fold respectively, when added to its luminal side. A similar increase in [3H]ryanodine binding, which reflects open probability of the channels, was also observed. This RyR1 activation is similar to activation induced by full length Trisk 95. Circular dichroism showed that both peptides were intrinsically disordered, suggesting a defined secondary structure is not necessary to mediate RyR1 activation. These data for the first time demonstrate that Trisk 95′s 200–231 region is responsible for RyR1 activation. Furthermore, it shows that no secondary structure is required to achieve this activation, the Trisk 95 residues themselves are critical for the Trisk 95-RyR1 interaction. PMID:22937102

  4. Interactions of the acidic domain and SRF interacting motifs with the NKX3.1 homeodomain.

    PubMed

    Ju, Jeong Ho; Maeng, Jin-Soo; Lee, Duck-Yeon; Piszczek, Grzegorz; Gelmann, Edward P; Gruschus, James M

    2009-11-10

    NKX3.1 is a prostate tumor suppressor belonging to the NK-2 family of homeodomain (HD) transcription factors. NK-2 family members often possess a stretch of 10-15 residues enriched in acidic amino acids, the acidic domain (AD), in the flexible, disordered region N-terminal to the HD. Interactions between the N-terminal region of NKX3.1 and its homeodomain affect protein stability and DNA binding. CD spectroscopy measuring the thermal unfolding of NKX3.1 constructs showed a 2 degrees C intramolecular stabilization of the HD by the N-terminal region containing the acidic domain (residues 85-96). CD of mixtures of various N-terminal peptides with a construct containing just the HD showed that the acidic domain and the following region, the SRF interacting (SI) motif (residues 99-105), was necessary for this stabilization. Phosphorylation of the acidic domain is known to slow proteasomal degradation of NKX3.1 in prostate cells, and NMR spectroscopy was used to measure and map the interaction of the HD with phosphorylated and nonphosphorylated forms of the AD peptide. The interaction with the phosphorylated AD peptide was considerably stronger (K(d) = 0.5 +/- 0.2 mM), resulting in large chemical shift perturbations for residues Ser150 and Arg175 in the HD, as well as a 2 degrees C increase in the HD thermal stability compared to that of the nonphosphorylated form. NKX3.1 constructs with AD phosphorylation site threonine residues (89 and 93) mutated to glutamate were 4 degrees C more stable than HD alone. Using polymer theory, effective concentrations for interactions between domains connected by flexible linkers are predicted to be in the millimolar range, and thus, the weak intramolecular interactions observed here could conceivably modulate or compete with stronger, intermolecular interactions with the NKX3.1 HD.

  5. STAC3 stably interacts through its C1 domain with CaV1.1 in skeletal muscle triads

    PubMed Central

    Campiglio, Marta; Flucher, Bernhard E.

    2017-01-01

    The adaptor protein STAC3 is essential for skeletal muscle excitation-contraction (EC) coupling and a mutation in the STAC3 gene has been linked to a severe muscle disease, Native American myopathy (NAM). However the function of STAC3, its interaction partner, and the mode of interaction within the EC-coupling complex remained elusive. Here we demonstrate that STAC3 forms a stable interaction with the voltage-sensor of EC-coupling, CaV1.1, and that this interaction depends on a hitherto unidentified protein-protein binding pocket in the C1 domain of STAC3. While the NAM mutation does not affect the stability of the STAC3-CaV1.1 interaction, mutation of two crucial residues in the C1 binding pocket increases the turnover of STAC3 in skeletal muscle triads. Thus, the C1 domain of STAC3 is responsible for its stable incorporation into the CaV1.1 complex, whereas the SH3 domain containing the NAM mutation site may be involved in low-affinity functional interactions in EC-coupling. PMID:28112192

  6. Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

    SciTech Connect

    Murphy, James M.; Korzhnev, Dmitry M.; Ceccarelli, Derek F.; Briant, Douglas J.; Zarrine-Afsar, Arash; Sicheri, Frank; Kay, Lewis E.; Pawson, Tony

    2012-10-23

    The Par-1/MARK protein kinases play a pivotal role in establishing cellular polarity. This family of kinases contains a unique domain architecture, in which a ubiquitin-associated (UBA) domain is located C-terminal to the kinase domain. We have used a combination of x-ray crystallography and NMR dynamics experiments to understand the interaction of the human (h) MARK3 UBA domain with the adjacent kinase domain as compared with ubiquitin. The x-ray crystal structure of the linked hMARK3 kinase and UBA domains establishes that the UBA domain forms a stable intramolecular interaction with the N-terminal lobe of the kinase domain. However, solution-state NMR studies of the isolated UBA domain indicate that it is highly dynamic, undergoing conformational transitions that can be explained by a folding-unfolding equilibrium. NMR titration experiments indicated that the hMARK3 UBA domain has a detectable but extremely weak affinity for mono ubiquitin, which suggests that conformational instability of the isolated hMARK3 UBA domain attenuates binding to ubiquitin despite the presence of residues typically involved in ubiquitin recognition. Our data identify a molecular mechanism through which the hMARK3 UBA domain has evolved to bind the kinase domain, in a fashion that stabilizes an open conformation of the N- and C-terminal lobes, at the expense of its capacity to engage ubiquitin. These results may be relevant more generally to the 30% of UBA domains that lack significant ubiquitin-binding activity, and they suggest a unique mechanism by which interaction domains may evolve new binding properties.

  7. Identification of a cytoplasmic interaction partner of the large regulatory proteins Rep78/Rep68 of adeno-associated virus type 2 (AAV-2)

    SciTech Connect

    Weger, Stefan . E-mail: stefan.weger@charite.de; Hammer, Eva; Goetz, Anne; Heilbronn, Regine

    2007-05-25

    Through yeast two-hybrid analysis and coimmunoprecipitation studies, we have identified a novel cellular AAV-2 Rep78/Rep68 interaction partner located predominantly in the cytoplasm. In public databases, it has been assigned as KCTD5, because of a region of high similarity to the cytoplasmic tetramerization domain of voltage-gated potassium channels. Whereas Rep/KCTD5 interaction relied on the region surrounding the Rep nuclear localization signal, nuclear accumulation of Rep was not required. Wildtype Rep78/Rep68 proteins induced the translocation of large portions of KCTD5 into the nucleus pointing to functional interactions both in the cytoplasm and the nucleus. In line with an anticipated functional interference in the cytoplasm, KCTD5 overexpression completely abrogated Rep68-mediated posttranscriptional activation of a HIV-LTR driven luciferase reporter gene. Our study expands the panel of already identified nuclear Rep interaction partners to a cytoplasmic protein, which raises the awareness that important steps in the AAV life cycle may be regulated in this compartment.

  8. PAIRpred: partner-specific prediction of interacting residues from sequence and structure.

    PubMed

    Minhas, Fayyaz ul Amir Afsar; Geiss, Brian J; Ben-Hur, Asa

    2014-07-01

    We present a novel partner-specific protein-protein interaction site prediction method called PAIRpred. Unlike most existing machine learning binding site prediction methods, PAIRpred uses information from both proteins in a protein complex to predict pairs of interacting residues from the two proteins. PAIRpred captures sequence and structure information about residue pairs through pairwise kernels that are used for training a support vector machine classifier. As a result, PAIRpred presents a more detailed model of protein binding, and offers state of the art accuracy in predicting binding sites at the protein level as well as inter-protein residue contacts at the complex level. We demonstrate PAIRpred's performance on Docking Benchmark 4.0 and recent CAPRI targets. We present a detailed performance analysis outlining the contribution of different sequence and structure features, together with a comparison to a variety of existing interface prediction techniques. We have also studied the impact of binding-associated conformational change on prediction accuracy and found PAIRpred to be more robust to such structural changes than existing schemes. As an illustration of the potential applications of PAIRpred, we provide a case study in which PAIRpred is used to analyze the nature and specificity of the interface in the interaction of human ISG15 protein with NS1 protein from influenza A virus. Python code for PAIRpred is available at http://combi.cs.colostate.edu/supplements/pairpred/. © 2013 Wiley Periodicals, Inc.

  9. Tropomyosin is an interaction partner of the Drosophila coiled coil protein yuri gagarin.

    PubMed

    Texada, Michael J; Simonette, Rebecca A; Deery, William J; Beckingham, Kathleen M

    2011-02-15

    The Drosophila gene yuri gagarin is a complex locus encoding three protein isoform classes that are ubiquitously expressed in the organism. Mutations to the gene affect processes as diverse as gravitactic behavior and spermatogenesis. The larger Yuri isoforms contain extensive coiled-coil regions. Our previous studies indicate that one of the large isoform classes (Yuri-65) is required for formation of specialized F-actin-containing structures generated during spermatogenesis, including the so-called actin "cones" that mediate spermatid individualization. We used the tandem affinity purification of a tagged version of Yuri-65 (the TAP-tagging technique) to identify proteins associated with Yuri-65 in the intact organism. Tropomyosin, primarily as the 284-residue isoform derived from the ubiquitously expressed Tropomyosin 1 gene was thus identified as a major Yuri interaction partner. Co-immunoprecipitation experiments confirmed this interaction. We have established that the stable F-actin cones of spermatogenesis contain Tropomyosin 1 (Tm1) and that in mutant yuri(F64), failure of F-actin cone formation is associated with failure of Tm1 to accumulate at the cone initiation sites. In investigating possible interactions of Tm1 and Yuri in other tissues, we discovered that Tm1 and Yuri frequently colocalize with the endoplasmic reticulum. Tropomyosin has been implicated in actin-mediated membrane trafficking activity in other systems. Our findings suggest that Yuri-Tm1 complexes participate in related functions.

  10. TROPOMYOSIN IS AN INTERACTION PARTNER OF THE DROSOPHILA COILED COIL PROTEIN YURI GAGARIN

    PubMed Central

    Texada, Michael J.; Simonette, Rebecca A.; Deery, William J.; Beckingham, Kathleen M.

    2011-01-01

    The Drosophila gene yuri gagarin is a complex locus encoding three protein isoform classes that are ubiquitously expressed in the organism. Mutations to the gene affect processes as diverse as gravitactic behavior and spermatogenesis. The larger Yuri isoforms contain extensive coiled-coil regions. Our previous studies indicate that one of the large isoform classes (Yuri-65) is required for formation of specialized F-actin-containing structures generated during spermatogenesis, including the so-called actin “cones” that mediate spermatid individualization. We used tandem affinity purification of a tagged version of Yuri-65 (the TAP-tagging technique) to identify proteins associated with Yuri-65 in the intact organism. Tropomyosin, primarily as the 284-residue isoform derived from the ubiquitously expressed Tropomyosin 1 gene was thus identified as a major Yuri interaction partner. Co-immunoprecipitation experiments confirmed this interaction. We have established that the stable F-actin cones of spermatogenesis contain Tropomyosin 1 (Tm1) and that in mutant yuriF64, failure of F-actin cone formation is associated with failure of Tm1 to accumulate at the cone initiation sites. In investigating possible interactions of Tm1 and Yuri in other tissues, we discovered that Tm1 and Yuri frequently colocalize with the endoplasmic reticulum. Tropomyosin has been implicated in actin-mediated membrane trafficking activity in other systems. Our findings suggest that Yuri-Tm1 complexes participate in related functions. PMID:21126519

  11. Microbes in the coral holobiont: partners through evolution, development, and ecological interactions

    PubMed Central

    Thompson, Janelle R.; Rivera, Hanny E.; Closek, Collin J.; Medina, Mónica

    2015-01-01

    In the last two decades, genetic and genomic studies have revealed the astonishing diversity and ubiquity of microorganisms. Emergence and expansion of the human microbiome project has reshaped our thinking about how microbes control host health—not only as pathogens, but also as symbionts. In coral reef environments, scientists have begun to examine the role that microorganisms play in coral life history. Herein, we review the current literature on coral-microbe interactions within the context of their role in evolution, development, and ecology. We ask the following questions, first posed by McFall-Ngai et al. (2013) in their review of animal evolution, with specific attention to how coral-microbial interactions may be affected under future environmental conditions: (1) How do corals and their microbiome affect each other's genomes? (2) How does coral development depend on microbial partners? (3) How is homeostasis maintained between corals and their microbial symbionts? (4) How can ecological approaches deepen our understanding of the multiple levels of coral-microbial interactions? Elucidating the role that microorganisms play in the structure and function of the holobiont is essential for understanding how corals maintain homeostasis and acclimate to changing environmental conditions. PMID:25621279

  12. Proliferating cell nuclear antigen structure and interactions: too many partners for one dancer?

    PubMed

    De Biasio, Alfredo; Blanco, Francisco J

    2013-01-01

    PCNA is the DNA sliding clamp found in eukaryotes and archaebacteria. Sliding clamps were first described as processivity factors in DNA replication. They consist of multimeric, toroidal-shaped structures with pseudo-sixfold symmetry that encircle the DNA duplex and tether the replicative polymerases to the genomic template. Later, it was found that PCNA serves as a docking platform where other proteins dock to carry out different DNA metabolic processes. The structure of the bacterial clamp bound to a short primed DNA shows a tilted duplex in the central channel, which is lined by α-helices with net positive charges. Many of the proteins reported to interact with PCNA do so via the PCNA Interaction Protein sequence (PIP-box). The structures of several proteins and peptides bound to PCNA show a common binding mode, but it is still unknown how the many different partners compete for binding and exert their enzymatic and regulatory functions. Furthermore, the literature contains many reports on proteins that directly bind to PCNA as detected by different methods, but only few of the putative complexes have been examined in detail by quantitative analytical techniques or high-resolution structural methods. Some of the reported interactions are not observed in solution using the pure proteins, indicating that the direct interaction is nonexistent or very weak and is likely mediated by other factors. We review here the current knowledge on PCNA interactions from a structural point of view, with a focus on human proteins and highlighting the questions that remain to be answered.

  13. Brain transcriptome-wide screen for HIV-1 Nef protein interaction partners reveals various membrane-associated proteins.

    PubMed

    Kammula, Ellen C; Mötter, Jessica; Gorgels, Alexandra; Jonas, Esther; Hoffmann, Silke; Willbold, Dieter

    2012-01-01

    HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

  14. Structure of the Brd4 ET domain bound to a C-terminal motif from γ-retroviral integrases reveals a conserved mechanism of interaction.

    PubMed

    Crowe, Brandon L; Larue, Ross C; Yuan, Chunhua; Hess, Sonja; Kvaratskhelia, Mamuka; Foster, Mark P

    2016-02-23

    The bromodomain and extraterminal domain (BET) protein family are promising therapeutic targets for a range of diseases linked to transcriptional activation, cancer, viral latency, and viral integration. Tandem bromodomains selectively tether BET proteins to chromatin by engaging cognate acetylated histone marks, and the extraterminal (ET) domain is the focal point for recruiting a range of cellular and viral proteins. BET proteins guide γ-retroviral integration to transcription start sites and enhancers through bimodal interaction with chromatin and the γ-retroviral integrase (IN). We report the NMR-derived solution structure of the Brd4 ET domain bound to a conserved peptide sequence from the C terminus of murine leukemia virus (MLV) IN. The complex reveals a protein-protein interaction governed by the binding-coupled folding of disordered regions in both interacting partners to form a well-structured intermolecular three-stranded β sheet. In addition, we show that a peptide comprising the ET binding motif (EBM) of MLV IN can disrupt the cognate interaction of Brd4 with NSD3, and that substitutions of Brd4 ET residues essential for binding MLV IN also impair interaction of Brd4 with a number of cellular partners involved in transcriptional regulation and chromatin remodeling. This suggests that γ-retroviruses have evolved the EBM to mimic a cognate interaction motif to achieve effective integration in host chromatin. Collectively, our findings identify key structural features of the ET domain of Brd4 that allow for interactions with both cellular and viral proteins.

  15. Designing Interaction as a Learning Process: Supporting Users' Domain Knowledge Development in Interaction

    ERIC Educational Resources Information Center

    Choi, Jung-Min

    2010-01-01

    The primary concern in current interaction design is focused on how to help users solve problems and achieve goals more easily and efficiently. While users' sufficient knowledge acquisition of operating a product or system is considered important, their acquisition of problem-solving knowledge in the task domain has largely been disregarded. As a…

  16. Designing Interaction as a Learning Process: Supporting Users' Domain Knowledge Development in Interaction

    ERIC Educational Resources Information Center

    Choi, Jung-Min

    2010-01-01

    The primary concern in current interaction design is focused on how to help users solve problems and achieve goals more easily and efficiently. While users' sufficient knowledge acquisition of operating a product or system is considered important, their acquisition of problem-solving knowledge in the task domain has largely been disregarded. As a…

  17. Experimental observation of the interaction of propagating spin waves with Néel domain walls in a Landau domain structure

    NASA Astrophysics Data System (ADS)

    Pirro, P.; Koyama, T.; Brächer, T.; Sebastian, T.; Leven, B.; Hillebrands, B.

    2015-06-01

    The interaction of propagating dipolar spin waves with magnetic domain walls is investigated in square-shaped microstructures patterned from the Heusler compound Co2Mn0.6Fe0.4Si. Using magnetic force microscopy, the reversible preparation of a Landau state with four magnetic domains separated by Néel domain walls is confirmed. A local spin-wave excitation using a microstructured antenna is realized in one of the domains. It is shown by Brillouin light scattering microscopy that the domain structure in the remanence state has a strong influence on the spin-wave excitation and propagation. The domain walls strongly reflect the spin waves and can be used as spin-wave reflectors. A comparison with micromagnetic simulations shows that the strong reflection is due to the long-range dipolar interaction which has important implications for the use of these spin waves for exerting an all-magnonic spin-transfer torque.

  18. Responses to intimate partner violence in Kakuma refugee camp: refugee interactions with agency systems.

    PubMed

    Horn, Rebecca

    2010-01-01

    Intimate partner violence (IPV) has been recognised as a significant problem amongst forcibly displaced communities, and great progress has been made by the United Nations High Commission for Refugees (UNHCR) in responding to IPV and other forms of sexual and gender based violence. However, they have not always effectively engaged refugee communities in these activities, with potentially negative consequences for the health and protection of women. This study was conducted in Kakuma refugee camp, north-west Kenya. Eighteen focus group discussions were conducted with 157 refugees from various nationalities, including Sudanese, Somali, Ethiopian, and Congolese. They focused on the nature and consequences of IPV in Kakuma. The aim of this paper is to explore how refugees in Kakuma talk about the ways that IPV is dealt with, focusing particularly on the ways that community responses are said to interact with formal response systems established by UNHCR and its implementing partners. Refugees talked about using a 'hierarchy of responses' to IPV, with only particularly serious or intransigent cases reaching UNHCR or its implementing agencies. Some male refugees described being mistrustful of agency responses, because agencies were believed to favour women and to prioritise protecting the woman at all costs, even if that means separating her from the family. Whilst community responses to IPV might often be appropriate and helpful, the findings of the current study suggest that in Kakuma they do not necessarily result in the protection of women. Yet women in Kakuma are reported to be reluctant to report their cases to UNHCR and its implementing agencies. A more effective protection response from UNHCR might involve closer co-operation with individuals and structures within the refugee communities to develop a co-ordinated response to IPV.

  19. Genetic exploration of interactive domains in RNA polymerase II subunits.

    PubMed Central

    Martin, C; Okamura, S; Young, R

    1990-01-01

    The two large subunits of RNA polymerase II, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpb1-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpb1-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpb1-1 suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed. Images PMID:2183012

  20. Interactions between tafenoquine and artemisinin-combination therapy partner drug in asexual and sexual stage Plasmodium falciparum.

    PubMed

    Kemirembe, Karen; Cabrera, Mynthia; Cui, Liwang

    2017-08-01

    The 8-aminoquinoline tafenoquine (TFQ), a primaquine derivative, is currently in late-stage clinical development for the radical cure of P. vivax. Here drug interactions between TFQ and chloroquine and six artemisinin-combination therapy (ACT) partner drugs in P. falciparum asexual stages and gametocytes were investigated. TFQ was mostly synergistic with the ACT-partner drugs in asexual parasites regardless of genetic backgrounds. However, at fixed ratios of 1:3, 1:1 and 3:1, TFQ only interacted synergistically with naphthoquine, pyronaridine and piperaquine in gametocytes. This study indicated that TFQ and ACT-partner drugs will likely have increased potency against asexual stages of the malaria parasites, whereas some drugs may interfere with each other against the P. falciparum gametocytes. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Adaptive multigrid domain decomposition solutions for viscous interacting flows

    NASA Technical Reports Server (NTRS)

    Rubin, Stanley G.; Srinivasan, Kumar

    1992-01-01

    Several viscous incompressible flows with strong pressure interaction and/or axial flow reversal are considered with an adaptive multigrid domain decomposition procedure. Specific examples include the triple deck structure surrounding the trailing edge of a flat plate, the flow recirculation in a trough geometry, and the flow in a rearward facing step channel. For the latter case, there are multiple recirculation zones, of different character, for laminar and turbulent flow conditions. A pressure-based form of flux-vector splitting is applied to the Navier-Stokes equations, which are represented by an implicit lowest-order reduced Navier-Stokes (RNS) system and a purely diffusive, higher-order, deferred-corrector. A trapezoidal or box-like form of discretization insures that all mass conservation properties are satisfied at interfacial and outflow boundaries, even for this primitive-variable, non-staggered grid computation.

  2. Structure determination of the functional domain interaction of a chimeric nonribosomal peptide synthetase from a challenging crystal with noncrystallographic translational symmetry

    SciTech Connect

    Sundlov, Jesse A.; Gulick, Andrew M.

    2013-08-01

    The structure of the functional interaction of NRPS adenylation and carrier protein domains, trapped with a mechanism-based inhibitor, is described. Crystals exhibit translational non-crystallographic symmetry, which challenged structure determination and refinement. The nonribosomal peptide synthetases (NRPSs) are a family of modular proteins that contain multiple catalytic domains joined in a single protein. Together, these domains work to produce chemically diverse peptides, including compounds with antibiotic activity or that play a role in iron acquisition. Understanding the structural mechanisms that govern the domain interactions has been a long-standing goal. During NRPS synthesis, amino-acid substrates are loaded onto integrated carrier protein domains through the activity of NRPS adenylation domains. The structures of two adenylation domain–carrier protein domain complexes have recently been determined in an effort that required the use of a mechanism-based inhibitor to trap the domain interaction. Here, the continued analysis of these proteins is presented, including a higher resolution structure of an engineered di-domain protein containing the EntE adenylation domain fused with the carrier protein domain of its partner EntB. The protein crystallized in a novel space group in which molecular replacement and refinement were challenged by noncrystallographic pseudo-translational symmetry. The structure determination and how the molecular packing impacted the diffraction intensities are reported. Importantly, the structure illustrates that in this new crystal form the functional interface between the adenylation domain and the carrier protein domain remains the same as that observed previously. At a resolution that allows inclusion of water molecules, additional interactions are observed between the two protein domains and between the protein and its ligands. In particular, a highly solvated region that surrounds the carrier protein cofactor is described.

  3. Structural organization and interactions of transmembrane domains in tetraspanin proteins

    PubMed Central

    Kovalenko, Oleg V; Metcalf, Douglas G; DeGrado, William F; Hemler, Martin E

    2005-01-01

    Background Proteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N- and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed. Results Among 28 human tetraspanin proteins, the TM1-3 sequences display a distinct heptad repeat motif (abcdefg)n. In TM1, position a is occupied by structurally conserved bulky residues and position d contains highly conserved Asn and Gly residues. In TM2, position a is occupied by conserved small residues (Gly/Ala/Thr), and position d has a conserved Gly and two bulky aliphatic residues. In TM3, three a positions of the heptad repeat are filled by two leucines and a glutamate/glutamine residue, and two d positions are occupied by either Phe/Tyr or Val/Ile/Leu residues. No heptad motif is apparent in TM4 sequences. Mutations of conserved glycines in human CD9 (Gly25 and Gly32 in TM1; Gly67 and Gly74 in TM2) caused aggregation of mutant proteins inside the cell. Modeling of the TM1-TM2 interface in CD9, using a novel algorithm, predicts tight packing of conserved bulky residues against conserved Gly residues along the two helices. The homodimeric interface of CD9 was mapped, by disulfide cross-linking of single-cysteine mutants, to the vicinity of residues Leu14 and Phe17 in TM1 (positions g and c) and Gly77, Gly80 and Ala81 in TM2 (positions d, g and a, respectively). Mutations of a and d residues in both TM1 and TM2 (Gly25, Gly32, Gly67 and Gly74), involved in intramolecular TM1-TM2 interaction, also strongly diminished intermolecular interaction, as assessed by cross-linking of Cys80. Conclusion Our results suggest that tetraspanin intra- and intermolecular interactions are mediated by conserved residues in adjacent, but distinct regions of TM1 and TM2. A key structural element that

  4. Lens Epithelium-derived Growth Factor/p75 Interacts with the Transposase-derived DDE Domain of PogZ*S⃞

    PubMed Central

    Bartholomeeusen, Koen; Christ, Frauke; Hendrix, Jelle; Rain, Jean-Christophe; Emiliani, Stéphane; Benarous, Richard; Debyser, Zeger; Gijsbers, Rik; De Rijck, Jan

    2009-01-01

    Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a prominent cellular interaction partner of human immunodeficiency virus-1 (HIV-1) integrase, tethering the preintegration complex to the host chromosome. In light of the development of LEDGF/p75-integrase interaction inhibitors, it is essential to understand the cell biology of LEDGF/p75. We identified pogZ as new cellular interaction partner of LEDGF/p75. Analogous to lentiviral integrase, pogZ, a domesticated transposase, carries a DDE domain, the major determinant for LEDGF/p75 interaction. Using different in vitro and in vivo approaches, we corroborated the interaction between the C terminus of LEDGF/p75 and the DDE domain of pogZ, revealing an overlap in the binding of pogZ and HIV-1 integrase. Competition experiments showed that integrase is efficient in displacing pogZ from LEDGF/p75. Moreover, pogZ does not seem to play a role as a restriction factor of HIV. The finding that LEDGF/p75 is capable of interacting with a DDE domain protein that is not a lentiviral integrase points to a profound role of LEDGF/p75 in DDE domain protein function. PMID:19244240

  5. Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats

    PubMed Central

    Pelassa, Ilaria; Fiumara, Ferdinando

    2015-01-01

    Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as “junk” sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in

  6. Human Peroxiredoxins 1 and 2 and Their Interacting Protein Partners; Through Structure Toward Functions of Biological Complexes.

    PubMed

    Bertoldi, Mariarita

    2016-01-01

    Since their discovery in the mid-nineties, peroxiredoxins have drawn much attention and the number of papers publications on different Prxs has been multiplied. The rise in interest in this topic is probably due, at least in part, to the large and further increasing functions attributed to the members of this family of ubiquitous proteins, including many redox and non-redox physiological functions. This review presents a Since their discovery in the mid-nineties, peroxiredoxins have drawn much attention and the number of publications on different Prxs has been multiplied. The rise in interest in this topic is probably due, at least in part, to the large and further increasing functions attributed to the members of this family of ubiquitous proteins, including many redox and non-redox physiological functions. This review presents a literature survey of the protein partners of the human Peroxiredoxin-1 and Peroxiredoxin- 2 of the Peroxiredoxin 1 subfamily, the most abundant class. Three sequence motifs, or combinations thereof, were found in the protein partners, namely, CXXC, PXXP, and LXXLL. These findings are discussed in light of i) protein partner localization, function and biological pathways and ii) the peroxiredoxins regions important for partner interaction, as revealed by the Peroxiredoxin-1-Sulfiredoxin-1 complex structure. The outcome of these analyses is expected to unravel some common molecular bases underlying peroxiredoxins propensity to bind a partner, as well as to propose a functional role for this interaction that could help to widen the biological role of this important class of enzymes.

  7. Screening for the interacting partners of the proteins MamK & MamJ by two-hybrid genomic DNA library of Magnetospirillum magneticum AMB-1.

    PubMed

    Pan, Weidong; Xie, Chunlan; Lv, Jing

    2012-06-01

    Magnetotactic bacteria are a group of prokaryotes capable of sensing and navigating along the earth's magnetic field. The linear alignment of magnetosomes, which acts as a compass needle for orientation, is dependent on the proteins MamJ (amb0964) & MamK (amb0965). We constructed Magnetospirillum magneticum AMB-1 two-hybrid DNA libraries by fusing the random genomic fragments of AMB-1 to the N-terminal domain of the α-subunit of RNA polymerase in vector pTRG and used as preys. The genes mamJ & mamK were cloned in frame with the λ repressor protein (λ cI) in vector pBT and used as baits for screening the binding partners. After preliminary screening, we further confirmed the candidate interactions between selected protein pairs. The results showed that there were relatively strong interactions between MamK versus Amb3498 (flagella motor switch protein fliM), versus Amb0854 MCPs (signal domain of methyl-accepting chemotaxis protein) and versus Amb3568 (GGDEF domain-containing protein), respectively. MamJ versus Amb1722 (hypothetical protein), MamJ versus MamK, and MamK versus Amb1807 (cation transport ATPase) exhibited low level of interaction. Although the TPR repeat protein MamA (amb0971) showed no interaction with either MamJ or MamK, the TPR repeat protein Amb0024 with more motif sequences exhibited relatively strong interaction with MamK. Among the identified proteins, all categorized as signal transduction-related displayed interaction only with MamK and without MamJ, suggesting that magnetotaxis via MamK in Magnetospirillum magneticum AMB-1 might be somehow concerned with the widely accepted chemotaxis mechanism in bacteria.

  8. Structure and functional interactions of the Tsg101 UEV domain

    PubMed Central

    Pornillos, Owen; Alam, Steven L.; Rich, Rebecca L.; Myszka, David G.; Davis, Darrell R.; Sundquist, Wesley I.

    2002-01-01

    Human Tsg101 plays key roles in HIV budding and in cellular vacuolar protein sorting (VPS). In performing these functions, Tsg101 binds both ubiquitin (Ub) and the PTAP tetrapeptide ‘late domain’ motif located within the viral Gag protein. These interactions are mediated by the N-terminal domain of Tsg101, which belongs to the catalytically inactive ubiquitin E2 variant (UEV) family. We now report the struc ture of Tsg101 UEV and chemical shift mapping of the Ub and PTAP binding sites. Tsg101 UEV resembles canonical E2 ubiquitin conjugating enzymes, but has an additional N-terminal helix, an extended β-hairpin that links strands 1 and 2, and lacks the two C-terminal helices normally found in E2 enzymes. PTAP-containing peptides bind in a hydrophobic cleft exposed by the absence of the C-terminal helices, whereas ubiquitin binds in a novel site surrounding the β-hairpin. These studies provide a structural framework for understanding how Tsg101 mediates the protein–protein interactions required for HIV budding and VPS. PMID:12006492

  9. Small molecule mimetics of an interferon-α receptor interacting domain.

    PubMed

    Bello, Angelica M; Wei, Lianhu; Majchrzak-Kita, Beata; Salum, Noruê; Purohit, Meena K; Fish, Eleanor N; Kotra, Lakshmi P

    2014-02-01

    Small molecules that mimic IFN-α epitopes that interact with the cell surface receptor, IFNAR, would be useful therapeutics. One such 8-amino acid region in IFN-α2, designated IRRP-1, was used to derive 11 chemical compounds that belong to 5 distinct chemotypes, containing the molecular features represented by the key residues Leu30, Arg33, and Asp35 in IRRP-1. Three of these compounds exhibited potential mimicry to IRRP-1 and, in cell based assays, as predicted, effectively inhibited IFNAR activation by IFN-α. Of these, compound 3 did not display cell toxicity and reduced IFN-α-inducible STAT1 phosphorylation and STAT-DNA binding. Based on physicochemical properties' analyses, our data suggest that moieties with acidic pKa on the small molecule may be a necessary element for mimicking the carboxyl group of Asp35 in IRRP-1. Our data confirm the relevance of this strategy of molecular mimicry of ligand-receptor interaction domains of protein partners for small molecule drug discovery.

  10. Brief report: expressive and collaborative relationship processes in observations of adolescents' interactions with parents and romantic partners.

    PubMed

    Madsen, Stephanie D; Andrew Collins, W

    2008-12-01

    This study examines observations of participants (N=64) interacting with family members at age 13 and romantic partners at age 20-21. Scales capturing expressive processes and collaborative processes were used to test and find support for the differential prediction that family collaborative processes at age 13 would predict both expressive processes and collaborative processes with romantic partners at age 20-21, whereas expressive processes at age 13 would predict only concurrent collaborative processes. Results suggest that continuity in relationship functioning is more differentiated than is commonly expected.

  11. Subtype-specific roles of phospholipase C-β via differential interactions with PDZ domain proteins.

    PubMed

    Kim, Jung Kuk; Lim, Seyoung; Kim, Jinho; Kim, Sanguk; Kim, Jae Ho; Ryu, Sung Ho; Suh, Pann-Ghill

    2011-01-01

    Since we first identified the PLC-β isozyme, enormous studies have been conducted to investigate the functional roles of this protein (Min et al., 1993; Suh et al.,1988). It is now well-known that the four PLC-β subtypes are major effector molecules in GPCR-mediated signaling, especially for intracellular Ca2+ signaling. Nonetheless, it is still poorly understood why multiple PLC-β subtype exist. Most cells express multiple subtypes of PLC-β in different combinations, and each subtype is involved in somewhat different signaling pathways. Therefore, studying the differential roles of each PLC-β subtype is a very interesting issue. In this regard, we focus here on PDZ domain proteins which are novel PLC-β interacting proteins. As scaffolders, PDZ domain proteins recruit various target proteins ranging from membrane receptors to cytoskeletal proteins to assemble highly organized signaling complexes; this can give rise to efficiency and diversity in cellular signaling. Because PLC-β subtypes have different PDZ-binding motifs, it is possible that they are engaged with different PDZ domain proteins, and in turn participate in distinct physiological responses. To date, several PDZ domain proteins, such as the NHERF family, Shank2, and Par-3, have been reported to selectively interact with certain PLC-β subtypes and GPCRs. Systematic predictions of potential binding partners also suggests differential binding properties between PLC-β subtypes. Furthermore, we elucidated parallel signaling processes for multiple PLC-β subtypes, which still perform distinct functions resulting from differential interactions with PDZ domain proteins within a single cell. Therefore, these results highlight the novel function of PDZ domain proteins as intermediaries in subtype-specific role of PLC-β in GPCR-mediated signaling. Future studies will focus on the physiological meanings of this signaling complex formation by different PDZ domain proteins and PLC-β subtypes. It has been

  12. 3did: a catalog of domain-based interactions of known three-dimensional structure.

    PubMed

    Mosca, Roberto; Céol, Arnaud; Stein, Amelie; Olivella, Roger; Aloy, Patrick

    2014-01-01

    The database of 3D interacting domains (3did, available online for browsing and bulk download at http://3did.irbbarcelona.org) is a catalog of protein-protein interactions for which a high-resolution 3D structure is known. 3did collects and classifies all structural templates of domain-domain interactions in the Protein Data Bank, providing molecular details for such interactions. The current version also includes a pipeline for the discovery and annotation of novel domain-motif interactions. For every interaction, 3did identifies and groups different binding modes by clustering similar interfaces into 'interaction topologies'. By maintaining a constantly updated collection of domain-based structural interaction templates, 3did is a reference source of information for the structural characterization of protein interaction networks. 3did is updated every 6 months.

  13. Resolution of Disagreements between Romantic Partners, among Adolescents, and Young Adults: Qualitative Analysis of Interaction Discourses

    ERIC Educational Resources Information Center

    Tuval-Mashiach, Rivka; Shulman, Shmuel

    2006-01-01

    The study was designed to explore qualitatively developmental differences in disagreement negotiation and resolution skills between adolescent and young adult romantic partners. Twenty adolescent and 20 young adult couples participated in the study. The Knox inventory was used to measure the level of disagreement between partners on ten domains…

  14. Resolution of Disagreements between Romantic Partners, among Adolescents, and Young Adults: Qualitative Analysis of Interaction Discourses

    ERIC Educational Resources Information Center

    Tuval-Mashiach, Rivka; Shulman, Shmuel

    2006-01-01

    The study was designed to explore qualitatively developmental differences in disagreement negotiation and resolution skills between adolescent and young adult romantic partners. Twenty adolescent and 20 young adult couples participated in the study. The Knox inventory was used to measure the level of disagreement between partners on ten domains…

  15. Proteins and Their Interacting Partners: An Introduction to Protein–Ligand Binding Site Prediction Methods

    PubMed Central

    Roche, Daniel Barry; Brackenridge, Danielle Allison; McGuffin, Liam James

    2015-01-01

    Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein–ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein–ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein–ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated Model EvaluatiOn (CAMEO) projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems. PMID:26694353

  16. A four-partner plant–virus interaction: enemies can also come from within.

    PubMed

    Iskra-Caruana, Marie-Line; Baurens, Franc-Christophe; Gayral, Philippe; Chabannes, Matthieu

    2010-11-01

    Plant viruses are disseminated by either vertical (vegetative multiplication or sexual reproduction) or horizontal (vector-mediated) propagation. Plant pararetroviruses—members of the Caulimoviridae family—have developed an alternative strategy for vertical propagation via integration within the host plant genome, although integration is not required for viral replication. Integrated endogenous pararetrovirus (EPRV) sequences have undergone extensive viral genome rearrangements and contain more than one copy of the viral genome. Furthermore, EPRV can become infectious upon spontaneous escape of active virus following stresses such as wounding, tissue culture, or interspecific crosses. Such infectious EPRV are of great importance, not only in terms of their ability to precipitate epidemic outbreaks but also because of their effect on breeding of numerous plant genomes in temperate and tropical crops. This is especially true for banana, a crop susceptible to banana streak viruses, the causative agents of banana streak disease. Thus, the classical three-component banana–Banana streak virus (BSV)–mealybug pathosystem can be expanded to include endogenous BSV as an alternative source of active virions. The BSV-banana pathosystem is one of only three pathosystems known to date to harbor this remarkable feature, and the present review focuses exclusively on it to illustrate this four-partner interaction.

  17. Diminished degradation of yeast cytochrome c by interactions with its physiological partners.

    PubMed Central

    Pearce, D A; Sherman, F

    1995-01-01

    The level and structure of yeast iso-1-cytochrome c and iso-2-cytochrome c, encoded by the nuclear genes CYC1 and CYC7, respectively, are normally not altered in rho- mutants, which completely lack the cytochromes a.a3 subunits and cytochrome b that are encoded by mitochondrial DNA. In contrast, iso-cytochromes c containing the amino acid change Thr-78-->Ile (T78I) were observed at the normal or near-normal wild-type level in rho+ strains but were completely absent in rho- mutants. We have demonstrated with the "global" suppressor mutation Asn-52-->Ile and by pulse-chase labeling that the T78I iso-1-cytochrome c undergoes rapid cellular degradation in rho- mutants. Furthermore, specific mutations revealed that the deficiency of T78I iso-1 cytochrome c can be caused by the lack of cytochrome a.a3 or cytochrome c1, but not by the lack of cytochrome b. Thus, this and certain other, but not all, labile forms of cytochrome c are protected from degradation by the interaction with its physiological partners. Images Fig. 2 Fig. 3 PMID:7731975

  18. Proteins and Their Interacting Partners: An Introduction to Protein-Ligand Binding Site Prediction Methods.

    PubMed

    Roche, Daniel Barry; Brackenridge, Danielle Allison; McGuffin, Liam James

    2015-12-15

    Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein-ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein-ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein-ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated Model EvaluatiOn (CAMEO) projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems.

  19. AIDA: ab initio domain assembly for automated multi-domain protein structure prediction and domain–domain interaction prediction

    PubMed Central

    Xu, Dong; Jaroszewski, Lukasz; Li, Zhanwen; Godzik, Adam

    2015-01-01

    Motivation: Most proteins consist of multiple domains, independent structural and evolutionary units that are often reshuffled in genomic rearrangements to form new protein architectures. Template-based modeling methods can often detect homologous templates for individual domains, but templates that could be used to model the entire query protein are often not available. Results: We have developed a fast docking algorithm ab initio domain assembly (AIDA) for assembling multi-domain protein structures, guided by the ab initio folding potential. This approach can be extended to discontinuous domains (i.e. domains with ‘inserted’ domains). When tested on experimentally solved structures of multi-domain proteins, the relative domain positions were accurately found among top 5000 models in 86% of cases. AIDA server can use domain assignments provided by the user or predict them from the provided sequence. The latter approach is particularly useful for automated protein structure prediction servers. The blind test consisting of 95 CASP10 targets shows that domain boundaries could be successfully determined for 97% of targets. Availability and implementation: The AIDA package as well as the benchmark sets used here are available for download at http://ffas.burnham.org/AIDA/. Contact: adam@sanfordburnham.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25701568

  20. Intrapolypeptide interactions between the GTPase effector domain (GED) and the GTPase domain form the bundle signaling element in dynamin dimers.

    PubMed

    Srinivasan, Saipraveen; Mattila, Juha-Pekka; Schmid, Sandra L

    2014-09-16

    Biochemical and structural studies of dynamin have shown that the C-terminus of the GTPase effector domain (GED) folds back and docks onto a platform created by the N- and C-terminal α-helices of the GTPase domain to form a three-helix bundle. While cross-linking studies suggested that insect cell-expressed dynamin existed as a domain-swapped dimer, X-ray structures of protein expressed in Escherichia coli failed to detect evidence of this domain swap. Here, by cross-linking several cysteine pair replacements and analyzing cross-linked species by matrix-assisted laser desorption ionization Mega time of flight, we conclude that dynamin is not domain-swapped and that GED-GTPase domain interactions occur in cis.

  1. Intrapolypeptide Interactions between the GTPase Effector Domain (GED) and the GTPase Domain Form the Bundle Signaling Element in Dynamin Dimers

    PubMed Central

    2015-01-01

    Biochemical and structural studies of dynamin have shown that the C-terminus of the GTPase effector domain (GED) folds back and docks onto a platform created by the N- and C-terminal α-helices of the GTPase domain to form a three-helix bundle. While cross-linking studies suggested that insect cell-expressed dynamin existed as a domain-swapped dimer, X-ray structures of protein expressed in Escherichia coli failed to detect evidence of this domain swap. Here, by cross-linking several cysteine pair replacements and analyzing cross-linked species by matrix-assisted laser desorption ionization Mega time of flight, we conclude that dynamin is not domain-swapped and that GED–GTPase domain interactions occur in cis. PMID:25171143

  2. Monitoring Protein-Protein Interactions between the Mammalian Integral Membrane Transporters and PDZ-interacting Partners Using a Modified Split-ubiquitin Membrane Yeast Two-hybrid System*S⃞

    PubMed Central

    Gisler, Serge M.; Kittanakom, Saranya; Fuster, Daniel; Wong, Victoria; Bertic, Mia; Radanovic, Tamara; Hall, Randy A.; Murer, Heini; Biber, Jürg; Markovich, Daniel; Moe, Orson W.; Stagljar, Igor

    2008-01-01

    PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this “MYTH 2.0” system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes. PMID:18407958

  3. SUMOylation of GTF2IRD1 Regulates Protein Partner Interactions and Ubiquitin-Mediated Degradation

    PubMed Central

    Widagdo, Jocelyn; Taylor, Kylie M.; Gunning, Peter W.; Hardeman, Edna C.; Palmer, Stephen J.

    2012-01-01

    GTF2IRD1 is one of the genes implicated in Williams-Beuren syndrome, a disease caused by haploinsufficiency of certain dosage-sensitive genes within a hemizygous microdeletion of chromosome 7. GTF2IRD1 is a prime candidate for some of the major features of the disease, presumably caused by abnormally reduced abundance of this putative transcriptional repressor protein. GTF2IRD1 has been shown to interact with the E3 SUMO ligase PIASxβ, but the significance of this relationship is largely unexplored. Here, we demonstrate that GTF2IRD1 can be SUMOylated by the SUMO E2 ligase UBC9 and the level of SUMOylation is enhanced by PIASxβ. A major SUMOylation site was mapped to lysine 495 within a conserved SUMO consensus motif. SUMOylation of GTF2IRD1 alters the affinity of the protein for binding partners that contain SUMO-interacting motifs, including a novel family member of the HDAC repressor complex, ZMYM5, and PIASxβ itself. In addition, we show that GTF2IRD1 is targeted for ubiquitination and proteasomal degradation. Cross regulation by SUMOylation modulates this process, thus potentially regulating the level of GTF2IRD1 protein in the cell. These findings, concerning post-translational control over the activity and stability of GTF2IRD1, together with previous work showing how GTF2IRD1 directly regulates its own transcription levels suggest an evolutionary requirement for fine control over GTF2IRD1 activity in the cell. PMID:23145142

  4. SUMOylation of GTF2IRD1 regulates protein partner interactions and ubiquitin-mediated degradation.

    PubMed

    Widagdo, Jocelyn; Taylor, Kylie M; Gunning, Peter W; Hardeman, Edna C; Palmer, Stephen J

    2012-01-01

    GTF2IRD1 is one of the genes implicated in Williams-Beuren syndrome, a disease caused by haploinsufficiency of certain dosage-sensitive genes within a hemizygous microdeletion of chromosome 7. GTF2IRD1 is a prime candidate for some of the major features of the disease, presumably caused by abnormally reduced abundance of this putative transcriptional repressor protein. GTF2IRD1 has been shown to interact with the E3 SUMO ligase PIASxβ, but the significance of this relationship is largely unexplored. Here, we demonstrate that GTF2IRD1 can be SUMOylated by the SUMO E2 ligase UBC9 and the level of SUMOylation is enhanced by PIASxβ. A major SUMOylation site was mapped to lysine 495 within a conserved SUMO consensus motif. SUMOylation of GTF2IRD1 alters the affinity of the protein for binding partners that contain SUMO-interacting motifs, including a novel family member of the HDAC repressor complex, ZMYM5, and PIASxβ itself. In addition, we show that GTF2IRD1 is targeted for ubiquitination and proteasomal degradation. Cross regulation by SUMOylation modulates this process, thus potentially regulating the level of GTF2IRD1 protein in the cell. These findings, concerning post-translational control over the activity and stability of GTF2IRD1, together with previous work showing how GTF2IRD1 directly regulates its own transcription levels suggest an evolutionary requirement for fine control over GTF2IRD1 activity in the cell.

  5. Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer.

    PubMed

    App, Christine; Knop, Jana; Huff, Thomas; Seebahn, Angela; Becker, Cord-Michael; Iavarone, Federica; Castagnola, Massimo; Hannappel, Ewald

    2014-07-01

    A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin β4 can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI-MS (electrospray ionization-mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin β4 did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin β4 as well as of other proteins and peptides.

  6. Interaction between the biotin carboxyl carrier domain and the biotin carboxylase domain in pyruvate carboxylase from Rhizobium etli.

    PubMed

    Lietzan, Adam D; Menefee, Ann L; Zeczycki, Tonya N; Kumar, Sudhanshu; Attwood, Paul V; Wallace, John C; Cleland, W Wallace; St Maurice, Martin

    2011-11-15

    Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family.

  7. Interaction Between the Biotin Carboxyl Carrier Domain and the Biotin Carboxylase Domain in Pyruvate Carboxylase from Rhizobium etli†

    PubMed Central

    Lietzan, Adam D.; Menefee, Ann L.; Zeczycki, Tonya N.; Kumar, Sudhanshu; Attwood, Paul V.; Wallace, John C.; Cleland, W. Wallace; Maurice, Martin St.

    2011-01-01

    Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family. PMID:21958016

  8. Tight coevolution of proliferating cell nuclear antigen (PCNA)-partner interaction networks in fungi leads to interspecies network incompatibility.

    PubMed

    Zamir, Lyad; Zaretsky, Marianna; Fridman, Yearit; Ner-Gaon, Hadas; Rubin, Eitan; Aharoni, Amir

    2012-02-14

    The structure and connectivity of protein-protein interaction (PPI) networks are maintained throughout evolution by coordinated changes (coevolution) of network proteins. Despite extensive research, relatively little is known regarding the molecular basis and functional implications of the coevolution of PPI networks. Here, we used proliferating cell nuclear antigen, a hub protein that mediates DNA replication and repair in eukaryotes, as a model system to study the coevolution of PPI networks in fungi. Using a combined bioinformatics and experimental approach, we discovered that PCNA-partner interactions tightly coevolved in fungal species, leading to specific modes of recognition. We found that fungal proliferating cell nuclear antigen-partner interaction networks diverged into two distinct groups as a result of such coevolution and that hybrid networks of these groups are functionally noncompatible in Saccharomyces cerevisiae. Our results indicate that the coevolution of PPI networks can form functional barriers between fungal species, and thus can promote and fix speciation.

  9. Experimental assessment of the contribution of electrodynamic interactions to long-distance recruitment of biomolecular partners: Theoretical basis

    NASA Astrophysics Data System (ADS)

    Preto, Jordane; Floriani, Elena; Nardecchia, Ilaria; Ferrier, Pierre; Pettini, Marco

    2012-04-01

    Highly specific spatiotemporal interactions between cognate molecular partners essentially sustain all biochemical transactions in living matter. That such an exquisite level of accuracy may result from encountering forces solely driven by thermal diffusive processes is unlikely. Here we propose a yet unexplored strategy to experimentally tackle the long-standing question of a possibly active recruitment at a distance of cognate partners of biomolecular reactions via the action of resonant electrodynamic interactions. We considered two simplified models for a preliminary feasibility investigation of the devised methodology. By taking advantage of advanced experimental techniques nowadays available, we propose to measure the characteristic encounter time scales of dually interacting biopartners and to compare them with theoretical predictions worked out in both the presence and absence of putative long-range electromagnetic forces.

  10. Strategic Sexual Signals: Women's Display versus Avoidance of the Color Red Depends on the Attractiveness of an Anticipated Interaction Partner.

    PubMed

    Niesta Kayser, Daniela; Agthe, Maria; Maner, Jon K

    2016-01-01

    The color red has special meaning in mating-relevant contexts. Wearing red can enhance perceptions of women's attractiveness and desirability as a potential romantic partner. Building on recent findings, the present study examined whether women's (N = 74) choice to display the color red is influenced by the attractiveness of an expected opposite-sex interaction partner. Results indicated that female participants who expected to interact with an attractive man displayed red (on clothing, accessories, and/or makeup) more often than a baseline consisting of women in a natural environment with no induced expectation. In contrast, when women expected to interact with an unattractive man, they eschewed red, displaying it less often than in the baseline condition. Findings are discussed with respect to evolutionary and cultural perspectives on mate evaluation and selection.

  11. Strategic Sexual Signals: Women's Display versus Avoidance of the Color Red Depends on the Attractiveness of an Anticipated Interaction Partner

    PubMed Central

    Maner, Jon K.

    2016-01-01

    The color red has special meaning in mating-relevant contexts. Wearing red can enhance perceptions of women’s attractiveness and desirability as a potential romantic partner. Building on recent findings, the present study examined whether women’s (N = 74) choice to display the color red is influenced by the attractiveness of an expected opposite-sex interaction partner. Results indicated that female participants who expected to interact with an attractive man displayed red (on clothing, accessories, and/or makeup) more often than a baseline consisting of women in a natural environment with no induced expectation. In contrast, when women expected to interact with an unattractive man, they eschewed red, displaying it less often than in the baseline condition. Findings are discussed with respect to evolutionary and cultural perspectives on mate evaluation and selection. PMID:26960135

  12. EHD4 and CDH23 are interacting partners in cochlear hair cells.

    PubMed

    Sengupta, Soma; George, Manju; Miller, Katharine K; Naik, Khurram; Chou, Jonathan; Cheatham, Mary Ann; Dallos, Peter; Naramura, Mayumi; Band, Hamid; Zheng, Jing

    2009-07-24

    Cadherin 23 (CDH23), a transmembrane protein localized near the tips of hair cell stereocilia in the mammalian inner ear, is important for delivering mechanical signals to the mechano-electric transducer channels. To identify CDH23-interacting proteins, a membrane-based yeast two-hybrid screen of an outer hair cell (OHC) cDNA library was performed. EHD4, a member of the C-terminal EH domain containing a protein family involved in endocytic recycling, was identified as a potential interactor. To confirm the interaction, we first demonstrated the EHD4 mRNA expression in hair cells using in situ hybridization. Next, we showed that EHD4 co-localizes and co-immunoprecipitates with CDH23 in mammalian cells. Interestingly, the co-immunoprecipitation was found to be calcium-sensitive. To investigate the role of EHD4 in hearing, compound action potentials were measured in EHD4 knock-out (KO) mice. Although EHD4 KO mice have normal hearing sensitivity, analysis of mouse cochlear lysates revealed a 2-fold increase in EHD1, but no increase in EHD2 or EHD3, in EHD4 KO cochleae compared with wild type, suggesting that a compensatory increase in EHD1 levels may account for the absence of a hearing defect in EHD4 KO mice. Taken together, these data indicate that EHD4 is a novel CDH23-interacting protein that could regulate CDH23 trafficking/localization in a calcium-sensitive manner.

  13. Polycomb purification by in vivo biotinylation tagging reveals cohesin and Trithorax group proteins as interaction partners

    PubMed Central

    Strübbe, Gero; Popp, Christian; Schmidt, Alexander; Pauli, Andrea; Ringrose, Leonie; Beisel, Christian; Paro, Renato

    2011-01-01

    The maintenance of specific gene expression patterns during cellular proliferation is crucial for the identity of every cell type and the development of tissues in multicellular organisms. Such a cellular memory function is conveyed by the complex interplay of the Polycomb and Trithorax groups of proteins (PcG/TrxG). These proteins exert their function at the level of chromatin by establishing and maintaining repressed (PcG) and active (TrxG) chromatin domains. Past studies indicated that a core PcG protein complex is potentially associated with cell type or even cell stage-specific sets of accessory proteins. In order to better understand the dynamic aspects underlying PcG composition and function we have established an inducible version of the biotinylation tagging approach to purify Polycomb and associated factors from Drosophila embryos. This system enabled fast and efficient isolation of Polycomb containing complexes under near physiological conditions, thereby preserving substoichiometric interactions. Novel interacting proteins were identified by highly sensitive mass spectrometric analysis. We found many TrxG related proteins, suggesting a previously unrecognized extent of molecular interaction of the two counteracting chromatin regulatory protein groups. Furthermore, our analysis revealed an association of PcG protein complexes with the cohesin complex and showed that Polycomb-dependent silencing of a transgenic reporter depends on cohesin function. PMID:21415365

  14. Interacting vs. non-interacting single domain behavior in natural and synthetic samples

    NASA Technical Reports Server (NTRS)

    Cisowski, S.

    1981-01-01

    The disparity in response to high alternating field (AF) demagnetization for samples containing fine magnetic carriers is apparently related to the degree of interactions between those carriers. The presence of interaction fields between single domain (SD) grains can be tested by plotting isothermal remanence (IRM) acquisition vs. saturation remanence (SIRM) demagnetization. For the case of noninteracting SD grains, the two curves will be symmetrical. For the interacting SD case, the acquisition curve will be steepest at higher field, and the demagnetization curve steepest at lower fields, resulting in nonsymmetry. The point of intersection of the two curves approximates the remanent coercive force (H sub RC) field for all cases. Minor hysteresis loops and anhysteretic remanence (ARM) acquisition curves are also strongly influenced by interaction fields. Because of the difficulty in dispersing strongly magnetic grains, fine grained synthetic samples made with highly magnetic materials will not display equivalent AF stability to natural samples with fine, dispersed grains.

  15. Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation

    PubMed Central

    Zokouri, Zina; Hürlemann, Samuel; Gerrits, Bertran; Ausländer, David; Britschgi, Adrian; Tschan, Mario P.; Simon, Hans-Uwe; Fussenegger, Martin

    2015-01-01

    Death-associated protein kinase 2 (DAPK2) is a Ca2+/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions. PMID:26483415

  16. Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation.

    PubMed

    Geering, Barbara; Zokouri, Zina; Hürlemann, Samuel; Gerrits, Bertran; Ausländer, David; Britschgi, Adrian; Tschan, Mario P; Simon, Hans-Uwe; Fussenegger, Martin

    2016-01-01

    Death-associated protein kinase 2 (DAPK2) is a Ca(2+)/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions.

  17. Interaction of myosin VI and its binding partner DOCK7 plays an important role in NGF-stimulated protrusion formation in PC12 cells.

    PubMed

    Sobczak, Magdalena; Chumak, Vira; Pomorski, Paweł; Wojtera, Emilia; Majewski, Łukasz; Nowak, Jolanta; Yamauchi, Junji; Rędowicz, Maria Jolanta

    2016-07-01

    DOCK7 (dedicator of cytokinesis 7) is a guanidine nucleotide exchange factor (GEF) for Rac1 GTPase that is involved in neuronal polarity and axon generation as well in Schwann cell differentiation and myelination. Recently, we identified DOCK7 as the binding partner of unconventional myosin VI (MVI) in neuronal-lineage PC12 cells and postulated that this interaction could be important in vivo [Majewski et al. (2012) Biochem Cell Biol., 90:565-574]. Herein, we found that MVI-DOCK7 interaction takes also place in other cell lines and demonstrated that MVI cargo domain via its RRL motif binds to DOCK7 C-terminal M2 and DHR2 domains. In MVI knockdown cells, lower Rac1 activity and a decrease of DOCK7 phosphorylation on Tyr1118 were observed, indicating that MVI could contribute to DOCK7 activity. MVI and DOCK7 co-localization was maintained during NGF-stimulated PC12 cell differentiation and observed also in the outgrowths. Also, during differentiation an increase in phosphorylation of DOCK7 as well as of its downstream effector JNK kinase was detected. Interestingly, overexpression of GFP-tagged MVI cargo domain (GFP-GT) impaired protrusion formation indicating that full length protein is important for this process. Moreover, a transient increase in Rac activity observed at 5min of NGF-stimulated differentiation of PC12 cells (overexpressing either GFP or GFP-MVI) was not detected in cells overexpressing the cargo domain. These data indicate that MVI-DOCK7 interaction could have functional implications in the protrusion outgrowth, and full length MVI seems to be important for delivery and maintenance of DOCK7 along the protrusions, and exerting its GEF activity.

  18. Jmjd6, a JmjC Dioxygenase with Many Interaction Partners and Pleiotropic Functions

    PubMed Central

    Kwok, Janice; O’Shea, Marie; Hume, David A.; Lengeling, Andreas

    2017-01-01

    Lysyl hydroxylation and arginyl demethylation are post-translational events that are important for many cellular processes. The jumonji domain containing protein 6 (JMJD6) has been reported to catalyze both lysyl hydroxylation and arginyl demethylation on diverse protein substrates. It also interacts directly with RNA. This review summarizes knowledge of JMJD6 functions that have emerged in the last 15 years and considers how a single Jumonji C (JmjC) domain-containing enzyme can target so many different substrates. New links and synergies between the three main proposed functions of Jmjd6 in histone demethylation, promoter proximal pause release of polymerase II and RNA splicing are discussed. The physiological context of the described molecular functions is considered and recently described novel roles for JMJD6 in cancer and immune biology are reviewed. The increased knowledge of JMJD6 functions has wider implications for our general understanding of the JmjC protein family of which JMJD6 is a member. PMID:28360925

  19. Characterization of domain-peptide interaction interface: a case study on the amphiphysin-1 SH3 domain.

    PubMed

    Hou, Tingjun; Zhang, Wei; Case, David A; Wang, Wei

    2008-02-29

    Many important protein-protein interactions are mediated by peptide recognition modular domains, such as the Src homology 3 (SH3), SH2, PDZ, and WW domains. Characterizing the interaction interface of domain-peptide complexes and predicting binding specificity for modular domains are critical for deciphering protein-protein interaction networks. Here, we propose the use of an energetic decomposition analysis to characterize domain-peptide interactions and the molecular interaction energy components (MIECs), including van der Waals, electrostatic, and desolvation energy between residue pairs on the binding interface. We show a proof-of-concept study on the amphiphysin-1 SH3 domain interacting with its peptide ligands. The structures of the human amphiphysin-1 SH3 domain complexed with 884 peptides were first modeled using virtual mutagenesis and optimized by molecular mechanics (MM) minimization. Next, the MIECs between domain and peptide residues were computed using the MM/generalized Born decomposition analysis. We conducted two types of statistical analyses on the MIECs to demonstrate their usefulness for predicting binding affinities of peptides and for classifying peptides into binder and non-binder categories. First, combining partial least squares analysis and genetic algorithm, we fitted linear regression models between the MIECs and the peptide binding affinities on the training data set. These models were then used to predict binding affinities for peptides in the test data set; the predicted values have a correlation coefficient of 0.81 and an unsigned mean error of 0.39 compared with the experimentally measured ones. The partial least squares-genetic algorithm analysis on the MIECs revealed the critical interactions for the binding specificity of the amphiphysin-1 SH3 domain. Next, a support vector machine (SVM) was employed to build classification models based on the MIECs of peptides in the training set. A rigorous training-validation procedure was

  20. Quantifying protein–protein interactions in high throughput using protein domain microarrays

    PubMed Central

    Kaushansky, Alexis; Allen, John E; Gordus, Andrew; Stiffler, Michael A; Karp, Ethan S; Chang, Bryan H; MacBeath, Gavin

    2011-01-01

    Protein microarrays provide an efficient way to identify and quantify protein–protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain–peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (KDs) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein–ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein–protein interaction networks. PMID:20360771

  1. Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein

    SciTech Connect

    Kellner, Julian N.; Meinhart, Anton

    2015-08-25

    The structure of the SPRY domain of the human RNA helicase DDX1 was determined at 2.0 Å resolution. The SPRY domain provides a putative protein–protein interaction platform within DDX1 that differs from other SPRY domains in its structure and conserved regions. The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein–protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein–protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.

  2. Interaction domains of p62: a bridge between p62 and selective autophagy.

    PubMed

    Lin, Xiaolong; Li, Shuang; Zhao, Yue; Ma, Xiaofeng; Zhang, Kai; He, Xinglan; Wang, Zuo

    2013-05-01

    p62 is a multidomain protein that contains different kinds of protein-protein interaction domains, including an N-terminal PB1 domain, a ZZ-type zinc finger domain, a nuclear localization signal (NLS), an export motif (NES), the LC3-interacting region (LIR), the KEAP1-interacting region (KIR), and a C-terminal Ub-associated domain (UBA). p62 is involved in the degradation of protein aggregates and cytoplasmic bodies via selective autophagy through its PB1, LIR, and UBA domains to maintain homeostasis in the cell. Moreover, NES, NLS, KIR, and ZZ domains have been found to be linked to ubiquitinated protein degradation by autophagy. Therefore, understanding the functional domains of p62 is important. In this review, we attempt to expound the mechanism of connection between p62 and selective autophagy to illustrate how the domains of p62 regulate selective autophagy, and to provide a new direction and perspective on selective autophagy research.

  3. DIMA 2.0—predicted and known domain interactions

    PubMed Central

    Pagel, Philipp; Oesterheld, Matthias; Tovstukhina, Oksana; Strack, Norman; Stümpflen, Volker; Frishman, Dmitrij

    2008-01-01

    DIMA—the domain interaction map has evolved from a simple web server for domain phylogenetic profiling into an integrative prediction resource combining both experimental data on domain–domain interactions and predictions from two different algorithms. With this update, DIMA obtains greatly improved coverage at the level of genomes and domains as well as with respect to available prediction approaches. The domain phylogenetic profiling method now uses SIMAP as its backend for exhaustive domain hit coverage: 7038 Pfam domains were profiled over 460 completely sequenced genomes.Domain pair exclusion predictions were produced from 83 969 distinct protein–protein interactions obtained from IntAct resulting in 21 513 domain pairs with significant domain pair exclusion algorithm scores. Additional predictions applying the same algorithm to predicted protein interactions from STRING yielded 2378 high-confidence pairs. Experimental data comes from iPfam (3074) and 3did (3034 pairs), two databases identifying domain contacts in solved protein structures. Taken together, these two resources yielded 3653 distinct interacting domain pairs. DIMA is available at http://mips.gsf.de/genre/proj/dima. PMID:17999995

  4. A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions.

    PubMed

    Peterson, A J; Kyba, M; Bornemann, D; Morgan, K; Brock, H W; Simon, J

    1997-11-01

    The Sex comb on midleg (Scm) and polyhomeotic (ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors. PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies. The Scm and ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, that is located at their respective C termini. Using the yeast two-hybrid system and in vitro protein-binding assays, we show that the SPM domain mediates direct interaction between Scm and ph. Binding studies with isolated SPM domains from Scm and ph show that the domain is sufficient for these protein interactions. These studies also show that the Scm-ph and Scm-Scm domain interactions are much stronger than the ph-ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants. Analysis of site-directed point mutations identifies residues that are important for SPM domain function. These binding properties, predicted alpha-helical secondary structure, and conservation of hydrophobic residues prompt comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators. In addition to in vitro studies, we show colocalization of the Scm and ph proteins at polytene chromosome sites in vivo. We discuss the possible roles of the SPM domain in the assembly or function of molecular complexes of PcG proteins.

  5. Candidate Cell and Matrix Interaction Domains on the Collagen Fibril, the Predominant Protein of Vertebrates*S⃞

    PubMed Central

    Sweeney, Shawn M.; Orgel, Joseph P.; Fertala, Andrzej; McAuliffe, Jon D.; Turner, Kevin R.; Di Lullo, Gloria A.; Chen, Steven; Antipova, Olga; Perumal, Shiamalee; Ala-Kokko, Leena; Forlino, Antonella; Cabral, Wayne A.; Barnes, Aileen M.; Marini, Joan C.; Antonio, James D. San

    2008-01-01

    Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The “cell interaction domain” is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The “matrix interaction domain” may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging. PMID:18487200

  6. Novel Potential Interacting Partners of Fibronectin in Spontaneous Animal Model of Interstitial Cystitis

    PubMed Central

    Treutlein, Gudrun; Dorsch, Roswitha; Euler, Kerstin N.; Hauck, Stefanie M.; Amann, Barbara; Hartmann, Katrin; Deeg, Cornelia A.

    2012-01-01

    Feline idiopathic cystitis (FIC) is the only spontaneous animal model for human interstitial cystitis (IC), as both possess a distinctive chronical and relapsing character. Underlying pathomechanisms of both diseases are not clearly established yet. We recently detected increased urine fibronectin levels in FIC cases. The purpose of this study was to gain further insight into the pathogenesis by assessing interacting partners of fibronectin in urine of FIC affected cats. Several candidate proteins were identified via immunoprecipitation and mass spectrometry. Considerable changes in FIC conditions compared to physiological expression of co-purified proteins were detected by Western blot and immunohistochemistry. Compared to controls, complement C4a and thioredoxin were present in higher levels in urine of FIC patients whereas loss of signal intensity was detected in FIC affected tissue. Galectin-7 was exclusively detected in urine of FIC cats, pointing to an important role of this molecule in FIC pathogenesis. Moderate physiological signal intensity of galectin-7 in transitional epithelium shifted to distinct expression in transitional epithelium under pathophysiological conditions. I-FABP expression was reduced in urine and urinary bladder tissue of FIC cats. Additionally, transduction molecules of thioredoxin, NF-κB p65 and p38 MAPK, were examined. In FIC affected tissue, colocalization of thioredoxin and NF-κB p65 could be demonstrated compared to absent coexpression of thioredoxin and p38 MAPK. These considerable changes in expression level and pattern point to an important role for co-purified proteins of fibronectin and thioredoxin-regulated signal transduction pathways in FIC pathogenesis. These results could provide a promising starting point for novel therapeutic approaches in the future. PMID:23236492

  7. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    PubMed

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-10-23

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  8. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

    PubMed Central

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-01-01

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

  9. Multiple interactions of the intrinsically disordered region between the helicase and nuclease domains of the archaeal Hef protein.

    PubMed

    Ishino, Sonoko; Yamagami, Takeshi; Kitamura, Makoto; Kodera, Noriyuki; Mori, Tetsuya; Sugiyama, Shyogo; Ando, Toshio; Goda, Natsuko; Tenno, Takeshi; Hiroaki, Hidekazu; Ishino, Yoshizumi

    2014-08-01

    Hef is an archaeal protein that probably functions mainly in stalled replication fork repair. The presence of an unstructured region was predicted between the two distinct domains of the Hef protein. We analyzed the interdomain region of Thermococcus kodakarensis Hef and demonstrated its disordered structure by CD, NMR, and high speed atomic force microscopy (AFM). To investigate the functions of this intrinsically disordered region (IDR), we screened for proteins interacting with the IDR of Hef by a yeast two-hybrid method, and 10 candidate proteins were obtained. We found that PCNA1 and a RecJ-like protein specifically bind to the IDR in vitro. These results suggested that the Hef protein interacts with several different proteins that work together in the pathways downstream from stalled replication fork repair by converting the IDR structure depending on the partner protein.

  10. Institutional, individual, and socio-cultural domains of partnerships: a typology of USDA Forest Service recreation partners

    Treesearch

    Erin Seekamp; Lee K. Cerveny; Allie. McCreary

    2011-01-01

    Federal land management agencies, such as the USDA Forest Service, have expanded the role of recreation partners reflecting constrained growth in appropriations and broader societal trends towards civic environmental governance. Partnerships with individual volunteers, service groups, commercial outfitters, and other government agencies provide the USDA Forest Service...

  11. Signal Activation and Inactivation by the Gα Helical Domain: A Long-Neglected Partner in G Protein Signaling

    PubMed Central

    Dohlman, Henrik G.; Jones, Janice C.

    2013-01-01

    Heterotrimeric guanine nucleotide–binding proteins (G proteins) are positioned at the top of many signal transduction pathways. The G protein α subunit is composed of two domains, one that resembles Ras and another that is composed entirely of α helices. Historically, most attention has focused on the Ras-like domain, but emerging evidence reveals that the helical domain is an active participant in G protein signaling. PMID:22649098

  12. A variably spliced region in the type 1 ryanodine receptor may participate in an inter-domain interaction.

    PubMed

    Kimura, Takashi; Pace, Suzy M; Wei, Lan; Beard, Nicole A; Dirksen, Robert T; Dulhunty, Angela F

    2007-01-01

    The aim of the present study was to examine residues that are variably spliced in the juvenile and adult isoforms of the skeletal-muscle RyR1 (type 1 ryanodine receptor). The juvenile ASI(-) splice variant is less active than the adult ASI(+) variant and is overexpressed in patients with DM (myotonic dystrophy) [Kimura, Nakamori, Lueck, Pouliquin, Aoike, Fujimura, Dirksen, Takahashi, Dulhunty and Sakoda (2005) Hum. Mol. Genet. 14, 2189-2200]. In the present study, we explore the ASI region using synthetic peptides corresponding to rabbit RyR1 residues Thr3471-Gly3500 either containing [PASI(+)] or lacking [PASI(-)] the ASI residues. Both peptides increased [3H]ryanodine binding to rabbit RyR1s, increased Ca2+ release from sarcoplasmic reti-culum vesicles and increased single RyR1 channel activity. The peptide PASI(-) was more active in each case than PASI(+). [3H]Ryanodine binding to recombinant ASI(+)RyR1 or ASI(-)-RyR1 was enhanced more by PASI(-) than PASI(+), with the greatest increase seen when PASI(-) was added to ASI(-)RyR1. The activation of the RyR channels is consistent with the hypo-thesis that the peptides interrupt an inhibitory inter-domain inter-action and that PASI(-) is more effective at interrupting this interaction than PASI(+). We therefore suggest that the ASI(-) sequence interacts more tightly than the ASI(+) sequence with its binding partner, so that the ASI(-)RyR1 is more strongly inhibited (less active) than the ASI(+)RyR1. Thus the affinity of the binding partners in this inter-domain interaction may deter-mine the activities of the mature and juvenile isoforms of RyR1 and the stronger inhibition in the juvenile isoform may contribute to the myopathy in DM.

  13. Interaction of ultrasonic waves with domain walls on nanocrystalline YIG.

    PubMed

    Murthy, S R

    2014-02-01

    The nanocrystalline YIG samples with different particle sizes (20-40 nm) has been prepared using microwave-hydrothermal method. As synthesized powders were characterized using XRD and TEM. The powders were pressed and sintered at three different temperatures i.e., 700 °C/30 min, 800 °C/30 min, 900 °C/30 min, using microwave furnace. The sintered samples were characterized using XRD and TEM. The sintered samples are monophasic in nature with average grain size ranging in between 72 nm and 90 nm. The thermal variation of ultrasonic velocities [longitudinal (V(l)) and transverse (V(S))] and longitudinal attenuation (α(l)) has been measured on sintered samples by the pulse transmissionmethod at 1 MHz, in the temperature range of 300-600 K. The room temperature velocity is found to be grain size dependent and decreases with increasing temperature, except near the Curie temperature, T(C), where a small anomaly is observed. The longitudinal attenuation (α(1)) at room temperature is also found to be more sample dependent. The temperature variation of ultrasonic longitudinal attenuation exhibits a sharp maximum just below Curie temperature (T(C)). The above observations were carried on in the demagnetized state, on the application of a saturation field of 380 mT, the anomaly observed in the thermal variation of velocities (longitudinal and transverse) and attenuation is found to disappears. The observed interaction of ultrasonic velocity with domain walls has been qualitatively explained with the help oftemperature variation of magneto-crystalline anisotropy constant (k(1)) and Landau's theory.

  14. Structural basis of interactions between epidermal growth factor receptor and SH2 domain proteins.

    PubMed

    Sierke, S L; Longo, G M; Koland, J G

    1993-02-26

    The structural basis of the interactions between the activated epidermal growth factor (EGF) receptor and SH2 domain proteins was investigated. The c-src SH2 domain (second domain of src homology) was expressed as a recombinant fusion protein, and an in vitro assay was developed to monitor EGF receptor/SH2 domain interactions. EGF receptor tyrosine kinase domain (TKD) forms expressed in the baculovirus/insect cell system were shown to bind to the SH2 domain when phosphorylated. These TKD/SH2 domain interactions were characterized by dissociation constants of 60-320 nM. Deletion analysis indicated that the entire SH2 domain was required for recognition of the phosphorylated TKD. The binding of a highly truncated TKD protein to the SH2 domain suggested that the sites recognized by the SH2 domain included the EGF receptor autophosphorylation site, tyr992. A phosphorylated EGF receptor peptide containing tyr992 was also shown to interact with the SH2 domain. This residue may therefore mediate interactions between the EGF receptor and tyrosine kinases in the src family.

  15. Rewiring of PDZ Domain-Ligand Interaction Network Contributed to Eukaryotic Evolution

    PubMed Central

    Kim, Jinho; Kim, Inhae; Yang, Jae-Seong; Shin, Young-Eun; Hwang, Jihye; Park, Solip; Choi, Yoon Sup; Kim, Sanguk

    2012-01-01

    PDZ domain-mediated interactions have greatly expanded during metazoan evolution, becoming important for controlling signal flow via the assembly of multiple signaling components. The evolutionary history of PDZ domain-mediated interactions has never been explored at the molecular level. It is of great interest to understand how PDZ domain-ligand interactions emerged and how they become rewired during evolution. Here, we constructed the first human PDZ domain-ligand interaction network (PDZNet) together with binding motif sequences and interaction strengths of ligands. PDZNet includes 1,213 interactions between 97 human PDZ proteins and 591 ligands that connect most PDZ protein-mediated interactions (98%) in a large single network via shared ligands. We examined the rewiring of PDZ domain-ligand interactions throughout eukaryotic evolution by tracing changes in the C-terminal binding motif sequences of the PDZ ligands. We found that interaction rewiring by sequence mutation frequently occurred throughout evolution, largely contributing to the growth of PDZNet. The rewiring of PDZ domain-ligand interactions provided an effective means of functional innovations in nervous system development. Our findings provide empirical evidence for a network evolution model that highlights the rewiring of interactions as a mechanism for the development of new protein functions. PDZNet will be a valuable resource to further characterize the organization of the PDZ domain-mediated signaling proteome. PMID:22346764

  16. Rab3A is a new interacting partner of synaptotagmin I and may modulate synaptic membrane fusion through a competitive mechanism

    SciTech Connect

    Xie, Chunliang; Li, Jianglin; Guo, Tianyao; Yan, Yizhong; Tang, Cheng; Wang, Ying; Chen, Ping; Wang, Xianchun; Liang, Songping

    2014-02-21

    Highlights: • Rab3A has been found to be a novel interacting protein of synaptotagmin I. • Rab3A binds to synaptotagmin I in a Ca{sup 2+}-independent manner. • KKKK motif in C2B domain of synaptotagmin I is a key site for Rab3A binding. • Rab3A competitively inhibits the binding of C2B in synaptotagmin I to syntaxin 1B. • Rab3A may regulate synaptic membrane fusion and exocytosis in a competitive manner. - Abstract: Rab3 and synaptotagmin have been reported to be the key proteins that have opposite actions but cooperatively play critical regulatory roles in selecting and limiting the number of vesicles released at central synapses. However, the exact mechanism has not been fully understood. In this study, Rab3A and synaptotagmin I, the most abundant isoforms of Rab3 and synaptotagmin, respectively, in brain were for the first time demonstrated to directly interact with each other in a Ca{sup 2+}-independent manner, and the KKKK motif in the C2B domain of synaptotagmin I was a key site for the Rab3A binding, which was further confirmed by the competitive inhibition of inositol hexakisphosphate. Further studies demonstrated that Rab3A competitively affected the synaptotagmin I interaction with syntaxin 1B that was involved in membrane fusion during the synaptic vesicle exocytosis. These data indicate that Rab3A is a new synaptotagmin I interacting partner and may participate in the regulation of synaptic membrane fusion and thus the vesicle exocytosis by competitively modulating the interaction of synaptotagmin with syntaxin of the t-SNARE complex in presynaptic membranes.

  17. A frequent kinase domain mutation that changes the interaction between PI3K[alpha] and the membrane

    SciTech Connect

    Mandelker, Diana; Gabelli, Sandra B.; Schmidt-Kittler, Oleg; Zhu, Jiuxiang; Cheong, Ian; Huang, Chuan-Hsiang; Kinzler, Kenneth W.; Vogelstein, Bert; Amzel, L. Mario

    2009-12-01

    Mutations in oncogenes often promote tumorigenesis by changing the conformation of the encoded proteins, thereby altering enzymatic activity. The PIK3CA oncogene, which encodes p110{alpha}, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3K{alpha}), is one of the two most frequently mutated oncogenes in human cancers. We report the structure of the most common mutant of p110{alpha} in complex with two interacting domains of its regulatory partner (p85{alpha}), both free and bound to an inhibitor (wortmannin). The N-terminal SH2 (nSH2) domain of p85{alpha} is shown to form a scaffold for the entire enzyme complex, strategically positioned to communicate extrinsic signals from phosphopeptides to three distinct regions of p110{alpha}. Moreover, we found that Arg-1047 points toward the cell membrane, perpendicular to the orientation of His-1047 in the WT enzyme. Surprisingly, two loops of the kinase domain that contact the cell membrane shift conformation in the oncogenic mutant. Biochemical assays revealed that the enzymatic activity of the p110{alpha} His1047Arg mutant is differentially regulated by lipid membrane composition. These structural and biochemical data suggest a previously undescribed mechanism for mutational activation of a kinase that involves perturbation of its interaction with the cellular membrane.

  18. Asymmetric effect of domain interactions on the kinetics of folding in yeast phosphoglycerate kinase.

    PubMed

    Osváth, Szabolcs; Köhler, Gottfried; Závodszky, Péter; Fidy, Judit

    2005-06-01

    The aim of this work is to shed more light on the effect of domain-domain interactions on the kinetics and the pathway of protein folding. A model protein system consisting of several single-tryptophan variants of the two-domain yeast phosphoglycerate kinase (PGK) and its individual domains was studied. Refolding was initiated from the guanidine-unfolded state by stopped-flow or manual mixing and monitored by tryptophan fluorescence from 1 msec to 1000 sec. Denaturant titrations of both individual domains showed apparent two-state unfolding transitions. Refolding kinetics of the individual domains from different denaturant concentrations, however, revealed the presence of intermediate structures during titration for both domains. Refolding of the same domains within the complete protein showed that domain-domain interactions direct the folding of both domains, but in an asymmetric way. Folding of the N domain was already altered within 1 msec, while detectable changes in the folding of the C domain occurred only 60-100 msec after initiating refolding. All mutants showed a hyperfluorescent kinetic intermediate. Both the disappearance of this intermediate and the completion of the folding were significantly faster in the individual N domain than in the complete protein. On the contrary, folding of the individual C domain was slower than in the complete protein. The presence of the C domain directs the refolding of the N domain along a completely different pathway than that of the individual N domain, while folding of the individual C domain follows the same path as within the complete protein.

  19. Identification of a multifunctional docking site on the catalytic unit of phosphodiesterase-4 (PDE4) that is utilised by multiple interaction partners

    PubMed Central

    Houslay, Kirsty F.; Christian, Frank; MacLeod, Ruth; Adams, David R.; Houslay, Miles D.

    2017-01-01

    Cyclic AMP (cAMP)-specific phosphodiesterase-4 (PDE4) enzymes underpin compartmentalised cAMP signalling by localising to distinct signalling complexes. PDE4 long isoforms can be phosphorylated by mitogen-activated protein kinase-activated protein kinase 2 (MK2), which attenuates activation of such enzymes through their phosphorylation by protein kinase A. Here we show that MK2 interacts directly with PDE4 long isoforms and define the sites of interaction. One is a unique site that locates within the regulatory upstream conserved region 1 (UCR1) domain and contains a core Phe141, Leu142 and Tyr143 (FLY) cluster (PDE4A5 numbering). Located with the second site is a critical core Phe693, Glu694, Phe695 (FQF) motif that is also employed in the sequestering of PDE4 long forms by an array of other signalling proteins, including the signalling scaffold β-arrestin, the tyrosyl kinase Lyn, the SUMOylation E2 ligase UBC9, the dynein regulator Lis1 (PAFAH1B1) and the protein kinase Erk. We propose that the FQF motif lies at the heart of a multifunctional docking (MFD) site located within the PDE4 catalytic unit. It is clear from our data that, as well as aiding fidelity of interaction, the MFD site confers exclusivity of binding between PDE4 and a single specific partner protein from the cohort of signalling proteins whose interaction with PDE4 involves the FQF motif. PMID:27993970

  20. Rab3A is a new interacting partner of synaptotagmin I and may modulate synaptic membrane fusion through a competitive mechanism.

    PubMed

    Xie, Chunliang; Li, Jianglin; Guo, Tianyao; Yan, Yizhong; Tang, Cheng; Wang, Ying; Chen, Ping; Wang, Xianchun; Liang, Songping

    2014-02-21

    Rab3 and synaptotagmin have been reported to be the key proteins that have opposite actions but cooperatively play critical regulatory roles in selecting and limiting the number of vesicles released at central synapses. However, the exact mechanism has not been fully understood. In this study, Rab3A and synaptotagmin I, the most abundant isoforms of Rab3 and synaptotagmin, respectively, in brain were for the first time demonstrated to directly interact with each other in a Ca(2+)-independent manner, and the KKKK motif in the C2B domain of synaptotagmin I was a key site for the Rab3A binding, which was further confirmed by the competitive inhibition of inositol hexakisphosphate. Further studies demonstrated that Rab3A competitively affected the synaptotagmin I interaction with syntaxin 1B that was involved in membrane fusion during the synaptic vesicle exocytosis. These data indicate that Rab3A is a new synaptotagmin I interacting partner and may participate in the regulation of synaptic membrane fusion and thus the vesicle exocytosis by competitively modulating the interaction of synaptotagmin with syntaxin of the t-SNARE complex in presynaptic membranes. Copyright © 2014. Published by Elsevier Inc.

  1. Tissue-specific amino acid transporter partners ACE2 and collectrin differentially interact with hartnup mutations.

    PubMed

    Camargo, Simone M R; Singer, Dustin; Makrides, Victoria; Huggel, Katja; Pos, Klaas M; Wagner, Carsten A; Kuba, Keiji; Danilczyk, Ursula; Skovby, Flemming; Kleta, Robert; Penninger, Josef M; Verrey, François

    2009-03-01

    Hartnup amino acid transporter B(0)AT1 (SLC6A19) is the major luminal sodium-dependent neutral amino acid transporter of small intestine and kidney proximal tubule. The expression of B(0)AT1 in kidney was recently shown to depend on its association with collectrin (Tmem27), a protein homologous to the membrane-anchoring domain of angiotensin-converting enzyme (ACE) 2. Because collectrin is almost absent from small intestine, we tested the hypothesis that it is ACE2 that interacts with B(0)AT1 in enterocytes. Furthermore, because B(0)AT1 expression depends on an associated protein, we tested the hypothesis that Hartnup-causing B(0)AT1 mutations differentially impact on B(0)AT1 interaction with intestinal and kidney accessory proteins. Immunofluorescence, coimmunoprecipitation, and functional experiments using wild-type and ace2-null mice showed that expression of B(0)AT1 in small intestine critically depends on ACE2. Coexpressing new and previously identified Hartnup disorder-causing missense mutations of B(0)AT1 with either collectrin or ACE2 in Xenopus laevis oocytes showed that the high-frequency D173N and the newly identified P265L mutant B(0)AT1 transporters can still be activated by ACE2 but not collectrin coexpression. In contrast, the human A69T and R240Q B(0)AT1 mutants cannot be activated by either of the associated proteins, although they function as wild-type B(0)AT1 when expressed alone. We thus show that ACE2 is necessary for the expression of the Hartnup transporter in intestine and suggest that the differential functional association of mutant B(0)AT1 transporters with ACE2 and collectrin in intestine and kidney, respectively, participates in the phenotypic heterogeneity of human Hartnup disorder.

  2. Experimental observation of the interaction of propagating spin waves with Néel domain walls in a Landau domain structure

    SciTech Connect

    Pirro, P.; Sebastian, T.; Leven, B.; Hillebrands, B.; Koyama, T.; Brächer, T.

    2015-06-08

    The interaction of propagating dipolar spin waves with magnetic domain walls is investigated in square-shaped microstructures patterned from the Heusler compound Co{sub 2}Mn{sub 0.6}Fe{sub 0.4}Si. Using magnetic force microscopy, the reversible preparation of a Landau state with four magnetic domains separated by Néel domain walls is confirmed. A local spin-wave excitation using a microstructured antenna is realized in one of the domains. It is shown by Brillouin light scattering microscopy that the domain structure in the remanence state has a strong influence on the spin-wave excitation and propagation. The domain walls strongly reflect the spin waves and can be used as spin-wave reflectors. A comparison with micromagnetic simulations shows that the strong reflection is due to the long-range dipolar interaction which has important implications for the use of these spin waves for exerting an all-magnonic spin-transfer torque.

  3. Identification of FUSE-binding proteins as interacting partners of TIA proteins

    SciTech Connect

    Rothe, Francoise; Gueydan, Cyril; Bellefroid, Eric; Huez, Georges; Kruys, Veronique . E-mail: vkruys@ulb.ac.be

    2006-04-28

    TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm.

  4. Energetics of Calmodulin Domain Interactions with the Calmodulin Binding Domain of CaMKII

    PubMed Central

    Evans, T. Idil Apak; Shea, Madeline A.

    2010-01-01

    Calmodulin (CaM) is an essential eukaryotic calcium receptor that regulates many kinases, including CaMKII. Calcium-depleted CaM does not bind to CaMKII under physiological conditions. However, binding of (Ca2+)4-CaM to a basic amphipathic helix in CaMKII releases auto-inhibition of the kinase. The crystal structure of CaM bound to CaMKIIp, a peptide representing the CaM-binding domain (CaMBD) of CaMKII, shows an anti-parallel interface: the C-domain of CaM primarily contacts the N-terminal half of the CaMBD. The two domains of calcium-saturated CaM are believed to play distinct roles in releasing auto-inhibition. To investigate the underlying mechanism of activation, calcium-dependent titrations of isolated domains of CaM binding to CaMKIIp were monitored using fluorescence anisotropy. The binding affinity of CaMKIIp for the domains of CaM increased upon saturation with calcium, with a 35-fold greater increase observed for the C-domain than the N-domain. Because the interdomain linker of CaM regulates calcium-binding affinity and contribute to conformational change, the role of each CaM domain was explored further by investigating effects of CaMKIIp on site-knockout mutants affecting the calcium-binding sites of a single domain. Investigation of the thermodynamic linkage between saturation of individual calcium-binding sites and CaM-domain binding to CaMKIIp showed that calcium binding to sites III and IV was sufficient to recapitulate the behavior of (Ca2+)4-CaM. The magnitude of favorable interdomain cooperativity varied depending on which of the four calcium-binding sites were mutated, emphasizing differential regulatory roles for the domains of CaM, despite the high degree of homology among the four EF-hands of CaM. PMID:19089983

  5. An Interactional Perspective on the Relationship of Immigration to Intimate Partner Violence in a Representative Sample of Help-Seeking Women

    ERIC Educational Resources Information Center

    Bo Vatnar, Solveig Karin; Bjorkly, Stal

    2010-01-01

    This article reports a study of the possible impact of immigration on interactional aspects of intimate partner violence (IPV) among help-seeking women. Are there differences concerning (a) IPV categories, (b) IPV severity, frequency, duration, regularity, and predictability, (c) guilt and shame, (d) partners' ethnicity, and (e) children being…

  6. Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins.

    PubMed

    Crisci, Angela; Raleff, Flore; Bagdiul, Ivona; Raabe, Monika; Urlaub, Henning; Rain, Jean-Christophe; Krämer, Angela

    2015-12-02

    Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3' splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein-protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins

    PubMed Central

    Crisci, Angela; Raleff, Flore; Bagdiul, Ivona; Raabe, Monika; Urlaub, Henning; Rain, Jean-Christophe; Krämer, Angela

    2015-01-01

    Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3′ splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein–protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions. PMID:26420826

  8. Remarriage Beliefs as Predictors of Marital Quality and Positive Interaction in Stepcouples: An Actor-Partner Interdependence Model.

    PubMed

    Garneau, Chelsea L; Higginbotham, Brian; Adler-Baeder, Francesca

    2015-12-01

    Using an Actor-Partner Interdependence Model, we examined remarriage beliefs as predictors of marital quality and positive interaction in a sample of 179 stepcouples. Three beliefs were measured using subscales from the Remarriage Belief Inventory (RMBI) including success is slim, children are the priority, and finances should be pooled. Several significant actor and partner effects were found for both wives' and husbands' beliefs. Wives' marital quality was positively associated with their own beliefs that finances should be pooled and negatively associated with their own beliefs that success is slim. Wives' reports of their own and spouses' positive interaction were both positively associated with their beliefs that finances should be pooled. Their reports of spouses' positive interaction were also negatively associated with husbands' beliefs that success is slim. Husbands' marital quality was positively associated with wives' beliefs that children are the priority, positively associated with their own beliefs that finances should be pooled, and negatively with success is slim. Positive interaction for husbands was positively associated with wives' beliefs that finances should be pooled and negatively associated with their own beliefs that success is slim. Finally, husbands' reports of positive interaction for their spouses were positively associated with wives' beliefs that finances should be pooled. Implications for future research utilizing dyadic data analysis with stepcouples are addressed.

  9. Identification of the WW domain-interaction sites in the unstructured N-terminal domain of EBV LMP 2A.

    PubMed

    Seo, Min-Duk; Park, Sung Jean; Kim, Hyun-Jung; Lee, Bong Jin

    2007-01-09

    Epstein-Barr virus latency is maintained by the latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. The cytoplasmic N-terminal domain of LMP2A is composed of 119 amino acids. The N-terminal domain of LMP2A (LMP2A NTD) contains two PY motifs (PPPPY) that interact with the WW domains of Nedd4 family ubiquitin-protein ligases. Based on our analysis of NMR data, we found that the LMP2A NTD adopts an overall random-coil structure in its native state. However, the region between residues 60 and 90 was relatively ordered, and seemed to form the hydrophobic core of the LMP2A NTD. This region resides between two PY motifs and is important for WW domain binding. Mapping of the residues involved in the interaction between the LMP2A NTD and WW domains was achieved by chemical shift perturbation, by the addition of WW2 and WW3 peptides. Interestingly, the binding of the WW domains mainly occurred in the hydrophobic core of the LMP2A NTD. In addition, we detected a difference in the binding modes of the two PY motifs against the two WW peptides. The binding of the WW3 peptide caused the resonances of five residues (Tyr(60), Glu(61), Asp(62), Trp(65), and Gly(66)) just behind the N-terminal PY motif of the LMP2A NTD to disappear. A similar result was obtained with WW2 binding. However, near the C-terminal PY motif, the chemical shift perturbation caused by WW2 binding was different from that due to WW3 binding, indicating that the residues near the PY motifs are involved in selective binding of WW domains. The present work represents the first structural study of the LMP2A NTD and provides fundamental structural information about its interaction with ubiquitin-protein ligase.

  10. Partner-Specific Interpretation of Maintained Referential Precedents during Interactive Dialog

    ERIC Educational Resources Information Center

    Brown-Schmidt, Sarah

    2009-01-01

    In dialog settings, conversational partners converge on similar names for referents. These "lexically entrained" terms [Garrod, S., & Anderson, A. (1987). "Saying what you mean in dialog: A study in conceptual and semantic co-ordination." "Cognition, 27," 181-218] are part of the common ground between the particular individuals who established the…

  11. Faculty and Staff Partnering with Student Activists: Unexplored Terrains of Interaction and Development

    ERIC Educational Resources Information Center

    Kezar, Adrianna

    2010-01-01

    In this study, we build on two recent works (Gaston-Gayles, Wolf-Wendel; Tuttle, Twombley, and Ward, 2004; Slocum & Rhoads, 2008) that examine faculty and staff work with student activists, but expand the scope to include new questions such as why and how they partner with students, the impact of institutional context, and what role it might play…

  12. Partner-Specific Interpretation of Maintained Referential Precedents during Interactive Dialog

    ERIC Educational Resources Information Center

    Brown-Schmidt, Sarah

    2009-01-01

    In dialog settings, conversational partners converge on similar names for referents. These "lexically entrained" terms [Garrod, S., & Anderson, A. (1987). "Saying what you mean in dialog: A study in conceptual and semantic co-ordination." "Cognition, 27," 181-218] are part of the common ground between the particular individuals who established the…

  13. Synaptotagmin-1 C2B domain interacts simultaneously with SNAREs and membranes to promote membrane fusion

    PubMed Central

    Wang, Shen; Li, Yun; Ma, Cong

    2016-01-01

    Synaptotagmin-1 (Syt1) acts as a Ca2+ sensor for neurotransmitter release through its C2 domains. It has been proposed that Syt1 promotes SNARE-dependent fusion mainly through its C2B domain, but the underlying mechanism is poorly understood. In this study, we show that the C2B domain interacts simultaneously with acidic membranes and SNARE complexes via the top Ca2+-binding loops, the side polybasic patch, and the bottom face in response to Ca2+. Disruption of the simultaneous interactions completely abrogates the triggering activity of the C2B domain in liposome fusion. We hypothesize that the simultaneous interactions endow the C2B domain with an ability to deform local membranes, and this membrane-deformation activity might underlie the functional significance of the Syt1 C2B domain in vivo. DOI: http://dx.doi.org/10.7554/eLife.14211.001 PMID:27083046

  14. Large-Scale Screening of Preferred Interactions of Human Src Homology-3 (SH3) Domains Using Native Target Proteins as Affinity Ligands.

    PubMed

    Kazlauskas, Arunas; Schmotz, Constanze; Kesti, Tapio; Hepojoki, Jussi; Kleino, Iivari; Kaneko, Tomonori; Li, Shawn S C; Saksela, Kalle

    2016-10-01

    The Src Homology-3 (SH3) domains are ubiquitous protein modules that mediate important intracellular protein interactions via binding to short proline-rich consensus motifs in their target proteins. The affinity and specificity of such core SH3 - ligand contacts are typically modest, but additional binding interfaces can give rise to stronger and more specific SH3-mediated interactions. To understand how commonly such robust SH3 interactions occur in the human protein interactome, and to identify these in an unbiased manner we have expressed 324 predicted human SH3 ligands as full-length proteins in mammalian cells, and screened for their preferred SH3 partners using a phage display-based approach. This discovery platform contains an essentially complete repertoire of the ∼300 human SH3 domains, and involves an inherent binding threshold that ensures selective identification of only SH3 interactions with relatively high affinity. Such strong and selective SH3 partners could be identified for only 19 of these 324 predicted ligand proteins, suggesting that the majority of human SH3 interactions are relatively weak, and thereby have capacity for only modest inherent selectivity. The panel of exceptionally robust SH3 interactions identified here provides a rich source of leads and hypotheses for further studies. However, a truly comprehensive characterization of the human SH3 interactome will require novel high-throughput methods based on function instead of absolute binding affinity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Interaction partners for human ZNF384/CIZ/NMP4-zyxin as a mediator for p130CAS signaling?

    SciTech Connect

    Janssen, Hilde; Marynen, Peter . E-mail: Peter.Marynen@med.kuleuven.be

    2006-04-15

    Transcription factor ZNF384/CIZ/NMP4 was first cloned in rat as a p130Cas-binding protein and has a role in bone metabolism and spermatogenesis. It is recurrently involved in translocations in acute lymphoblastic leukemia. Translocations t(12;17) and t(12;22) fuse ZNF384 to RNA-binding proteins TAF15 and EWSR1, while a translocation t(12;19) generates an E2A/ZNF384 fusion. We screened for ZNF384 interacting proteins using yeast two-hybrid technology. In contrast to its rat homolog, human ZNF384 does not interact with p130CAS. Zyxin, PCBP1, and vimentin, however, were identified as ZNF384-binding partners. Given the interaction between human zyxin and p130CAS, these results suggest that zyxin indirectly enables the interaction of ZNF384 with p130CAS which is described in rat.

  16. Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle.

    PubMed

    Katzemich, Anja; West, Ryan J H; Fukuzawa, Atsushi; Sweeney, Sean T; Gautel, Mathias; Sparrow, John; Bullard, Belinda

    2015-09-15

    Drosophila obscurin (Unc-89) is a titin-like protein in the M-line of the muscle sarcomere. Obscurin has two kinase domains near the C-terminus, both of which are predicted to be inactive. We have identified proteins binding to the kinase domains. Kinase domain 1 bound Bällchen (Ball, an active kinase), and both kinase domains 1 and 2 bound MASK (a 400-kDa protein with ankyrin repeats). Ball was present in the Z-disc and M-line of the indirect flight muscle (IFM) and was diffusely distributed in the sarcomere. MASK was present in both the M-line and the Z-disc. Reducing expression of Ball or MASK by siRNA resulted in abnormalities in the IFM, including missing M-lines and multiple Z-discs. Obscurin was still present, suggesting that the kinase domains act as a scaffold binding Ball and MASK. Unlike obscurin in vertebrate skeletal muscle, Drosophila obscurin is necessary for the correct assembly of the IFM sarcomere. We show that Ball and MASK act downstream of obscurin, and both are needed for development of a well defined M-line and Z-disc. The proteins have not previously been identified in Drosophila muscle.

  17. Staphylococcus aureus domain V functions in Escherichia coli ribosomes provided a conserved interaction with domain IV is restored.

    PubMed Central

    Thompson, J; Tapprich, W E; Munger, C; Dahlberg, A E

    2001-01-01

    Domain V of Escherichia coli 23 S rRNA (residues 2023-2630) was replaced by that from Staphylococcus aureus, thereby introducing 132 changes in the rRNA sequence. The resulting ribosomal mutant was unable to support cell growth. The mutant was rescued, however, by restoring an interaction between domains IV and V (residues 1782 and 2586). Although the importance of this interaction, U/U in E. coli, C/C in S. aureus, is therefore demonstrated, it cannot be the only tertiary interaction important for ribosomal function as the rescued hybrid grew more slowly than the wild type. Additionally, although the single-site mutations U1782C and U2586C in E. coli are viable, the double mutant is lethal. PMID:11497427

  18. OsSRO1a Interacts with RNA Binding Domain-Containing Protein (OsRBD1) and Functions in Abiotic Stress Tolerance in Yeast

    PubMed Central

    Sharma, Shweta; Kaur, Charanpreet; Singla-Pareek, Sneh L.; Sopory, Sudhir K.

    2016-01-01

    SRO1 is an important regulator of stress and hormonal response in plants and functions by interacting with transcription factors and several other proteins involved in abiotic stress response. In the present study, we report OsRBD1, an RNA binding domain 1- containing protein as a novel interacting partner of OsSRO1a from rice. The interaction of OsSRO1a with OsRBD1 was shown in yeast as well as in planta. Domain–domain interaction study revealed that C-terminal RST domain of OsSRO1a interacts with the N-terminal RRM1 domain of OsRBD1 protein. Both the proteins were found to co-localize in nucleus. Transcript profiling under different stress conditions revealed co-regulation of OsSRO1a and OsRBD1 expression under some abiotic stress conditions. Further, co-transformation of both OsSRO1a and OsRBD1 in yeast conferred enhanced tolerance toward salinity, osmotic, and methylglyoxal treatments. Our study suggests that the interaction of OsSRO1a with OsRBD1 confers enhanced stress tolerance in yeast and may play an important role under abiotic stress responses in plants. PMID:26870074

  19. Interactions between the S-Domain Receptor Kinases and AtPUB-ARM E3 Ubiquitin Ligases Suggest a Conserved Signaling Pathway in Arabidopsis1[W][OA

    PubMed Central

    Samuel, Marcus A.; Mudgil, Yashwanti; Salt, Jennifer N.; Delmas, Frédéric; Ramachandran, Shaliny; Chilelli, Andrea; Goring, Daphne R.

    2008-01-01

    The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses. PMID:18552232

  20. Autoinhibitory structure of the WW domain of HYPB/SETD2 regulates its interaction with the proline-rich region of huntingtin.

    PubMed

    Gao, Yong-Guang; Yang, Hui; Zhao, Jian; Jiang, Ya-Jun; Hu, Hong-Yu

    2014-03-04

    Huntington's disease (HD) is an autosomally dominant neurodegenerative disorder caused by expansion of polyglutamine (polyQ) in the huntingtin (Htt) protein. Htt yeast two-hybrid protein B (HYPB/SETD2), a histone methyltransferase, directly interacts with Htt and is involved in HD pathology. Using NMR techniques, we characterized a polyproline (polyP) stretch at the C terminus of HYPB, which directly interacts with the following WW domain and leads this domain predominantly to be in a closed conformational state. The solution structure shows that the polyP stretch extends from the back and binds to the WW core domain in a typical binding mode. This autoinhibitory structure regulates interaction between the WW domain of HYPB and the proline-rich region (PRR) of Htt, as evidenced by NMR and immunofluorescence techniques. This work provides structural and mechanistic insights into the intramolecular regulation of the WW domain in Htt-interacting partners and will be helpful for understanding the pathology of HD.

  1. Smooth Muscle Titin Zq Domain Interaction with the Smooth Muscle α-Actinin Central Rod*

    PubMed Central

    Chi, Richard J.; Simon, Alanna R.; Bienkiewicz, Ewa A.; Felix, Augustine; Keller, Thomas C. S.

    2008-01-01

    Actin-myosin II filament-based contractile structures in striated muscle, smooth muscle, and nonmuscle cells contain the actin filament-cross-linking protein α-actinin. In striated muscle Z-disks, α-actinin interacts with N-terminal domains of titin to provide a structural linkage crucial for the integrity of the sarcomere. We previously discovered a long titin isoform, originally smitin, hereafter sm-titin, in smooth muscle and demonstrated that native sm-titin interacts with C-terminal EF hand region and central rod R2-R3 spectrin-like repeat region sites in α-actinin. Reverse transcription-PCR analysis of RNA from human adult smooth muscles and cultured rat smooth muscle cells and Western blot analysis with a domain-specific antibody presented here revealed that sm-titin contains the titin gene-encoded Zq domain that may bind to the α-actinin R2-R3 central rod domain as well as Z-repeat domains that bind to the EF hand region. We investigated whether the sm-titin Zq domain binds to α-actinin R2 and R3 spectrin repeat-like domain loops that lie in proximity with two-fold symmetry on the surface of the central rod. Mutations in α-actinin R2 and R3 domain loop residues decreased interaction with expressed sm-titin Zq domain in glutathione S-transferase pull-down and solid phase binding assays. Alanine mutation of a region of the Zq domain with high propensity for α-helix formation decreased apparent Zq domain dimer formation and decreased Zq interaction with the α-actinin R2-R3 region in surface plasmon resonance assays. We present a model in which two sm-titin Zq domains interact with each other and with the two R2-R3 sites in the α-actinin central rod. PMID:18519573

  2. Evolutionarily Conserved Paired Immunoglobulin-like Receptor α (PILRα) Domain Mediates Its Interaction with Diverse Sialylated Ligands

    PubMed Central

    Sun, Yonglian; Senger, Kate; Baginski, Tomasz K.; Mazloom, Anita; Chinn, Yvonne; Pantua, Homer; Hamidzadeh, Kajal; Ramani, Sree Ranjani; Luis, Elizabeth; Tom, Irene; Sebrell, Andrew; Quinones, Gabriel; Ma, Yan; Mukhyala, Kiran; Sai, Tao; Ding, Jiabing; Haley, Benjamin; Shadnia, Hooman; Kapadia, Sharookh B.; Gonzalez, Lino C.; Hass, Philip E.; Zarrin, Ali A.

    2012-01-01

    Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed. PMID:22396535

  3. The intercropping partner affects arbuscular mycorrhizal fungi and Fusarium oxysporum f. sp. lycopersici interactions in tomato.

    PubMed

    Hage-Ahmed, Karin; Krammer, Johannes; Steinkellner, Siegrid

    2013-10-01

    Arbuscular mycorrhizal fungi (AMF) and their bioprotective aspects are of great interest in the context of sustainable agriculture. Combining the benefits of AMF with the utilisation of plant species diversity shows great promise for the management of plant diseases in environmentally compatible agriculture. In the present study, AMF were tested against Fusarium oxysporum f. sp. lycopersici with tomato intercropped with either leek, cucumber, basil, fennel or tomato itself. Arbuscular mycorrhizal (AM) root colonisation of tomato was clearly affected by its intercropping partners. Tomato intercropped with leek showed even a 20 % higher AM colonisation rate than tomato intercropped with tomato. Positive effects of AMF expressed as an increase of tomato biomass compared to the untreated control treatment could be observed in root as well as in shoot weights. A compensation of negative effects of F. oxysporum f. sp. lycopersici on tomato biomass by AMF was observed in the tomato/leek combination. The intercropping partners leek, cucumber, basil and tomato had no effect on F. oxysporum f. sp. lycopersici disease incidence or disease severity indicating no allelopathic suppression; however, tomato co-cultivated with tomato clearly showed a negative effect on one plant/pot with regard to biomass and disease severity of F. oxysporum f. sp. lycopersici. Nonetheless, bioprotective effects of AMF resulting in the decrease of F. oxysporum f. sp. lycopersici disease severity were evident in treatments with AMF and F. oxysporum f. sp. lycopersici co-inoculation. However, these bioprotective effects depended on the intercropping partner since these effects were only observed in the tomato/leek and tomato/basil combination and for the better developed plant of tomato/tomato. In conclusion, the effects of the intercropping partner on AMF colonisation of tomato are of great interest for crop plant communities and for the influences on each other. The outcome of the bioprotective

  4. Functional Complementation Studies Reveal Different Interaction Partners of Escherichia coli IscS and Human NFS1.

    PubMed

    Bühning, Martin; Friemel, Martin; Leimkühler, Silke

    2017-08-29

    The trafficking and delivery of sulfur to cofactors and nucleosides is a highly regulated and conserved process among all organisms. All sulfur transfer pathways generally have an l-cysteine desulfurase as an initial sulfur-mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of sulfur-containing biomolecules like iron-sulfur (Fe-S) clusters, thiamine, biotin, lipoic acid, the molybdenum cofactor (Moco), and thiolated nucleosides in tRNA. The human l-cysteine desulfurase NFS1 and the Escherichia coli homologue IscS share a level of amino acid sequence identity of ∼60%. While E. coli IscS has a versatile role in the cell and was shown to have numerous interaction partners, NFS1 is mainly localized in mitochondria with a crucial role in the biosynthesis of Fe-S clusters. Additionally, NFS1 is also located in smaller amounts in the cytosol with a role in Moco biosynthesis and mcm(5)s(2)U34 thio modifications of nucleosides in tRNA. NFS1 and IscS were conclusively shown to have different interaction partners in their respective organisms. Here, we used functional complementation studies of an E. coli iscS deletion strain with human NFS1 to dissect their conserved roles in the transfer of sulfur to a specific target protein. Our results show that human NFS1 and E. coli IscS share conserved binding sites for proteins involved in Fe-S cluster assembly like IscU, but not with proteins for tRNA thio modifications or Moco biosynthesis. In addition, we show that human NFS1 was almost fully able to complement the role of IscS in Moco biosynthesis when its specific interaction partner protein MOCS3 from humans was also present.

  5. Cartilage oligomeric matrix protein and its binding partners in the cartilage extracellular matrix: interaction, regulation and role in chondrogenesis.

    PubMed

    Acharya, Chitrangada; Yik, Jasper H N; Kishore, Ashleen; Van Dinh, Victoria; Di Cesare, Paul E; Haudenschild, Dominik R

    2014-07-01

    Thrombospondins (TSPs) are widely known as a family of five calcium-binding matricellular proteins. While these proteins belong to the same family, they are encoded by different genes, regulate different cellular functions and are localized to specific regions of the body. TSP-5 or Cartilage Oligomeric Matrix Protein (COMP) is the only TSP that has been associated with skeletal disorders in humans, including pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). The pentameric structure of COMP, the evidence that it interacts with multiple cellular proteins, and the recent reports of COMP acting as a 'lattice' to present growth factors to cells, inspired this review of COMP and its interacting partners. In our review, we have compiled the interactions of COMP with other proteins in the cartilage extracellular matrix and summarized their importance in maintaining the structural integrity of cartilage as well as in regulating cellular functions.

  6. GAIA: a gram-based interaction analysis tool – an approach for identifying interacting domains in yeast

    PubMed Central

    Zhang, Kelvin X; Ouellette, BF Francis

    2009-01-01

    Background Protein-Protein Interactions (PPIs) play important roles in many biological functions. Protein domains, which are defined as independently folding structural blocks of proteins, physically interact with each other to perform these biological functions. Therefore, the identification of Domain-Domain Interactions (DDIs) is of great biological interests because it is generally accepted that PPIs are mediated by DDIs. As a result, much effort has been put on the prediction of domain pair interactions based on computational methods. Many DDI prediction tools using PPIs network and domain evolution information have been reported. However, tools that combine the primary sequences, domain annotations, and structural annotations of proteins have not been evaluated before. Results In this study, we report a novel approach called Gram-bAsed Interaction Analysis (GAIA). GAIA extracts peptide segments that are composed of fixed length of continuous amino acids, called n-grams (where n is the number of amino acids), from the annotated domain and DDI data set in Saccharomyces cerevisiae (budding yeast) and identifies a list of n-grams that may contribute to DDIs and PPIs based on the frequencies of their appearance. GAIA also reports the coordinate position of gram pairs on each interacting domain pair. We demonstrate that our approach improves on other DDI prediction approaches when tested against a gold-standard data set and achieves a true positive rate of 82% and a false positive rate of 21%. We also identify a list of 4-gram pairs that are significantly over-represented in the DDI data set and may mediate PPIs. Conclusion GAIA represents a novel and reliable way to predict DDIs that mediate PPIs. Our results, which show the localizations of interacting grams/hotspots, provide testable hypotheses for experimental validation. Complemented with other prediction methods, this study will allow us to elucidate the interactome of cells. PMID:19208164

  7. Social and emotional relevance in face processing: happy faces of future interaction partners enhance the late positive potential.

    PubMed

    Bublatzky, Florian; Gerdes, Antje B M; White, Andrew J; Riemer, Martin; Alpers, Georg W

    2014-01-01

    Human face perception is modulated by both emotional valence and social relevance, but their interaction has rarely been examined. Event-related brain potentials (ERP) to happy, neutral, and angry facial expressions with different degrees of social relevance were recorded. To implement a social anticipation task, relevance was manipulated by presenting faces of two specific actors as future interaction partners (socially relevant), whereas two other face actors remained non-relevant. In a further control task all stimuli were presented without specific relevance instructions (passive viewing). Face stimuli of four actors (2 women, from the KDEF) were randomly presented for 1s to 26 participants (16 female). Results showed an augmented N170, early posterior negativity (EPN), and late positive potential (LPP) for emotional in contrast to neutral facial expressions. Of particular interest, face processing varied as a function of experimental tasks. Whereas task effects were observed for P1 and EPN regardless of instructed relevance, LPP amplitudes were modulated by emotional facial expression and relevance manipulation. The LPP was specifically enhanced for happy facial expressions of the anticipated future interaction partners. This underscores that social relevance can impact face processing already at an early stage of visual processing. These findings are discussed within the framework of motivated attention and face processing theories.

  8. Social and emotional relevance in face processing: happy faces of future interaction partners enhance the late positive potential

    PubMed Central

    Bublatzky, Florian; Gerdes, Antje B. M.; White, Andrew J.; Riemer, Martin; Alpers, Georg W.

    2014-01-01

    Human face perception is modulated by both emotional valence and social relevance, but their interaction has rarely been examined. Event-related brain potentials (ERP) to happy, neutral, and angry facial expressions with different degrees of social relevance were recorded. To implement a social anticipation task, relevance was manipulated by presenting faces of two specific actors as future interaction partners (socially relevant), whereas two other face actors remained non-relevant. In a further control task all stimuli were presented without specific relevance instructions (passive viewing). Face stimuli of four actors (2 women, from the KDEF) were randomly presented for 1s to 26 participants (16 female). Results showed an augmented N170, early posterior negativity (EPN), and late positive potential (LPP) for emotional in contrast to neutral facial expressions. Of particular interest, face processing varied as a function of experimental tasks. Whereas task effects were observed for P1 and EPN regardless of instructed relevance, LPP amplitudes were modulated by emotional facial expression and relevance manipulation. The LPP was specifically enhanced for happy facial expressions of the anticipated future interaction partners. This underscores that social relevance can impact face processing already at an early stage of visual processing. These findings are discussed within the framework of motivated attention and face processing theories. PMID:25076881

  9. The PUB domain: a putative protein-protein interaction domain implicated in the ubiquitin-proteasome pathway.

    PubMed

    Suzuki, T; Park, H; Till, E A; Lennarz, W J

    2001-10-12

    Cytoplasmic peptide:N-glycanase (PNGase) is a de-N-glycosylating enzyme which may be involved in the proteasome-dependent pathway for degradation of misfolded glycoproteins formed in the endoplasmic reticulum (ER) that are exported into the cytoplasm. A cytoplasmic PNGase found in Saccharomyces cerevisiae, Png1p, is widely distributed in higher eukaryotes as well as in yeast (Suzuki, T., et al. J. Cell Biol. 149, 1039-1051, 2000). The recently uncovered complete genome sequence of Arabidopsis thaliana prompted us to search for the protein homologue of Png1p in this organism. Interestingly, when the mouse Png1p homologue sequence was used as a query, not only a Png1p homologue containing a transglutaminase-like domain that is believed to contain a catalytic triad for PNGase activity, but also four proteins which had a domain of 46 amino acids in length that exhibited significant similarity to the N-terminus of mouse Png1p were identified. Moreover, three of these homologous proteins were also found to possess a UBA or UBX domain, which are found in various proteins involved in the ubiquitin-related pathway. We name this newly found homologous region the PUB (Peptide:N-glycanase/UBA or UBX-containing proteins) domain and propose that this domain may mediate protein-protein interactions.

  10. Role of Domain Interactions in the Collective Motion of Phosphoglycerate Kinase

    PubMed Central

    Schay, Gusztáv; Herényi, Levente; Fidy, Judit; Osváth, Szabolcs

    2013-01-01

    Protein function is governed by the underlying conformational dynamics of the molecule. The experimental and theoretical work leading to contemporary understanding of enzyme dynamics was mostly restricted to the large-scale movements of single-domain proteins. Collective movements resulting from a regulatory interplay between protein domains is often crucial for enzymatic activity. It is not clear, however, how our knowledge could be extended to describe collective near-equilibrium motions of multidomain enzymes. We examined the effect of domain interactions on the low temperature near equilibrium dynamics of the native state, using phosphoglycerate kinase as model protein. We measured thermal activation of tryptophan phosphorescence quenching to explore millisecond-range protein motions. The two protein domains of phosphoglycerate kinase correspond to two dynamic units, but interdomain interactions link the motion of the two domains. The effect of the interdomain interactions on the activation of motions in the individual domains is asymmetric. As the temperature of the frozen protein is increased from the cryogenic, motions of the N domain are activated first. This is a partial activation, however, and the full dynamics of the domain becomes activated only after the activation of the C domain. PMID:23442918

  11. Role of domain interactions in the collective motion of phosphoglycerate kinase.

    PubMed

    Schay, Gusztáv; Herényi, Levente; Fidy, Judit; Osváth, Szabolcs

    2013-02-05

    Protein function is governed by the underlying conformational dynamics of the molecule. The experimental and theoretical work leading to contemporary understanding of enzyme dynamics was mostly restricted to the large-scale movements of single-domain proteins. Collective movements resulting from a regulatory interplay between protein domains is often crucial for enzymatic activity. It is not clear, however, how our knowledge could be extended to describe collective near-equilibrium motions of multidomain enzymes. We examined the effect of domain interactions on the low temperature near equilibrium dynamics of the native state, using phosphoglycerate kinase as model protein. We measured thermal activation of tryptophan phosphorescence quenching to explore millisecond-range protein motions. The two protein domains of phosphoglycerate kinase correspond to two dynamic units, but interdomain interactions link the motion of the two domains. The effect of the interdomain interactions on the activation of motions in the individual domains is asymmetric. As the temperature of the frozen protein is increased from the cryogenic, motions of the N domain are activated first. This is a partial activation, however, and the full dynamics of the domain becomes activated only after the activation of the C domain. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  12. Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes

    PubMed Central

    Andreu-Fernández, Vicente; Sancho, Mónica; Genovés, Ainhoa; Lucendo, Estefanía; Todt, Franziska; Lauterwasser, Joachim; Funk, Kathrin; Jahreis, Günther; Pérez-Payá, Enrique; Mingarro, Ismael; Edlich, Frank; Orzáez, Mar

    2017-01-01

    The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions were implicated recently in Bax pore formation. Here, we show that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity. Our results suggest that interactions between the transmembrane domains of Bax and antiapoptotic Bcl-2 proteins represent a previously unappreciated level of apoptosis regulation. PMID:28028215

  13. Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes.

    PubMed

    Andreu-Fernández, Vicente; Sancho, Mónica; Genovés, Ainhoa; Lucendo, Estefanía; Todt, Franziska; Lauterwasser, Joachim; Funk, Kathrin; Jahreis, Günther; Pérez-Payá, Enrique; Mingarro, Ismael; Edlich, Frank; Orzáez, Mar

    2017-01-10

    The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions were implicated recently in Bax pore formation. Here, we show that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity. Our results suggest that interactions between the transmembrane domains of Bax and antiapoptotic Bcl-2 proteins represent a previously unappreciated level of apoptosis regulation.

  14. Proteome scanning to predict PDZ domain interactions using support vector machines

    PubMed Central

    2010-01-01

    Background PDZ domains mediate protein-protein interactions involved in important biological processes through the recognition of short linear motifs in their target proteins. Two recent independent studies have used protein microarray or phage display technology to detect PDZ domain interactions with peptide ligands on a large scale. Several computational predictors of PDZ domain interactions have been developed, however they are trained using only protein microarray data and focus on limited subsets of PDZ domains. An accurate predictor of genomic PDZ domain interactions would allow the proteomes of organisms to be scanned for potential binders. Such an application would require an accurate and precise predictor to avoid generating too many false positive hits given the large amount of possible interactors in a given proteome. Once validated these predictions will help to increase the coverage of current PDZ domain interaction networks and further our understanding of the roles that PDZ domains play in a variety of biological processes. Results We developed a PDZ domain interaction predictor using a support vector machine (SVM) trained with both protein microarray and phage display data. In order to use the phage display data for training, which only contains positive interactions, we developed a method to generate artificial negative interactions. Using cross-validation and a series of independent tests, we showed that our SVM successfully predicts interactions in different organisms. We then used the SVM to scan the proteomes of human, worm and fly to predict binders for several PDZ domains. Predictions were validated using known genomic interactions and published protein microarray experiments. Based on our results, new protein interactions potentially associated with Usher and Bardet-Biedl syndromes were predicted. A comparison of performance measures (F1 measure and FPR) for the SVM and published predictors demonstrated our SVM's improved accuracy and

  15. Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein.

    PubMed

    Kellner, Julian N; Meinhart, Anton

    2015-09-01

    The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein-protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein-protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.

  16. Label-Free Proteomic Identification of Endogenous, Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1

    NASA Astrophysics Data System (ADS)

    Geetha, Thangiah; Langlais, Paul; Luo, Moulun; Mapes, Rebekka; Lefort, Natalie; Chen, Shu-Chuan; Mandarino, Lawrence J.; Yi, Zhengping

    2011-03-01

    Protein-protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein-protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein-protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.

  17. Correlation between magnetic interactions and domain structure in A1 FePt ferromagnetic thin films

    NASA Astrophysics Data System (ADS)

    Álvarez, N.; Sallica Leva, E.; Valente, R. C.; Vásquez Mansilla, M.; Gómez, J.; Milano, J.; Butera, A.

    2014-02-01

    We have investigated the relationship between the domain structure and the magnetic interactions in a series of FePt ferromagnetic thin films of varying thickness. As-made films grow in the magnetically soft and chemically disordered A1 phase that may have two distinct domain structures. Above a critical thickness dcr ˜ 30 nm the presence of an out of plane anisotropy induces the formation of stripes, while for d < dcr planar domains occur. Magnetic interactions have been characterized using the well known DC demagnetization - isothermal remanent magnetization remanence protocols, δM plots, and magnetic viscosity measurements. We have observed a strong correlation between the domain configuration and the sign of the magnetic interactions. Planar domains are associated with positive exchange-like interactions, while stripe domains have a strong negative dipolar-like contribution. In this last case we have found a close correlation between the interaction parameter and the surface dipolar energy of the stripe domain structure. Using time dependent magnetic viscosity measurements, we have also estimated an average activation volume for magnetic reversal, ⟨Vac⟩˜1.37×104 nm3, which is approximately independent of the film thickness or the stripe period.

  18. Multiple protein domains mediate interaction between Bcl10 and MALT1.

    PubMed

    Langel, Felicia D; Jain, Nidhi A; Rossman, Jeremy S; Kingeter, Lara M; Kashyap, Anuj K; Schaefer, Brian C

    2008-11-21

    Bcl10 and MALT1 are essential mediators of NF-kappaB activation in response to the triggering of a diverse array of transmembrane receptors, including antigen receptors. Additionally, both proteins are translocation targets in MALT lymphoma. Thus, a detailed understanding of the interaction between these mediators is of considerable biological importance. Previous studies have indicated that a 13-amino acid region downstream of the Bcl10 caspase recruitment domain (CARD) is responsible for interacting with the immunoglobulin-like domains of MALT1. We now provide evidence that the death domain of MALT1 and the CARD of Bcl10 also contribute to Bcl10-MALT1 interactions. Although a direct interaction between the MALT1 death domain and Bcl10 cannot be detected via immunoprecipitation, FRET data strongly suggest that the death domain of MALT1 contributes significantly to the association between Bcl10 and MALT1 in T cells in vivo. Furthermore, analysis of point mutants of conserved residues of Bcl10 shows that the Bcl10 CARD is essential for interaction with the MALT1 N terminus. Mutations that disrupt proper folding of the Bcl10 CARD strongly impair Bcl10-MALT1 interactions. Molecular modeling and functional analyses of Bcl10 point mutants suggest that residues Asp(80) and Glu(84) of helix 5 of the Bcl10 CARD directly contact MALT1. Together, these data demonstrate that the association between Bcl10 and MALT1 involves a complex interaction between multiple protein domains. Moreover, the Bcl10-MALT1 interaction is the second reported example of interactions between a CARD and a non-CARD protein region, which suggests that many signaling cascades may utilize CARD interactions with non-CARD domains.

  19. Enhanced sampling simulations to construct free-energy landscape of protein-partner substrate interaction.

    PubMed

    Ikebe, Jinzen; Umezawa, Koji; Higo, Junichi

    2016-03-01

    Molecular dynamics (MD) simulations using all-atom and explicit solvent models provide valuable information on the detailed behavior of protein-partner substrate binding at the atomic level. As the power of computational resources increase, MD simulations are being used more widely and easily. However, it is still difficult to investigate the thermodynamic properties of protein-partner substrate binding and protein folding with conventional MD simulations. Enhanced sampling methods have been developed to sample conformations that reflect equilibrium conditions in a more efficient manner than conventional MD simulations, thereby allowing the construction of accurate free-energy landscapes. In this review, we discuss these enhanced sampling methods using a series of case-by-case examples. In particular, we review enhanced sampling methods conforming to trivial trajectory parallelization, virtual-system coupled multicanonical MD, and adaptive lambda square dynamics. These methods have been recently developed based on the existing method of multicanonical MD simulation. Their applications are reviewed with an emphasis on describing their practical implementation. In our concluding remarks we explore extensions of the enhanced sampling methods that may allow for even more efficient sampling.

  20. Southwestern Cooperative Educational Laboratory Interaction Observation Schedule (SCIOS): A System for Analyzing Teacher-Pupil Interaction in the Affective Domain.

    ERIC Educational Resources Information Center

    Bemis, Katherine A.; Liberty, Paul G.

    The Southwestern Cooperative Interaction Observation Schedule (SCIOS) is a classroom observation instrument designed to record pupil-teacher interaction. The classification of pupil behavior is based on Krathwohl's (1964) theory of the three lowest levels of the affective domain. The levels are (1) receiving: the learner should be sensitized to…

  1. Defining subdomains of the K domain important for protein-protein interactions of plant MADS proteins.

    PubMed

    Yang, Yingzhen; Jack, Thomas

    2004-05-01

    The MADS proteins APETALA3 (AP3), PISTILLATA (PI), SEPALLATAI (SEPI), SEP2, SEP3, AGAMOUS, and APETALA are required for proper floral organ identity in Arabidopsis flowers. All of these floral MADS proteins conserve two domains: the MADS domain that mediates DNA binding and dimerization, and the K domain that mediates protein protein interaction. The K domain is postulated to form a several amphipathic c-helices referred to as K1, K2, and K3. The K1 and K2 helicies are located entirely within the K domain while the K3 helix spans the K domain-C domain boundary. Here we report on our studies on the interactions of the B class MADS proteins AP3 and PI with the E class MADS proteins SEP1, SEP2, and SEP3. A comparative analysis of mutants in the K domain reveals that the subdomains mediating the PI/AP3 interaction are different from the subdomains mediating the PI/SEP3 (or PI/SEP1) interaction. The strong PI/SEP3 (or PI/SEP1) interaction requires K2, part of K3, and the interhelical region between K1 and K2. By contrast, K1, K2 and the region between K1 and K2 are important for strong AP3/PI interaction. Most of the K3 helix does not appear to be important for either the PI/AP3 or the PI/SEP3 (or PI/SEP1) interaction. Conserved hydrophobic positions are most important for the strength of both PI/AP3 and PI/SEP3 dimerization, though ionic and/or polar interactions appear to play a secondary role.

  2. Domain interactions direct misfolding and amyloid formation of yeast phosphoglycerate kinase.

    PubMed

    Osváth, Szabolcs; Jäckel, Márta; Agócs, Gergely; Závodszky, Péter; Köhler, Gottfried; Fidy, Judit

    2006-03-01

    There are proteins that are built of two structural domains and are deposited full-length in amyloid plaques formed in various diseases. In spite of the known differences in the mechanisms of folding of single- and multidomain proteins, no published studies can be found that address the role of the domain-domain interactions during misfolding and amyloid formation. By the discovery of the role of domain-domain interactions, here we provide important insight in the submolecular mechanism of amyloid formation. A model system based on yeast phosphoglycerate kinase was designed. This system includes the wild-type yeast phosphoglycerate kinase and single-tryptophan mutants of the individual N and C terminal domains and the complete protein. Electron microscopic measurements proved that amyloid fibrils grow from all mutants under identical conditions as for the wild-type protein. Misfolding and amyloid formation was followed in stopped-flow and manual mixing experiments on the 1 ms to 4 days timescale. Tryptophan fluorescence was used for selective detection of conformational changes accompanying the formation of the amyloidogenic intermediates and the growth of amyloid fibrils. The interactions between the polypeptide chains of the two domains direct the misfolding process from the early steps to the amyloid formation, and influence the final structure. The kinetics of misfolding is different for the individual domains, pointing to the significance of the amino acid sequence. Misfolding of the domains within the complete protein is synchronized indicating that domain-domain interactions direct the misfolding and amyloid formation mechanism. 2006 Wiley-Liss, Inc.

  3. Evolution of domain-peptide interactions to coadapt specificity and affinity to functional diversity.

    PubMed

    Kelil, Abdellali; Levy, Emmanuel D; Michnick, Stephen W

    2016-07-05

    Evolution of complexity in eukaryotic proteomes has arisen, in part, through emergence of modular independently folded domains mediating protein interactions via binding to short linear peptides in proteins. Over 30 years, structural properties and sequence preferences of these peptides have been extensively characterized. Less successful, however, were efforts to establish relationships between physicochemical properties and functions of domain-peptide interactions. To our knowledge, we have devised the first strategy to exhaustively explore the binding specificity of protein domain-peptide interactions. We applied the strategy to SH3 domains to determine the properties of their binding peptides starting from various experimental data. The strategy identified the majority (∼70%) of experimentally determined SH3 binding sites. We discovered mutual relationships among binding specificity, binding affinity, and structural properties and evolution of linear peptides. Remarkably, we found that these properties are also related to functional diversity, defined by depth of proteins within hierarchies of gene ontologies. Our results revealed that linear peptides evolved to coadapt specificity and affinity to functional diversity of domain-peptide interactions. Thus, domain-peptide interactions follow human-constructed gene ontologies, which suggest that our understanding of biological process hierarchies reflect the way chemical and thermodynamic properties of linear peptides and their interaction networks, in general, have evolved.

  4. Calcium-Dependent Protein Kinase in Ginger Binds with Importin-α through Its Junction Domain for Nuclear Localization, and Further Interacts with NAC Transcription Factor

    PubMed Central

    Vivek, Padmanabhan Jayanthi; Resmi, Mohankumar Saraladevi; Sreekumar, Sweda; Sivakumar, K. C.; Tuteja, Narendra; Soniya, Eppurathu Vasudevan

    2017-01-01

    Calcium-dependent protein kinases (CDPKs) are important sensors of Ca2+ elevations in plant cells regulating the gene expression linked with various cellular processes like stress response, growth and development, metabolism, and cytoskeleton dynamics. Ginger is an extensively used spice due to its unique flavor and immense medicinal value. The two major threats that interfere with the large scale production of ginger are the salinity and drought stress. ZoCDPK1 (Zingiber officinale Calcium-dependent protein kinase 1) is a salinity and drought-inducible CDPK gene isolated from ginger and undergoes dynamic subcellular localization during stress conditions. ZoCDPK1, with signature features of a typical Ca2+ regulated kinase, also possesses a bipartite nuclear localization sequence (NLS) in its junction domain (JD). A striking feature in ZoCDPK1 is the rare occurrence of a coupling between the NLS in JD and consensus sequences in regulatory domain. Here, we further identified its nature of nuclear localization and its interaction partners. In the homology model generated for ZoCDPK1, the regulatory domain mimics the crystal structure of the regulatory domain in Arabidopsis CDPK1. Molecular docking simulation of importin (ZoIMPα), an important protein involved in nuclear translocation, into the NLS of ZoCDPK1 was well-visualized. Furthermore, the direct interaction of ZoCDPK1 and ZoIMPα proteins was studied by the yeast 2-hybrid (Y2H) system, which confirmed that junction domain (JD) is an important interaction module required for ZoCDPK1 and ZoIMPα binding. The probable interacting partners of ZoCDPK1 were also identified using Y2H experiment. Of the 10 different stress-related interacting partners identified for ZoCDPK1, NAC transcription factor (TF) needs special mention, especially in the context of ZoCDPK1 function. The interaction between ZoCDPK1 and NAC TF, in fact, corroborate with the results of gene expression and over-expression studies of ZoCDPK1. Hence

  5. Nephrocystin-1 Forms a Complex with Polycystin-1 via a Polyproline Motif/SH3 Domain Interaction and Regulates the Apoptotic Response in Mammals

    PubMed Central

    Wodarczyk, Claas; Bricoli, Barbara; Muorah, Mordi; Spitaleri, Andrea; Mannella, Valeria; Ricchiuto, Piero; Pema, Monika; Castelli, Maddalena; Casanova, Ariel E.; Mollica, Luca; Banzi, Manuela; Boca, Manila; Antignac, Corinne; Saunier, Sophie; Musco, Giovanna; Boletta, Alessandra

    2010-01-01

    Mutations in PKD1, the gene encoding for the receptor Polycystin-1 (PC-1), cause autosomal dominant polycystic kidney disease (ADPKD). The cytoplasmic C-terminus of PC-1 contains a coiled-coil domain that mediates an interaction with the PKD2 gene product, Polycystin-2 (PC-2). Here we identify a novel domain in the PC-1 C-terminal tail, a polyproline motif mediating an interaction with Src homology domain 3 (SH3). A screen for interactions using the PC-1 C-terminal tail identified the SH3 domain of nephrocystin-1 (NPHP1) as a potential binding partner of PC-1. NPHP1 is the product of a gene that is mutated in a different form of renal cystic disease, nephronophthisis (NPHP). We show that in vitro pull-down assays and NMR structural studies confirmed the interaction between the PC-1 polyproline motif and the NPHP1 SH3 domain. Furthermore, the two full-length proteins interact through these domains; using a recently generated model system allowing us to track endogenous PC-1, we confirm the interaction between the endogenous proteins. Finally, we show that NPHP1 trafficking to cilia does not require PC-1 and that PC-1 may require NPHP1 to regulate resistance to apoptosis, but not to regulate cell cycle progression. In line with this, we find high levels of apoptosis in renal specimens of NPHP patients. Our data uncover a link between two different ciliopathies, ADPKD and NPHP, supporting the notion that common pathogenetic defects, possibly involving de-regulated apoptosis, underlie renal cyst formation. PMID:20856870

  6. Nephrocystin-1 forms a complex with polycystin-1 via a polyproline motif/SH3 domain interaction and regulates the apoptotic response in mammals.

    PubMed

    Wodarczyk, Claas; Distefano, Gianfranco; Rowe, Isaline; Gaetani, Massimiliano; Bricoli, Barbara; Muorah, Mordi; Spitaleri, Andrea; Mannella, Valeria; Ricchiuto, Piero; Pema, Monika; Castelli, Maddalena; Casanova, Ariel E; Mollica, Luca; Banzi, Manuela; Boca, Manila; Antignac, Corinne; Saunier, Sophie; Musco, Giovanna; Boletta, Alessandra

    2010-09-14

    Mutations in PKD1, the gene encoding for the receptor Polycystin-1 (PC-1), cause autosomal dominant polycystic kidney disease (ADPKD). The cytoplasmic C-terminus of PC-1 contains a coiled-coil domain that mediates an interaction with the PKD2 gene product, Polycystin-2 (PC-2). Here we identify a novel domain in the PC-1 C-terminal tail, a polyproline motif mediating an interaction with Src homology domain 3 (SH3). A screen for interactions using the PC-1 C-terminal tail identified the SH3 domain of nephrocystin-1 (NPHP1) as a potential binding partner of PC-1. NPHP1 is the product of a gene that is mutated in a different form of renal cystic disease, nephronophthisis (NPHP). We show that in vitro pull-down assays and NMR structural studies confirmed the interaction between the PC-1 polyproline motif and the NPHP1 SH3 domain. Furthermore, the two full-length proteins interact through these domains; using a recently generated model system allowing us to track endogenous PC-1, we confirm the interaction between the endogenous proteins. Finally, we show that NPHP1 trafficking to cilia does not require PC-1 and that PC-1 may require NPHP1 to regulate resistance to apoptosis, but not to regulate cell cycle progression. In line with this, we find high levels of apoptosis in renal specimens of NPHP patients. Our data uncover a link between two different ciliopathies, ADPKD and NPHP, supporting the notion that common pathogenetic defects, possibly involving de-regulated apoptosis, underlie renal cyst formation.

  7. A Time Domain Analysis of Gust-Cascade Interaction Noise

    NASA Technical Reports Server (NTRS)

    Nallasamy, M.; Hixon, R.; Sawyer, S. D.; Dyson, R. W.

    2003-01-01

    The gust response of a 2 D cascade is studied by solving the full nonlinear Euler equations employing higher order accurate spatial differencing and time stepping techniques. The solutions exhibit the exponential decay of the two circumferential mode orders of the cutoff blade passing frequency (BPF) tone and propagation of one circumferential mode order at 2BPF, as would be expected for the flow configuration considered. Two frequency excitations indicate that the interaction between the frequencies and the self interaction contribute to the amplitude of the propagating mode.

  8. CC chemokine receptor 10 cell surface presentation in melanocytes is regulated by the novel interaction partner S100A10

    PubMed Central

    Hessner, F.; Dlugos, C. P.; Chehab, T.; Schaefer, C.; Homey, B.; Gerke, V.; Weide, T.; Pavenstädt, H.; Rescher, U.

    2016-01-01

    The superfamily of G-protein-coupled receptors (GPCR) conveys signals in response to various endogenous and exogenous stimuli. Consequently, GPCRs are the most important drug targets. CCR10, the receptor for the chemokines CCL27/CTACK and CCL28/MEC, belongs to the chemokine receptor subfamily of GPCRs and is thought to function in immune responses and tumour progression. However, there is only limited information on the intracellular regulation of CCR10. We find that S100A10, a member of the S100 family of Ca2+ binding proteins, binds directly to the C-terminal cytoplasmic tail of CCR10 and that this interaction regulates the CCR10 cell surface presentation. This identifies S100A10 as a novel interaction partner and regulator of CCR10 that might serve as a target for therapeutic intervention. PMID:26941067

  9. Comparative structural and energetic analysis of WW domain-peptide interactions.

    PubMed

    Schleinkofer, Karin; Wiedemann, Urs; Otte, Livia; Wang, Ting; Krause, Gerd; Oschkinat, Hartmut; Wade, Rebecca C

    2004-11-26

    WW domains are small globular protein interaction modules found in a wide spectrum of proteins. They recognize their target proteins by binding specifically to short linear peptide motifs that are often proline-rich. To infer the determinants of the ligand binding propensities of WW domains, we analyzed 42 WW domains. We built models of the 3D structures of the WW domains and their peptide complexes by comparative modeling supplemented with experimental data from peptide library screens. The models provide new insights into the orientation and position of the peptide in structures of WW domain-peptide complexes that have not yet been determined experimentally. From a protein interaction property similarity analysis (PIPSA) of the WW domain structures, we show that electrostatic potential is a distinguishing feature of WW domains and we propose a structure-based classification of WW domains that expands the existent ligand-based classification scheme. Application of the comparative molecular field analysis (CoMFA), GRID/GOLPE and comparative binding energy (COMBINE) analysis methods permitted the derivation of quantitative structure-activity relationships (QSARs) that aid in identifying the specificity-determining residues within WW domains and their ligand-recognition motifs. Using these QSARs, a new group-specific sequence feature of WW domains that target arginine-containing peptides was identified. Finally, the QSAR models were applied to the design of a peptide to bind with greater affinity than the known binding peptide sequences of the yRSP5-1 WW domain. The prediction was verified experimentally, providing validation of the QSAR models and demonstrating the possibility of rationally improving peptide affinity for WW domains. The QSAR models may also be applied to the prediction of the specificity of WW domains with uncharacterized ligand-binding properties.

  10. Interaction between categorical knowledge and episodic memory across domains

    PubMed Central

    Hemmer, Pernille; Persaud, Kimele

    2014-01-01

    Categorical knowledge and episodic memory have traditionally been viewed as separate lines of inquiry. Here, we present a perspective on the interrelatedness of categorical knowledge and reconstruction from memory. We address three underlying questions: what knowledge do people bring to the task of remembering? How do people integrate that knowledge with episodic memory? Is this the optimal way for the memory system to work? In the review of five studies spanning four category domains (discrete, continuous, temporal, and linguistic), we evaluate the relative contribution and the structure of influence of categorical knowledge on long-term episodic memory. These studies suggest a robustness of peoples’ knowledge of the statistical regularities of the environment, and provide converging evidence of the quality and influence of category knowledge on reconstructive memory. Lastly, we argue that combining categorical knowledge and episodic memory is an efficient strategy of the memory system. PMID:24966848

  11. Bilingualism interacts with domain in a working memory task: evidence from aging.

    PubMed

    Luo, Lin; Craik, Fergus I M; Moreno, Sylvain; Bialystok, Ellen

    2013-03-01

    Younger and older adults who were either monolingual or bilingual were tested with verbal and spatial working memory (WM) span tasks. Aging was associated with a greater decline in spatial WM than in verbal WM, but the age-related declines were equivalent in both language groups. The bilingual participants outperformed the monolinguals in spatial WM, but achieved lower levels of performance than monolinguals in verbal WM. This interaction between bilingualism and WM domain was also consistent across the adult life span. These results are discussed in terms of the interactions between a domain-general executive processing advantage for bilinguals and the domain-specific content of particular WM tasks.

  12. Phosphopeptide interactions with BRCA1 BRCT domains: More than just a motif.

    PubMed

    Wu, Qian; Jubb, Harry; Blundell, Tom L

    2015-03-01

    BRCA1 BRCT domains function as phosphoprotein-binding modules for recognition of the phosphorylated protein-sequence motif pSXXF. While the motif interaction interface provides strong anchor points for binding, protein regions outside the motif have recently been found to be important for binding affinity. In this review, we compare the available structural data for BRCA1 BRCT domains in complex with phosphopeptides in order to gain a more complete understanding of the interaction between phosphopeptides and BRCA1-BRCT domains.

  13. Forms of Social Interaction and Domains of Social Understanding.

    ERIC Educational Resources Information Center

    Nucci, Larry P.

    The five observational studies reported in this paper provide consistent and interlocking testimony for the view that moral events differ qualitatively from social conventional events, and that these two aspects of the social world are associated with qualitatively differing individual-environment interactions. Each of the five studies focuses on…

  14. A Brownian Dynamics Study of the Effects of Cytochrome f Structure and Deletion of Its Small Domain in Interactions with Cytochrome c6 and Plastocyanin in Chlamydomonas reinhardtii

    PubMed Central

    Haddadian, Esmael J.; Gross, Elizabeth L.

    2006-01-01

    The availability of seven different structures of cytochrome f (cyt f) from Chlamydomonas reinhardtii allowed us, using Brownian dynamics simulations, to model interactions between these molecules and their redox partners, plastocyanin (PC) and cytochrome c6 (cyt c6) in the same species to study the effect of cyt f structure on its function. Our results showed that different cyt f structures, which are very similar, produced different reaction rates in interactions with PC and cyt c6. We were able to attribute this to structural differences among these molecules, particularly to a small flexible loop between A-184 and G-191 (which has some of the highest crystallographic temperature factors in all of the cyt f structures) on the cyt f small domain. We also showed that deletion of the cyt f small domain affected cyt c6 more than PC, due to their different binding positions on cyt f. One function of the small domain in cyt f may be to guide PC or cyt c6 to a uniform dock with cyt f, especially due to electrostatic interactions with K-188 and K-189 on this domain. Our results could serve as a good guide for future experimental work on these proteins to understand better the electron transfer process between them. Also, these results demonstrated the sensitivity and the power of the Brownian dynamics simulations in the study of molecular interactions. PMID:16239335

  15. Impact of interaction with a partner or friend on the exposure effects of pornography and erotica.

    PubMed

    Senn, Charlene Y; Desmarais, Serge

    2004-12-01

    Past studies on the effects of sexually explicit materials on women have tended to study them alone, in pairs, or in groups of strangers. By contrast, our study randomly assigned women to bring either a same-sex friend or a male partner to reflect more natural viewing conditions. Discussion between the participant and her companion followed exposure to the sexual images. Women who viewed pornography maintained their (quite high) level of negative mood, whereas women who viewed erotica experienced a substantial improvement in mood. The sex of the companion did not have a direct influence on participants' mood, with discussion improving mood across the board. However, participants' ratings of their satisfaction with the discussion were significantly influenced by the sex of their companion. We suggest that future research should focus more on the interpersonal aspects of male-female relationships when exploring the effects of sexually explicit materials on heterosexual women.

  16. Molecular Basis of Interactions Between SH3 Domain-Containing Proteins and the Proline-Rich Region of the Ubiquitin Ligase Itch.

    PubMed

    Desrochers, Guillaume; Cappadocia, Laurent; Lussier-Price, Mathieu; Ton, Anh-Tien; Ayoubi, Riham; Serohijos, Adrian; Omichinski, James G; Angers, Annie

    2017-02-24

    The ligase Itch plays major roles in signalling pathways by inducing ubiquitylation-dependent degradation of several substrates. Substrate recognition and binding is critical for the regulation of this reaction. Like closely related ligases, Itch can interact with proteins containing a PPxY motif via its WW domains. In addition to these WW domains, Itch possesses a proline-rich region (PRR) that has been shown to interact with several Src Homology 3 (SH3) domain-containing proteins. We have previously established that despite the apparent surface uniformity and conserved fold of SH3 domains, they display different binding mechanisms and affinities for their interaction with the PRR of Itch. Here, we attempt to determine the molecular bases underlying the wide range of binding properties of the Itch PRR. Using pull-down assays combined with mass spectrometry analysis, we show that the Itch PRR preferentially forms complexes with Endophilins, Amphyphisins and Pacsins, but can also target a variety of other SH3 domain-containing proteins. In addition, we map the binding sites of these proteins using a combination of PRR sub-sequences and mutants. We find that different SH3 domains target distinct proline-rich sequences overlapping significantly. We also structurally analyze these protein complexes using crystallography and molecular modelling. These structures depict the position of Itch PRR engaged in a 1:2 protein complex with β-PIX and a 1:1 complex with the other SH3 domain-containing proteins. Taken together, these results reveal the binding preferences of the Itch PRR towards its most common SH3 domain-containing partners, and demonstrate that the PRR region is sufficient for binding.

  17. Inositol Pentakisphosphate Isomers Bind PH Domains with Varying Specificity and Inhibit Phosphoinositide Interactions

    SciTech Connect

    S Jackson; S Al-Saigh; C Schultz; M Junop

    2011-12-31

    PH domains represent one of the most common domains in the human proteome. These domains are recognized as important mediators of protein-phosphoinositide and protein-protein interactions. Phosphoinositides are lipid components of the membrane that function as signaling molecules by targeting proteins to their sites of action. Phosphoinositide based signaling pathways govern a diverse range of important cellular processes including membrane remodeling, differentiation, proliferation and survival. Myo-Inositol phosphates are soluble signaling molecules that are structurally similar to the head groups of phosphoinositides. These molecules have been proposed to function, at least in part, by regulating PH domain-phosphoinositide interactions. Given the structural similarity of inositol phosphates we were interested in examining the specificity of PH domains towards the family of myo-inositol pentakisphosphate isomers. In work reported here we demonstrate that the C-terminal PH domain of pleckstrin possesses the specificity required to discriminate between different myo-inositol pentakisphosphate isomers. The structural basis for this specificity was determined using high-resolution crystal structures. Moreover, we show that while the PH domain of Grp1 does not possess this high degree of specificity, the PH domain of protein kinase B does. These results demonstrate that some PH domains possess enough specificity to discriminate between myo-inositol pentakisphosphate isomers allowing for these molecules to differentially regulate interactions with phosphoinositides. Furthermore, this work contributes to the growing body of evidence supporting myo-inositol phosphates as regulators of important PH domain-phosphoinositide interactions. Finally, in addition to expanding our knowledge of cellular signaling, these results provide a basis for developing tools to probe biological pathway.

  18. Identification of five B-type response regulators as members of a multistep phosphorelay system interacting with histidine-containing phosphotransfer partners of Populus osmosensor

    PubMed Central

    2012-01-01

    Background In plants, the multistep phosphorelay signaling pathway mediates responses to environmental factors and plant hormones. This system is composed of three successive partners: hybrid Histidine-aspartate Kinases (HKs), Histidine-containing Phosphotransfer proteins (HPts), and Response Regulators (RRs). Among the third partners, B-type RR family members are the final output elements of the pathway; they act as transcription factors and clearly play a pivotal role in the early response to cytokinin in Arabidopsis. While interactions studies between partners belonging to the multistep phosphorelay system are mainly focused on protagonists involved in cytokinin or ethylene pathways, very few reports are available concerning partners of osmotic stress signaling pathway. Results In Populus, we identified eight B-type RR proteins, RR12-16, 19, 21 and 22 in the Dorskamp genotype. To assess HPt/B-type RR interactions and consequently determine potential third partners in the osmosensing multistep phosphorelay system, we performed global yeast two-hybrid (Y2H) assays in combination with Bimolecular Fluorescence Complementation (BiFC) assays in plant cells. We found that all B-type RRs are able to interact with HPt predominant partners (HPt2, 7 and 9) of HK1, which is putatively involved in the osmosensing pathway. However, different profiles of interaction are observed depending on the studied HPt. HPt/RR interactions displayed a nuclear localization, while the nuclear and cytosolic localization of HPt and nuclear localization of RR proteins were validated. Although the nuclear localization of HPt/RR interaction was expected, this work constitutes the first evidence of such an interaction in plants. Furthermore, the pertinence of this partnership is reinforced by highlighting a co-expression of B-type RR transcripts and the other partners (HK1 and HPts) belonging to a potential osmosensing pathway. Conclusion Based on the interaction studies between identified B

  19. Mg2+ mediates interaction between the voltage sensor and cytosolic domain to activate BK channels.

    PubMed

    Yang, Huanghe; Hu, Lei; Shi, Jingyi; Delaloye, Kelli; Horrigan, Frank T; Cui, Jianmin

    2007-11-13

    The voltage-sensor domain (VSD) of voltage-dependent ion channels and enzymes is critical for cellular responses to membrane potential. The VSD can also be regulated by interaction with intracellular proteins and ligands, but how this occurs is poorly understood. Here, we show that the VSD of the BK-type K(+) channel is regulated by a state-dependent interaction with its own tethered cytosolic domain that depends on both intracellular Mg(2+) and the open state of the channel pore. Mg(2+) bound to the cytosolic RCK1 domain enhances VSD activation by electrostatic interaction with Arg-213 in transmembrane segment S4. Our results demonstrate that a cytosolic domain can come close enough to the VSD to regulate its activity electrostatically, thereby elucidating a mechanism of Mg(2+)-dependent activation in BK channels and suggesting a general pathway by which intracellular factors can modulate the function of voltage-dependent proteins.

  20. Interaction dynamics of multiple autonomous mobile robots in bounded spatial domains

    NASA Technical Reports Server (NTRS)

    Wang, P. K. C.

    1989-01-01

    A general navigation strategy for multiple autonomous robots in a bounded domain is developed analytically. Each robot is modeled as a spherical particle (i.e., an effective spatial domain about the center of mass); its interactions with other robots or with obstacles and domain boundaries are described in terms of the classical many-body problem; and a collision-avoidance strategy is derived and combined with homing, robot-robot, and robot-obstacle collision-avoidance strategies. Results from homing simulations involving (1) a single robot in a circular domain, (2) two robots in a circular domain, and (3) one robot in a domain with an obstacle are presented in graphs and briefly characterized.

  1. Interaction dynamics of multiple autonomous mobile robots in bounded spatial domains

    NASA Technical Reports Server (NTRS)

    Wang, P. K. C.

    1989-01-01

    A general navigation strategy for multiple autonomous robots in a bounded domain is developed analytically. Each robot is modeled as a spherical particle (i.e., an effective spatial domain about the center of mass); its interactions with other robots or with obstacles and domain boundaries are described in terms of the classical many-body problem; and a collision-avoidance strategy is derived and combined with homing, robot-robot, and robot-obstacle collision-avoidance strategies. Results from homing simulations involving (1) a single robot in a circular domain, (2) two robots in a circular domain, and (3) one robot in a domain with an obstacle are presented in graphs and briefly characterized.

  2. Tetramer formation in Arabidopsis MADS domain proteins: analysis of a protein-protein interaction network

    PubMed Central

    2014-01-01

    Background MADS domain proteins are transcription factors that coordinate several important developmental processes in plants. These proteins interact with other MADS domain proteins to form dimers, and it has been proposed that they are able to associate as tetrameric complexes that regulate transcription of target genes. Whether the formation of functional tetramers is a widespread property of plant MADS domain proteins, or it is specific to few of these transcriptional regulators remains unclear. Results We analyzed the structure of the network of physical interactions among MADS domain proteins in Arabidopsis thaliana. We determined the abundance of subgraphs that represent the connection pattern expected for a MADS domain protein heterotetramer. These subgraphs were significantly more abundant in the MADS domain protein interaction network than in randomized analogous networks. Importantly, these subgraphs are not significantly frequent in a protein interaction network of TCP plant transcription factors, when compared to expectation by chance. In addition, we found that MADS domain proteins in tetramer-like subgraphs are more likely to be expressed jointly than proteins in other subgraphs. This effect is mainly due to proteins in the monophyletic MIKC clade, as there is no association between tetramer-like subgraphs and co-expression for proteins outside this clade. Conclusions Our results support that the tendency to form functional tetramers is widespread in the MADS domain protein-protein interaction network. Our observations also suggest that this trend is prevalent, or perhaps exclusive, for proteins in the MIKC clade. Because it is possible to retrodict several experimental results from our analyses, our work can be an important aid to make new predictions and facilitates experimental research on plant MADS domain proteins. PMID:24468197

  3. One-step affinity purification of recombinant TATA binding proteins utilizing a modular protein interaction partner.

    PubMed

    Shooltz, Dean D; Alberts, Glen L; Triezenberg, Steven J

    2008-06-01

    We describe a rapid and effective procedure for purifying recombinant eukaryotic TATA binding protein (TBP) from Escherichia coli. The method employs an affinity ligand comprising glutathione-S-transferase fused to the carboxyl-terminal activation domain of the transcriptional activator VP16 and an amino-terminal domain (TAND2) of the yeast TBP-associated factor TAF1. TBP can be purified without the need for extrinsic affinity tags, subsequent proteolysis, or downstream clean-up steps. This TBP purification process is rapid (requiring about 4h after bacterial harvest) and does not require sophisticated chromatographic equipment. The resulting material is monodisperse, structured, and functionally active. We demonstrate the efficacy of this method for purifying recombinant full-length or TBP core fragments encoded by yeast, humans and Arabidopsis.

  4. Systematic Analysis Reveals Elongation Factor 2 and α-Enolase as Novel Interaction Partners of AKT2

    PubMed Central

    Bottermann, Katharina; Reinartz, Michael; Barsoum, Marian; Kötter, Sebastian; Gödecke, Axel

    2013-01-01

    AKT2 is one of the three isoforms of the protein kinase AKT being involved in the modulation of cellular metabolism. Since protein-protein interactions are one possibility to convey specificity in signal transduction, we performed AKT2-protein interaction analysis to elucidate their relevance for AKT2-dependent cellular functions. We identified heat shock protein 90 kDa (HSP90), Cdc37, heat shock protein 70 kDa (HSP70), 78 kDa glucose regulated protein (GRP78), tubulin, GAPDH, α-enolase and elongation factor 2 (EF2) as AKT2-interacting proteins by a combination of tandem affinity purification and mass spectrometry in HEK293T cells. Quantitative MS-analysis using stable isotope labeling by amino acids in cell culture (SILAC) revealed that only HSP90 and Cdc37 interact stably with AKT2, whereas the other proteins interact with low affinity with AKT2. The interactions of AKT2 with α-enolase and EF2 were further analyzed in order to uncover the functional relevance of these newly discovered binding partners. Despite the interaction of AKT2 and α-enolase, which was additionally validated by proximity ligation assay (PLA), no significant impact of AKT on α-enolase activity was detected in activity measurements. AKT stimulation via insulin and/or inhibition with the ATP-competitive inhibitor CCT128930 did not alter enzymatic activity of α-enolase. Interestingly, the direct interaction of AKT2 and EF2 was found to be dynamically regulated in embryonic rat cardiomyocytes. Treatment with the PI3-kinase inhibitor LY294002 before stimulation with several hormones stabilized the complex, whereas stimulation alone led to complex dissociation which was analyzed in situ with PLA. Taken together, these findings point to new aspects of AKT2-mediated signal transduction in protein synthesis and glucose metabolism. PMID:23823123

  5. 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*

    PubMed Central

    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

  6. 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.

    PubMed

    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

    2011-11-11

    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.

  7. Domain structures and inter-domain interactions defining the holoenzyme architecture of archaeal d-family DNA polymerase.

    PubMed

    Matsui, Ikuo; Matsui, Eriko; Yamasaki, Kazuhiko; Yokoyama, Hideshi

    2013-07-05

    Archaea-specific D-family DNA polymerase (PolD) forms a dimeric heterodimer consisting of two large polymerase subunits and two small exonuclease subunits. According to the protein-protein interactions identified among the domains of large and small subunits of PolD, a symmetrical model for the domain topology of the PolD holoenzyme is proposed. The experimental evidence supports various aspects of the model. The conserved amphipathic nature of the N-terminal putative α-helix of the large subunit plays a key role in the homodimeric assembly and the self-cyclization of the large subunit and is deeply involved in the archaeal PolD stability and activity. We also discuss the evolutional transformation from archaeal D-family to eukaryotic B-family polymerase on the basis of the structural information.

  8. Domain Structures and Inter-Domain Interactions Defining the Holoenzyme Architecture of Archaeal D-Family DNA Polymerase

    PubMed Central

    Matsui, Ikuo; Matsui, Eriko; Yamasaki, Kazuhiko; Yokoyama, Hideshi

    2013-01-01

    Archaea-specific D-family DNA polymerase (PolD) forms a dimeric heterodimer consisting of two large polymerase subunits and two small exonuclease subunits. According to the protein-protein interactions identified among the domains of large and small subunits of PolD, a symmetrical model for the domain topology of the PolD holoenzyme is proposed. The experimental evidence supports various aspects of the model. The conserved amphipathic nature of the N-terminal putative α-helix of the large subunit plays a key role in the homodimeric assembly and the self-cyclization of the large subunit and is deeply involved in the archaeal PolD stability and activity. We also discuss the evolutional transformation from archaeal D-family to eukaryotic B-family polymerase on the basis of the structural information. PMID:25369811

  9. Does It Make Any Difference if She Is a Mother? An Interactional Perspective on Intimate Partner Violence with a Focus on Motherhood and Pregnancy

    ERIC Educational Resources Information Center

    Vatnar, Solveig Karin Bo; Bjorkly, Stal

    2010-01-01

    The authors report on the impact of motherhood and pregnancy on interactional aspects of intimate partner violence (IPV) among help-seeking women. Is having children a protective or a risk factor for IPV severity, injury, duration, frequency, and mortal danger, controlling for sociodemographics? Regarding interactional aspects of IPV, do survivors…

  10. Does It Make Any Difference if She Is a Mother? An Interactional Perspective on Intimate Partner Violence with a Focus on Motherhood and Pregnancy

    ERIC Educational Resources Information Center

    Vatnar, Solveig Karin Bo; Bjorkly, Stal

    2010-01-01

    The authors report on the impact of motherhood and pregnancy on interactional aspects of intimate partner violence (IPV) among help-seeking women. Is having children a protective or a risk factor for IPV severity, injury, duration, frequency, and mortal danger, controlling for sociodemographics? Regarding interactional aspects of IPV, do survivors…

  11. Interacting domain-specific languages with biological problem solving environments

    NASA Astrophysics Data System (ADS)

    Cickovski, Trevor M.

    Iteratively developing a biological model and verifying results with lab observations has become standard practice in computational biology. This process is currently facilitated by biological Problem Solving Environments (PSEs), multi-tiered and modular software frameworks which traditionally consist of two layers: a computational layer written in a high level language using design patterns, and a user interface layer which hides its details. Although PSEs have proven effective, they still enforce some communication overhead between biologists refining their models through repeated comparison with experimental observations in vitro or in vivo, and programmers actually implementing model extensions and modifications within the computational layer. I illustrate the use of biological Domain-Specific Languages (DSLs) as a middle-level PSE tier to ameliorate this problem by providing experimentalists with the ability to iteratively test and develop their models using a higher degree of expressive power compared to a graphical interface, while saving the requirement of general purpose programming knowledge. I develop two radically different biological DSLs: XML-based BIOLOGO will model biological morphogenesis using a cell-centered stochastic cellular automaton and translate into C++ modules for an object-oriented PSE C OMPUCELL3D, and MDLab will provide a set of high-level Python libraries for running molecular dynamics simulations, using wrapped functionality from the C++ PSE PROTOMOL. I describe each language in detail, including its its roles within the larger PSE and its expressibility in terms of representable phenomena, and a discussion of observations from users of the languages. Moreover I will use these studies to draw general conclusions about biological DSL development, including dependencies upon the goals of the corresponding PSE, strategies, and tradeoffs.

  12. Pinning induced by inter-domain wall interactions in planar magnetic nanowires

    SciTech Connect

    Hayward, T.J.; Bryan, M.T.; Fry, P.W.; Fundi, P.M.; Gibbs, M.R.J.; Allwood, D.A.; Im, M.-Y.; Fischer, P.

    2009-10-30

    We have investigated pinning potentials created by inter-domain wall magnetostatic interactions in planar magnetic nanowires. We show that these potentials can take the form of an energy barrier or an energy well depending on the walls' relative monopole moments, and that the applied magnetic fields required to overcome these potentials are significant. Both transverse and vortex wall pairs are investigated and it is found that transverse walls interact more strongly due to dipolar coupling between their magnetization structures. Simple analytical models which allow the effects of inter-domain wall interactions to be estimated are also presented.

  13. DEP domains: structurally similar but functionally different.

    PubMed

    Consonni, Sarah V; Maurice, Madelon M; Bos, Johannes L

    2014-05-01

    The Dishevelled, EGL-10 and pleckstrin (DEP) domain is a globular protein domain that is present in about ten human protein families with well-defined structural features. A picture is emerging that DEP domains mainly function in the spatial and temporal control of diverse signal transduction events by recruiting proteins to the plasma membrane. DEP domains can interact with various partners at the membrane, including phospholipids and membrane receptors, and their binding is subject to regulation.

  14. Hsp70A and GlsA interact as partner chaperones to regulate asymmetric division in Volvox.

    PubMed

    Cheng, Qian; Pappas, Valeria; Hallmann, Armin; Miller, Stephen M

    2005-10-15

    GlsA, a J-protein chaperone, is required for the asymmetric divisions that set aside germ and somatic cell precursors during embryogenesis in Volvox carteri, and previous evidence indicated that this function requires an intact Hsp70-binding site. To determine if Hsp70A, the only known cytoplasmic Hsp70 in V. carteri, is the chaperone partner of GlsA, we investigated the localization of the two proteins during critical stages of embryogenesis and tested their capacity to interact. We found that a substantial fraction of Hsp70A co-localizes with GlsA, both in interphase and mitotic blastomeres. In addition, Hsp70A coimmunoprecipitated with GlsA, and co-expression of GlsA and Hsp70A variants partially rescued the Gls phenotype of a glsA mutant, whereas neither variant by itself rescued the mutant phenotype. Immunofluorescence analysis demonstrated that GlsA is about equally abundant in all blastomeres at all cleavage stages examined but that Hsp70A is more abundant in anterior (asymmetrically dividing) blastomeres than in posterior (symmetrically dividing) blastomeres during the period of asymmetric division. We conclude that Hsp70A and GlsA function as chaperone partners that regulate asymmetric division and that the relative abundance of Hsp70A in asymmetrically dividing embryos may determine which blastomeres divide asymmetrically and which do not.

  15. When training with a partner is inferior to training alone: the importance of dyad type and interaction quality.

    PubMed

    Crook, Amy E; Beier, Margaret E

    2010-12-01

    Dyad training, where trainees learn in pairs but ultimately perform individually, has been shown to be an effective method for training some skills. The effectiveness of this approach, however, may be tied to the type of task to be trained and the quality of the interaction in the dyad. We report two studies on the effectiveness of dyad training and the role of metacognitive activity for learning a software program. In Study 1, participants completed training alone or with a partner. Performance was assessed individually immediately after training and again after a 1-week nonuse interval. Results of Study 1 suggested that learning retention is superior when people are trained individually. Study 2 examined performance for individuals, task-switching dyads, and interdependent dyads. Results also showed that performance for individuals was superior to dyads and that the type of dyad collaboration did not affect performance. However, partner-prompted metacognitive activity was helpful for interdependent dyads and harmful for task-switching dyads, suggesting that the quality of collaboration varies by dyad type. Our findings suggest that dyad training may not be effective for all types of tasks. Possible boundary conditions for effective dyad training are discussed.

  16. Structural Evidence for Direct Interactions Between the BRCT Domains of Human BRCA1 and a Phospho-Peptide from Human ACC1

    SciTech Connect

    Shen,Y.; Tong, L.

    2008-01-01

    The tandem BRCA1 C-terminal (BRCT) domains are phospho-serine/threonine recognition modules essential for the function of BRCA1. Recent studies suggest that acetyl-CoA carboxylase 1 (ACC1), an enzyme with crucial roles in de novo fatty acid biosynthesis and lipogenesis and essential for cancer cell survival, may be a novel binding partner for BRCA1, through interactions with its BRCT domains. We report here the crystal structure at 3.2 Angstroms resolution of human BRCA1 BRCT domains in complex with a phospho-peptide from human ACC1 (p-ACC1 peptide, with the sequence 1258-DSPPQ-pS-PTFPEAGH-1271), which provides molecular evidence for direct interactions between BRCA1 and ACC1. The p-ACC1 peptide is bound in an extended conformation, located in a groove between the tandem BRCT domains. There are recognizable and significant structural differences to the binding modes of two other phospho-peptides to these domains, from BACH1 and CtIP, even though they share a conserved pSer-Pro-(Thr/Val)-Phe motif. Our studies establish a framework for understanding the regulation of lipid biosynthesis by BRCA1 through its inhibition of ACC1 activity, which could be a novel tumor suppressor function of BRCA1.

  17. The immunoglobulin-like domain is involved in interaction of Neuregulin1 with ErbB

    SciTech Connect

    Eto, Ko . E-mail: etoko@gpo.kumamoto-u.ac.jp; Eda, Kazufumi; Kanemoto, Shintaro; Abe, Shin-ichi

    2006-11-17

    Neuregulin1 (NRG1) is a growth factor that signals through the interaction of the epidermal growth factor (EGF)-like domain with ErbB receptors. An immunoglobulin (Ig)-like domain is contained together with EGF-like domain in the ectodomain of some isoforms generated by alternative splicing, but its role in NRG1 signaling remained unclear. In the present study, we identified a novel isoform of NRG1 containing an Ig-like domain conserved among species from adult Xenopus laevis, which is predominantly expressed in the testis and brain. We generated recombinant proteins for the whole ectodomain and EGF-like domain alone of the isoform to compare their effects on cell proliferation, and phosphorylation of and their association with ErbB receptor, demonstrating that the ectodomain had {approx}10{sup 3}-fold higher abilities than the EGF-like domain. Therefore, the Ig-like domain is probably essential for efficient interaction of an EGF-like domain with ErbB receptors.

  18. An Efficient Semi-supervised Learning Approach to Predict SH2 Domain Mediated Interactions.

    PubMed

    Kundu, Kousik; Backofen, Rolf

    2017-01-01

    Src homology 2 (SH2) domain is an important subclass of modular protein domains that plays an indispensable role in several biological processes in eukaryotes. SH2 domains specifically bind to the phosphotyrosine residue of their binding peptides to facilitate various molecular functions. For determining the subtle binding specificities of SH2 domains, it is very important to understand the intriguing mechanisms by which these domains recognize their target peptides in a complex cellular environment. There are several attempts have been made to predict SH2-peptide interactions using high-throughput data. However, these high-throughput data are often affected by a low signal to noise ratio. Furthermore, the prediction methods have several additional shortcomings, such as linearity problem, high computational complexity, etc. Thus, computational identification of SH2-peptide interactions using high-throughput data remains challenging. Here, we propose a machine learning approach based on an efficient semi-supervised learning technique for the prediction of 51 SH2 domain mediated interactions in the human proteome. In our study, we have successfully employed several strategies to tackle the major problems in computational identification of SH2-peptide interactions.

  19. Membrane interaction of the factor VIIIa discoidin domains in atomistic detail

    PubMed Central

    Madsen, Jesper J.; Ohkubo, Y. Zenmei; Peters, Günther H.; Faber, Johan H.; Tajkhorshid, Emad; Olsen, Ole H.

    2016-01-01

    A recently developed membrane-mimetic model was applied to study membrane interaction and binding of the two anchoring C2-like discoidin domains of human coagulation factor (F)VIIIa, the C1 and C2 domains. Both individual domains, FVIII C1 and FVIII C2, were observed to bind the phospholipid membrane by partial or full insertion of their extruding loops (the spikes). However, the two domains adopted different molecular orientations in their membrane-bound states; FVIII C2 roughly positioned normal to the membrane plane, while FVIII C1 displayed a multitude of tilted orientations. The results indicate that FVIII C1 may be important in modulating the orientation of the FVIIIa molecule to optimize the interaction with FIXa, which is anchored to the membrane via its γ-carboxyglutamic acid-rich (Gla)-domain. Additionally, a structural change was observed in FVIII C1 in the coiled main chain leading the first spike. A tight interaction with one lipid per domain, similar to what has been suggested for the homologous FVa C2, is characterized. Finally, we rationalize known FVIII antibody epitopes and the scarcity of documented hemophilic missense mutations related to improper membrane binding of FVIIIa, based on the prevalent non-specificity of ionic interactions in the simulated membrane-bound states of FVIII C1 and FVIII C2. PMID:26346528

  20. Cryptic protein-protein interaction motifs in the cytoplasmic domain of MHCI proteins.

    PubMed

    Frietze, Karla K; Pappy, Adlai L; Melson, Jack W; O'Driscoll, Emily E; Tyler, Carolyn M; Perlman, David H; Boulanger, Lisa M

    2016-07-19

    Major histocompatibility complex class I (MHCI) proteins present antigenic peptides for immune surveillance and play critical roles in nervous system development and plasticity. Most MHCI are transmembrane proteins. The extracellular domain of MHCI interacts with immunoreceptors, peptides, and co-receptors to mediate immune signaling. While the cytoplasmic domain also plays important roles in endocytic trafficking, cross-presentation of extracellularly derived antigens, and CTL priming, the molecular mediators of cytoplasmic signaling by MHCI remain largely unknown. Here we show that the cytoplasmic domain of MHCI contains putative protein-protein interaction domains known as PDZ (PSD95/disc large/zonula occludens-1) ligands. PDZ ligands are motifs that bind to PDZ domains to organize and mediate signaling at cell-cell contacts. PDZ ligands are short, degenerate motifs, and are therefore difficult to identify via sequence homology alone, but several lines of evidence suggest that putative PDZ ligand motifs in MHCI are under positive selective pressure. Putative PDZ ligands are found in all of the 99 MHCI proteins examined from diverse species, and are enriched in the cytoplasmic domain, where PDZ interactions occur. Both the position of the PDZ ligand and the class of ligand motif are conserved across species, as well as among genes within a species. Non-synonymous substitutions, when they occur, frequently preserve the motif. Of the many specific possible PDZ ligand motifs, a handful are strikingly and selectively overrepresented in MHCI's cytoplasmic domain, but not elsewhere in the same proteins. Putative PDZ ligands in MHCI encompass conserved serine and tyrosine residues that are targets of phosphorylation, a post-translational modification that can regulate PDZ interactions. Finally, proof-of-principle in vitro interaction assays demonstrate that the cytoplasmic domains of particular MHCI proteins can bind directly and specifically to PDZ1 and PDZ4&5 of MAGI

  1. Calsequestrin interacts directly with the cardiac ryanodine receptor luminal domain

    PubMed Central

    Handhle, Ahmed; Ormonde, Chloe E.; Thomas, N. Lowri; Bralesford, Catherine; Williams, Alan J.; Lai, F. Anthony

    2016-01-01

    ABSTRACT Cardiac muscle contraction requires sarcoplasmic reticulum (SR) Ca2+ release mediated by the quaternary complex comprising the ryanodine receptor 2 (RyR2), calsequestrin 2 (CSQ2), junctin (encoded by ASPH) and triadin. Here, we demonstrate that a direct interaction exists between RyR2 and CSQ2. Topologically, CSQ2 binding occurs at the first luminal loop of RyR2. Co-expression of RyR2 and CSQ2 in a human cell line devoid of the other quaternary complex proteins results in altered Ca2+-release dynamics compared to cells expressing RyR2 only. These findings provide a new perspective for understanding the SR luminal Ca2+ sensor and its involvement in cardiac physiology and disease. PMID:27609834

  2. Higher risk of death among MEN1 patients with mutations in the JunD interacting domain: a Groupe d'etude des Tumeurs Endocrines (GTE) cohort study.

    PubMed

    Thevenon, Julien; Bourredjem, Abderrahmane; Faivre, Laurence; Cardot-Bauters, Catherine; Calender, Alain; Murat, Arnaud; Giraud, Sophie; Niccoli, Patricia; Odou, Marie-Françoise; Borson-Chazot, Françoise; Barlier, Anne; Lombard-Bohas, Catherine; Clauser, Eric; Tabarin, Antoine; Parfait, Béatrice; Chabre, Olivier; Castermans, Emilie; Beckers, Albert; Ruszniewski, Philippe; Le Bras, Morgane; Delemer, Brigitte; Bouchard, Philippe; Guilhem, Isabelle; Rohmer, Vincent; Goichot, Bernard; Caron, Philippe; Baudin, Eric; Chanson, Philippe; Groussin, Lionel; Du Boullay, Hélène; Weryha, Georges; Lecomte, Pierre; Penfornis, Alfred; Bihan, Hélène; Archambeaud, Françoise; Kerlan, Véronique; Duron, Françoise; Kuhn, Jean-Marc; Vergès, Bruno; Rodier, Michel; Renard, Michel; Sadoul, Jean-Louis; Binquet, Christine; Goudet, Pierre

    2013-05-15

    Multiple endocrine neoplasia syndrome type 1 (MEN1), which is secondary to mutation of the MEN1 gene, is a rare autosomal-dominant disease that predisposes mutation carriers to endocrine tumors. Although genotype-phenotype studies have so far failed to identify any statistical correlations, some families harbor recurrent tumor patterns. The function of MENIN is unclear, but has been described through the discovery of its interacting partners. Mutations in the interacting domains of MENIN functional partners have been shown to directly alter its regulation abilities. We report on a cohort of MEN1 patients from the Groupe d'étude des Tumeurs Endocrines. Patients with a molecular diagnosis and a clinical follow-up, totaling 262 families and 806 patients, were included. Associations between mutation type, location or interacting factors of the MENIN protein and death as well as the occurrence of MEN1-related tumors were tested using a frailty Cox model to adjust for potential heterogeneity across families. Accounting for the heterogeneity across families, the overall risk of death was significantly higher when mutations affected the JunD interacting domain (adjusted HR = 1.88: 95%-CI = 1.15-3.07). Patients had a higher risk of death from cancers of the MEN1 spectrum (HR = 2.34; 95%-CI = 1.23-4.43). This genotype-phenotype correlation study confirmed the lack of direct genotype-phenotype correlations. However, patients with mutations affecting the JunD interacting domain had a higher risk of death secondary to a MEN1 tumor and should thus be considered for surgical indications, genetic counseling and follow-up.

  3. bHLH05 Is an Interaction Partner of MYB51 and a Novel Regulator of Glucosinolate Biosynthesis in Arabidopsis1[W][OPEN

    PubMed Central

    Gigolashvili, Tamara

    2014-01-01

    By means of yeast (Saccharomyces cerevisiae) two-hybrid screening, we identified basic helix-loop-helix transcription factor05 (bHLH05) as an interacting partner of MYB51, the key regulator of indolic glucosinolates (GSLs) in Arabidopsis (Arabidopsis thaliana). Furthermore, we show that bHLH04, bHLH05, and bHLH06/MYC2 also interact with other R2R3-MYBs regulating GSL biosynthesis. Analysis of bhlh loss-of-function mutants revealed that the single bhlh mutants retained GSL levels that were similar to those in wild-type plants, whereas the triple bhlh04/05/06 mutant was depleted in the production of GSL. Unlike bhlh04/06 and bhlh05/06 mutants, the double bhlh04/05 mutant was strongly affected in the production of GSL, pointing to a special role of bHLH04 and bHLH05 in the control of GSL levels in the absence of jasmonic acid. The combination of two specific gain-of-function alleles of MYB and bHLH proteins had an additive effect on GSL levels, as demonstrated by the analysis of the double MYB34-1D bHLH05D94N mutant, which produces 20-fold more indolic GSLs than bHLH05D94N and ecotype Columbia-0 of Arabidopsis. The amino acid substitution D94N in bHLH05D94N negatively affects the interaction with JASMONATE-ZIM DOMAIN protein, thereby resulting in constitutive activation of bHLH05 and mimicking jasmonic acid treatment. Our study revealed the bHLH04, bHLH05, and bHLH06/MYC2 factors as novel regulators of GSL biosynthesis in Arabidopsis. PMID:25049362

  4. Structural interactions between lipids, water and S1-S4 voltage-sensing domains

    PubMed Central

    Krepkiy, Dmitriy; Gawrisch, Klaus; Swartz, Kenton J.

    2012-01-01

    Membrane proteins serve crucial signaling and transport functions, yet relatively little is known about their structures in membrane environments or how lipids interact with these proteins. For voltage-activated ion channels, X-ray structures suggest that the mobile voltage-sensing S4 helix would be exposed to the membrane, and functional studies reveal that lipid modification can profoundly alter channel activity. Here we use solid-state NMR to investigate structural interactions of lipids and water with S1-S4 voltage-sensing domains, and to explore whether lipids influence the structure of the protein. Our results demonstrate that S1-S4 domains exhibit extensive interactions with lipids, and that these domains are heavily hydrated when embedded in a membrane. We also find evidence for preferential interactions of anionic lipids with S1-S4 domains, and that these interactions have lifetimes on the timescale of 10−3s. Arg residues within S1-S4 domains are well-hydrated and are positioned in close proximity to lipids, exhibiting local interactions with both lipid head groups and acyl chains. Comparative studies with a positively charged lipid lacking a phosphodiester group reveal that this lipid modification has only modest effects on the structure and hydration of S1-S4 domains. Taken together, our results demonstrate that Arg residues in S1-S4 voltage-sensing domains reside in close proximity to the hydrophobic interior of the membrane, yet are well-hydrated, a requirement for carrying charge and driving protein motions in response to changes in membrane voltage. PMID:22858867

  5. Simultaneous prediction of binding free energy and specificity for PDZ domain-peptide interactions

    NASA Astrophysics Data System (ADS)

    Crivelli, Joseph J.; Lemmon, Gordon; Kaufmann, Kristian W.; Meiler, Jens

    2013-12-01

    Interactions between protein domains and linear peptides underlie many biological processes. Among these interactions, the recognition of C-terminal peptides by PDZ domains is one of the most ubiquitous. In this work, we present a mathematical model for PDZ domain-peptide interactions capable of predicting both affinity and specificity of binding based on X-ray crystal structures and comparative modeling with R osetta. We developed our mathematical model using a large phage display dataset describing binding specificity for a wild type PDZ domain and 91 single mutants, as well as binding affinity data for a wild type PDZ domain binding to 28 different peptides. Structural refinement was carried out through several R osetta protocols, the most accurate of which included flexible peptide docking and several iterations of side chain repacking and backbone minimization. Our findings emphasize the importance of backbone flexibility and the energetic contributions of side chain-side chain hydrogen bonds in accurately predicting interactions. We also determined that predicting PDZ domain-peptide interactions became increasingly challenging as the length of the peptide increased in the N-terminal direction. In the training dataset, predicted binding energies correlated with those derived through calorimetry and specificity switches introduced through single mutations at interface positions were recapitulated. In independent tests, our best performing protocol was capable of predicting dissociation constants well within one order of magnitude of the experimental values and specificity profiles at the level of accuracy of previous studies. To our knowledge, this approach represents the first integrated protocol for predicting both affinity and specificity for PDZ domain-peptide interactions.

  6. HIV-1 p6-Another viral interaction partner to the host cellular protein cyclophilin A.

    PubMed

    Solbak, Sara M Ø; Reksten, Tove R; Röder, Rene; Wray, Victor; Horvli, Ole; Raae, Arnt J; Henklein, Petra; Henklein, Peter; Fossen, Torgils

    2012-04-01

    The 52-amino acid human immunodeficiency virus type 1 (HIV-1) p6 protein has previously been recognized as a docking site for several cellular and viral binding factors and is important for the formation of infectious viruses. A particular structural feature of p6 is the notably high relative content of proline residues, located at positions 5, 7, 10, 11, 24, 30, 37 and 49 in the sequence. Proline cis/trans isomerism was detected for all these proline residues to such an extent that more than 40% of all p6 molecules contain at least one proline in a cis conformation. 2D (1)H nuclear magnetic resonance analysis of full-length HIV-1 p6 and p6 peptides established that cyclophilin A (CypA) interacts as a peptidyl-prolyl cis/trans isomerase with all proline residues of p6. Only catalytic amounts of CypA were necessary for the interaction with p6 to occur, strongly suggesting that the observed interaction is highly relevant in vivo. In addition, surface plasmon resonance studies revealed binding of full-length p6 to CypA, and that this binding was significantly stronger than any of its N- or C-terminal peptides. This study demonstrates the first identification of an interaction between HIV-1 p6 and the host cellular protein CypA. The mode of interaction involves both transient enzyme-substrate interactions and a more stable binding. The binding motifs of p6 to Tsg-101, ALIX and Vpr coincide with binding regions and catalytic sites of p6 to CypA, suggesting a potential role of CypA in modulating functional interactions of HIV-1.

  7. Apolipoprotein E4 domain interaction accelerates diet-induced atherosclerosis in hypomorphic Arg-61 Apoe mice

    PubMed Central

    Eberlé, Delphine; Kim, Roy Y.; Luk, Fu Sang; de Mochel, Nabora Soledad Reyes; Gaudreault, Nathalie; Olivas, Victor R.; Kumar, Nikit; Posada, Jessica M.; Birkeland, Andrew C.; Rapp, Joseph H.; Raffai, Robert L.

    2012-01-01

    Objective Apolipoprotein (apo) E4 is an established risk factor for atherosclerosis, but the structural components underlying this association remain unclear. ApoE4 is characterized by two biophysical properties: domain interaction and molten globule state. Substituting Arg-61 for Thr-61 in mouse apoE introduces domain interaction without molten globule state, allowing us to delineate potential pro-atherogenic effects of domain interaction in vivo. Methods and Results We studied atherosclerosis susceptibility of hypomorphic Apoe mice expressing either Thr-61 or Arg-61 apoE (ApoeTh/h or ApoeRh/h mice). On a chow diet, both mouse models were normo-lipidemic with similar levels of plasma apoE and lipoproteins. However, on a high cholesterol diet, ApoeRh/h mice displayed increased levels of total plasma cholesterol and VLDL as well as larger atherosclerotic plaques in the aortic root, arch and descending aorta compared to ApoeTh/h mice. In addition, evidence of cellular dysfunction was identified in peritoneal ApoeRh/h macrophages which released lower amounts of apoE in culture medium and displayed increased expression of MHC class II molecules. Conclusions These data indicate that domain interaction mediates pro-atherogenic effects of apoE4 in part by modulating lipoprotein metabolism and macrophage biology. Pharmaceutical targeting of domain interaction could lead to new treatments for atherosclerosis in apoE4 individuals. PMID:22441102

  8. Novel modular domain PB1 recognizes PC motif to mediate functional protein–protein interactions

    PubMed Central

    Ito, Takashi; Matsui, Yasushi; Ago, Tetsuro; Ota, Kazuhisa; Sumimoto, Hideki

    2001-01-01

    Modular domains mediating specific protein–protein interactions play central roles in the formation of complex regulatory networks to execute various cellular activities. Here we identify a novel domain PB1 in the budding yeast protein Bem1p, which functions in polarity establishment, and mammalian p67phox, which activates the microbicidal phagocyte NADPH oxidase. Each of these specifically recognizes an evolutionarily conserved PC motif to interact directly with Cdc24p (an essential protein for cell polarization) and p40phox (a component of the signaling complex for the oxidase), respectively. Swapping the PB1 domain of Bem1p with that of p67phox, which abolishes its interaction with Cdc24p, confers on cells temperature- sensitive growth and a bilateral mating defect. These phenotypes are suppressed by a mutant Cdc24p harboring the PC motif-containing region of p40phox, which restores the interaction with the altered Bem1p. This domain-swapping experiment demonstrates that Bem1p function requires interaction with Cdc24p, in which the PB1 domain and the PC motif participate as responsible modules. PMID:11483497

  9. The Janus Kinase (JAK) FERM and SH2 Domains: Bringing Specificity to JAK–Receptor Interactions

    PubMed Central

    Ferrao, Ryan; Lupardus, Patrick J.

    2017-01-01

    The Janus kinases (JAKs) are non-receptor tyrosine kinases essential for signaling in response to cytokines and interferons and thereby control many essential functions in growth, development, and immune regulation. JAKs are unique among tyrosine kinases for their constitutive yet non-covalent association with class I and II cytokine receptors, which upon cytokine binding bring together two JAKs to create an active signaling complex. JAK association with cytokine receptors is facilitated by N-terminal FERM and SH2 domains, both of which are classical mediators of peptide interactions. Together, the JAK FERM and SH2 domains mediate a bipartite interaction with two distinct receptor peptide motifs, the proline-rich “Box1” and hydrophobic “Box2,” which are present in the intracellular domain of cytokine receptors. While the general sidechain chemistry of Box1 and Box2 peptides is conserved between receptors, they share very weak primary sequence homology, making it impossible to posit why certain JAKs preferentially interact with and signal through specific subsets of cytokine receptors. Here, we review the structure and function of the JAK FERM and SH2 domains in light of several recent studies that reveal their atomic structure and elucidate interaction mechanisms with both the Box1 and Box2 receptor motifs. These crystal structures demonstrate how evolution has repurposed the JAK FERM and SH2 domains into a receptor-binding module that facilitates interactions with multiple receptors possessing diverse primary sequences. PMID:28458652

  10. Gene by Social-Context Interactions for Number of Sexual Partners Among White Male Youths: Genetics-informed Sociology

    PubMed Central

    Guo, Guang; Tong, Yuying; Cai, Tianji

    2010-01-01

    In this study, we set out to investigate whether introducing molecular genetic measures into an analysis of sexual partner variety will yield novel sociological insights. The data source is the white male DNA sample in the National Longitudinal Study of Adolescent Health. Our empirical analysis has produced a robust protective effect of the 9R/9R genotype relative to the Any10R genotype in the dopamine transporter gene (DAT1). The gene-environment interaction analysis demonstrates that the protective effect of 9R/9R tends to be lost in schools in which higher proportions of students start having sex early or among those with relatively low levels of cognitive ability. Our genetics-informed sociological analysis suggests that the “one size” of a single social theory may not fit all. Explaining a human trait or behavior may require a theory that accommodates the complex interplay between social contextual and individual influences and genetic predispositions. PMID:19569400

  11. Identification of extracellular signal-regulated kinase 3 as a new interaction partner of cyclin D3

    SciTech Connect

    Sun Maoyun; Wei Yuanyan; Yao Luyang; Xie Jianhui; Chen Xiaoning; Wang Hanzhou; Jiang Jianhai; Gu Jianxin . E-mail: jxgu@shmu.edu.cn

    2006-02-03

    Cyclin D3, like cyclin D1 and D2 isoforms, is a crucial component of the core cell cycle machinery in mammalian cells. It also exhibits its unique properties in many other physiological processes. In the present study, using yeast two-hybrid screening, we identified ERK3, an atypical mitogen-activated protein kinase (MAPK), as a cyclin D3 binding partner. GST pull-down assays showed that cyclin D3 interacts directly and specifically with ERK3 in vitro. The binding of cyclin D3 and ERK3 was further confirmed in vivo by co-immunoprecipitation assay and confocal microscopic analysis. Moreover, carboxy-terminal extension of ERK3 was responsible for its association with intact cyclin D3. These findings further expand distinct roles of cyclin D3 and suggest the potential activity of ERK3 in cell proliferation.

  12. Gene by social context interactions for number of sexual partners among white male youths: genetics-informed sociology.

    PubMed

    Guo, Guang; Tong, Yuying; Cai, Tianji

    2008-01-01

    This study sets out to investigate whether introducing molecular genetic measures into an analysis of sexual partner variety will yield novel sociological insights. The data source is the white male DNA sample in the National Longitudinal Study of Adolescent Health. The authors' empirical gene-environment interaction analysis produces a robust protective effect of the 9R/9R genotype relative to the Any10R genotype in the dopamine transporter gene (DAT1). This protective effect tends to be lost in schools in which higher proportions of students start having sex early, as well as in individuals with relatively low cognitive ability. The genetics-informed socio logical analysis here suggests that explaining a human trait or behavior may require a theory that accommodates the complex interplay between social contextual and individual influences and genetic predispositions.

  13. Application of histone modification-specific interaction domains as an alternative to antibodies

    PubMed Central

    Kungulovski, Goran; Kycia, Ina; Tamas, Raluca; Jurkowska, Renata Z.; Kudithipudi, Srikanth; Henry, Chisato; Reinhardt, Richard; Labhart, Paul

    2014-01-01

    Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies. PMID:25301795

  14. Structures of and Interactions Between Domains of Trigger Factor from Thermotoga maritima

    SciTech Connect

    Martinez-Hackert,E.; Hendrickson, W.

    2007-01-01

    Trigger factor (TF) is a eubacterial chaperone that associates with ribosomes at the peptide-exit tunnel and also occurs in excess free in the cytosol. TF is a three-domain protein that appears to exist in a dynamic equilibrium of oligomerization states and interdomain conformations. X-ray crystallography and chemical cross-linking were used to study the roles of the N- and C-terminal domains of Thermotoga maritima TF in TF oligomerization and chaperone activity. The structural conservation of both the N- and C-terminal TF domains was unambiguously established. The biochemical and crystallographic data reveal a tendency for these domains to partake in diverse and apparently nonspecific protein-protein interactions. It is found that the T. maritima and Escherichia coli TF surfaces lack evident exposed hydrophobic patches. Taken together, these data suggest that TF chaperones could interact with nascent proteins via hydrophilic surfaces.

  15. The role of DNA helicases and their interaction partners in genome stability and meiotic recombination in plants.

    PubMed

    Knoll, Alexander; Puchta, Holger

    2011-03-01

    DNA helicases are enzymes that are able to unwind DNA by the use of the energy-equivalent ATP. They play essential roles in DNA replication, DNA repair, and DNA recombination in all organisms. As homologous recombination occurs in somatic and meiotic cells, the same proteins may participate in both processes, albeit not necessarily with identical functions. DNA helicases involved in genome stability and meiotic recombination are the focus of this review. The role of these enzymes and their characterized interaction partners in plants will be summarized. Although most factors are conserved in eukaryotes, plant-specific features are becoming apparent. In the RecQ helicase family, Arabidopsis thaliana RECQ4A has been shown before to be the functional homologue of the well-researched baker's yeast Sgs1 and human BLM proteins. It was surprising to find that its interaction partners AtRMI1 and AtTOP3α are absolutely essential for meiotic recombination in plants, where they are central factors of a formerly underappreciated dissolution step of recombination intermediates. In the expanding group of anti-recombinases, future analysis of plant helicases is especially promising. While no FBH1 homologue is present, the Arabidopsis genome contains homologues of both SRS2 and RTEL1. Yeast and mammals, on the other hand. only possess homologues of either one or the other of these helicases. Plants also contain several other classes of helicases that are known from other organisms to be involved in the preservation of genome stability: FANCM is conserved with parts of the human Fanconi anaemia proteins, as are homologues of the Swi2/Snf2 family and of PIF1.

  16. A physical model describing the interaction of nuclear transport receptors with FG nucleoporin domain assemblies.

    PubMed

    Zahn, Raphael; Osmanović, Dino; Ehret, Severin; Araya Callis, Carolina; Frey, Steffen; Stewart, Murray; You, Changjiang; Görlich, Dirk; Hoogenboom, Bart W; Richter, Ralf P

    2016-04-08

    The permeability barrier of nuclear pore complexes (NPCs) controls bulk nucleocytoplasmic exchange. It consists of nucleoporin domains rich in phenylalanine-glycine motifs (FG domains). As a bottom-up nanoscale model for the permeability barrier, we have used planar films produced with three different end-grafted FG domains, and quantitatively analyzed the binding of two different nuclear transport receptors (NTRs), NTF2 and Importin β, together with the concomitant film thickness changes. NTR binding caused only moderate changes in film thickness; the binding isotherms showed negative cooperativity and could all be mapped onto a single master curve. This universal NTR binding behavior - a key element for the transport selectivity of the NPC - was quantitatively reproduced by a physical model that treats FG domains as regular, flexible polymers, and NTRs as spherical colloids with a homogeneous surface, ignoring the detailed arrangement of interaction sites along FG domains and on the NTR surface.

  17. Integrating natural language processing and web GIS for interactive knowledge domain visualization

    NASA Astrophysics Data System (ADS)

    Du, Fangming

    Recent years have seen a powerful shift towards data-rich environments throughout society. This has extended to a change in how the artifacts and products of scientific knowledge production can be analyzed and understood. Bottom-up approaches are on the rise that combine access to huge amounts of academic publications with advanced computer graphics and data processing tools, including natural language processing. Knowledge domain visualization is one of those multi-technology approaches, with its aim of turning domain-specific human knowledge into highly visual representations in order to better understand the structure and evolution of domain knowledge. For example, network visualizations built from co-author relations contained in academic publications can provide insight on how scholars collaborate with each other in one or multiple domains, and visualizations built from the text content of articles can help us understand the topical structure of knowledge domains. These knowledge domain visualizations need to support interactive viewing and exploration by users. Such spatialization efforts are increasingly looking to geography and GIS as a source of metaphors and practical technology solutions, even when non-georeferenced information is managed, analyzed, and visualized. When it comes to deploying spatialized representations online, web mapping and web GIS can provide practical technology solutions for interactive viewing of knowledge domain visualizations, from panning and zooming to the overlay of additional information. This thesis presents a novel combination of advanced natural language processing - in the form of topic modeling - with dimensionality reduction through self-organizing maps and the deployment of web mapping/GIS technology towards intuitive, GIS-like, exploration of a knowledge domain visualization. A complete workflow is proposed and implemented that processes any corpus of input text documents into a map form and leverages a web

  18. Introduction of human apolipoprotein E4 "domain interaction" into mouse apolipoprotein E.

    PubMed

    Raffai, R L; Dong, L M; Farese, R V; Weisgraber, K H

    2001-09-25

    Human apolipoprotein E4 (apoE4) binds preferentially to lower density lipoproteins, including very low density lipoproteins, and is associated with increased risk of atherosclerosis and neurodegenerative disorders, including Alzheimer's disease. This binding preference is the result of the presence of Arg-112, which causes Arg-61 in the amino-terminal domain to interact with Glu-255 in the carboxyl-terminal domain. ApoE2 and apoE3, which have Cys-112, bind preferentially to high density lipoproteins (HDL) and do not display apoE4 domain interaction. Mouse apoE, like apoE4, contains the equivalent of Arg-112 and Glu-255, but lacks the critical Arg-61 equivalent (it contains Thr-61). Thus, mouse apoE does not display apoE4 domain interaction and, as a result, behaves like human apoE3, including preferential binding to HDL. To assess the potential role of apoE4 domain interaction in atherosclerosis and neurodegeneration, we sought to introduce apoE4 domain interaction into mouse apoE. Replacing Thr-61 in mouse apoE with arginine converted the binding preference from HDL to very low density lipoproteins in vitro, suggesting that apoE4 domain interaction could be introduced into mouse apoE in vivo. Using gene targeting in embryonic stem cells, we created mice expressing Arg-61 apoE. Heterozygous Arg-61/wild-type apoE mice displayed two phenotypes found in human apoE4/E3 heterozygotes: preferential binding to lower density lipoproteins and reduced abundance of Arg-61 apoE in the plasma, reflecting its more rapid catabolism. These findings demonstrate the successful introduction of apoE4 domain interaction into mouse apoE in vivo. The Arg-61 apoE mouse model will allow the effects of apoE4 domain interaction in lipoprotein metabolism, atherosclerosis, and neurodegeneration to be determined.

  19. Crystal structure and interaction studies of the human FBxo3 ApaG domain

    PubMed Central

    Krzysiak, Troy C.; Chen, Bill B.; Lear, Travis; Mallampalli, Rama K.; Gronenborn, Angela M.

    2016-01-01

    Transcriptional activation of proinflammatory cytokines, mediated by tumor necrosis factor receptor-associated factors (TRAFs), is in part triggered by the degradation of the F-box protein, FBxl2, via an E3 ligase that contains another F-box protein, FBxo3. The ApaG domain of FBxo3 is required for the interaction with and degradation of FBxl2 [Mallampalli RK et al., (2013) J Immunol 191, 5247–5255]. Here, we report the X-ray structure of the human FBxo3 ApaG domain, residues 278–407, at 2.0 Å resolution. Like bacterial ApaG proteins, this domain is characterized by a classic Immunoglobin/Fibronectin III-type fold, comprising a seven-stranded β-sheet core, surrounded by four extended loops. Although cation binding had been proposed for bacterial ApaG proteins, no interactions with Mg2+ or Co2+ were detected for the human ApaG domain. In addition, dinucleotide polyphosphates, which have been reported to be second messengers in the inflammation response and targets of the bacterial apaG-containing operon, are not bound by the human ApaG domain. In the context of the full-length protein, loop 1, comprising residues 294–303, is critical for the interaction with FBxl2. However, titration of the individual ApaG domain with a 15-mer FBxl2 peptide that was phosphorylated on the crucial T404, as well as the inability of the ApaG domain to interact with full-length FBxl2, assessed by coimmunoprecipitation, indicate that the ApaG domain alone is necessary, but not sufficient for binding and degradation of FBxl2. PMID:27010866

  20. Investigating the Role of Large-Scale Domain Dynamics in Protein-Protein Interactions

    PubMed Central

    Delaforge, Elise; Milles, Sigrid; Huang, Jie-rong; Bouvier, Denis; Jensen, Malene Ringkjøbing; Sattler, Michael; Hart, Darren J.; Blackledge, Martin

    2016-01-01

    Intrinsically disordered linkers provide multi-domain proteins with degrees of conformational freedom that are often essential for function. These highly dynamic assemblies represent a significant fraction of all proteomes, and deciphering the physical basis of their interactions represents a considerable challenge. Here we describe the difficulties associated with mapping the large-scale domain dynamics and describe two recent examples where solution state methods, in particular NMR spectroscopy, are used to investigate conformational exchange on very different timescales. PMID:27679800

  1. Spontaneous domain nucleation under in-plane fields in ultrathin films with Dzyaloshinskii-Moriya interaction

    NASA Astrophysics Data System (ADS)

    Agrawal, Parnika; Bauer, Uwe; Beach, Geoffrey S. D.

    2015-05-01

    In this paper, we show that applying a hard axis field reduces the energy barrier for the spontaneous formation of a multi-domain state in magnetic ultrathin films sandwiched between a heavy metal and an oxide. This provides a simple technique to generate a metastable multi-domain state in magnetic films where the ground state is uniform. This approach could be particularly interesting in materials with strong Dzyaloshinskii-Moriya interaction as a means to realize metastable chiral textures.

  2. Comparing domain interactions within antibody Fabs with kappa and lambda light chains

    PubMed Central

    Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W.; Dickey, Mark; Froning, Karen; Conner, Elaine M.; Cujec, Thomas P.; Demarest, Stephen J.

    2016-01-01

    ABSTRACT IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates. PMID:27454112

  3. Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

    PubMed

    Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

    2016-10-01

    IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

  4. Dynamic combinatorial interactions of RUNX1 and cooperating partners regulates megakaryocytic differentiation in cell line models.

    PubMed

    Pencovich, Niv; Jaschek, Ram; Tanay, Amos; Groner, Yoram

    2011-01-06

    Specific interactions of transcription factors (TFs) with their targets are crucial for specifying gene expression programs during cell differentiation. How specificity is maintained despite limited selectivity of individual TF-DNA interactions is not fully understood. RUNX1 TF is among the most frequently mutated genes in human leukemia and an important regulator of megakaryopoiesis. We used megakaryocytic cell lines to characterize the network of RUNX1 targets and cooperating TFs in differentiating megakaryocytes and demonstrated how dynamic partnerships between RUNX1 and cooperating TFs facilitated regulatory plasticity and specificity during this process. After differentiation onset, RUNX1 directly activated a large number of genes through interaction with preexisting and de novo binding sites. Recruitment of RUNX1 to de novo occupied sites occurred at H3K4me1-marked preprogrammed enhancers. A significant number of these de novo bound sites lacked RUNX motif but were occupied by AP-1 TFs. Reciprocally, AP-1 TFs were up-regulated by RUNX1 after 12-O-tetradecanoylphorbol-13-acetate induction and recruited to RUNX1-occupied sites lacking AP-1 motifs. At other differentiation stages, additional combinatorial interactions occurred between RUNX1 and its coregulators, GATA1 and ETS. The findings suggest that in differentiating megakaryocytic cell lines, RUNX1 cooperates with GATA1, AP-1, and ETS to orchestrate cell-specific transcription programs through dynamic TF partnerships.

  5. The interplay of communication device output mode and interaction style between nonspeaking persons and their speaking partners.

    PubMed

    Higginbotham, D J

    1989-08-01

    This study sought to determine how augmentative communication device output modes differentially affected various aspects of interactions between nonspeaking persons (NSPs) and their speaking partners (SPs). It was hypothesized that when an electronic output display (EOD) was added to a communication board, the semipermanent display of information would lessen the dyad's need to adopt specialized turn and message formulation conventions, permitting the NSP to construct more complex messages with fewer communication breakdowns. A series of 10 interactional teaching tasks were recorded for two adult male nonhandicapped dyads performing under the two output conditions (+/- EOD). Interaction transcripts were analyzed with regard to quantitative differences within and between dyads with respect to turn taking, message formulation, propositional content, and several types of insertion sequences (guessing, confirmation queries, message reformulations). With the exception of message reformulation, changes due to output mode were nonexistent or inconsistent for the variables measured within and across dyads. The addition of the EOD significantly lowered the rate of message reformulation and the total number of reformulation-related turns. Results are discussed with regard to research and clinical implications for augmentative communication.

  6. Establishment of a Protein Frequency Library and Its Application in the Reliable Identification of Specific Protein Interaction Partners*

    PubMed Central

    Boulon, Séverine; Ahmad, Yasmeen; Trinkle-Mulcahy, Laura; Verheggen, Céline; Cobley, Andy; Gregor, Peter; Bertrand, Edouard; Whitehorn, Mark; Lamond, Angus I.

    2010-01-01

    The reliable identification of protein interaction partners and how such interactions change in response to physiological or pathological perturbations is a key goal in most areas of cell biology. Stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry has been shown to provide a powerful strategy for characterizing protein complexes and identifying specific interactions. Here, we show how SILAC can be combined with computational methods drawn from the business intelligence field for multidimensional data analysis to improve the discrimination between specific and nonspecific protein associations and to analyze dynamic protein complexes. A strategy is shown for developing a protein frequency library (PFL) that improves on previous use of static “bead proteomes.” The PFL annotates the frequency of detection in co-immunoprecipitation and pulldown experiments for all proteins in the human proteome. It can provide a flexible and objective filter for discriminating between contaminants and specifically bound proteins and can be used to normalize data values and facilitate comparisons between data obtained in separate experiments. The PFL is a dynamic tool that can be filtered for specific experimental parameters to generate a customized library. It will be continuously updated as data from each new experiment are added to the library, thereby progressively enhancing its utility. The application of the PFL to pulldown experiments is especially helpful in identifying either lower abundance or less tightly bound specific components of protein complexes that are otherwise lost among the large, nonspecific background. PMID:20023298

  7. The measles virus phosphoprotein interacts with the linker domain of STAT1

    SciTech Connect

    Devaux, Patricia Priniski, Lauren; Cattaneo, Roberto

    2013-09-15

    The measles virus (MV) phosphoprotein (P) and V proteins block the interferon (IFN) response by impeding phosphorylation of the signal transducer and activator of transcription 1 (STAT1) by the Janus kinase 1 (JAK1). We characterized how STAT1 mutants interact with P and JAK1 phosphorylation. Certain mutants of the linker, the Src-homology 2 domain (SH2), or the transactivation domain had reduced or abolished phosphorylation through JAK1 after IFN treatment. Other mutants, mainly localized in the linker, failed to interact with P as documented by the lack of interference with nuclear translocation. Thus the functional footprint of P on STAT1 localizes mainly to the linker domain; there is also some overlap with the STAT1 phosphorylation functional footprint on the SH2 domain. Based on these observations, we discuss how the MV-P might operate to inhibit the JAK/STAT pathway. - Highlights: • Residue in the linker and SH2 domains of STAT1 are important for MV-P interaction. • Residue in the linker and SH2 domains of STAT1 are important for STAT1 phosphorylation. • Residues interferring with both functions have similar location on STAT1. • The viral P and V proteins may operate in concert to inhibit the JAK/STAT pathway.

  8. Interacting partners of macrophage-secreted cathepsin B contribute to HIV-induced neuronal apoptosis

    PubMed Central

    CANTRES-ROSARIO, Yisel M.; HERNANDEZ, Natalia; NEGRON, Karla; PEREZ-LASPIUR, Juliana; LESZYK, John; SHAFFER, Scott A.; MELENDEZ, Loyda M.

    2015-01-01

    Objective HIV-1 infection of macrophages increases cathepsin B secretion and induces neuronal apoptosis, but the molecular mechanism remains unclear. Design We identified macrophage secreted cathepsin B protein interactions extracellularly and their contribution to neuronal death in vitro. Methods Cathepsin B was immunoprecipitated from monocyte-derived macrophage supernatants after 12 days post-infection. The cathepsin B interactome was quantified by label-free tandem mass spectrometry and compared to uninfected supernatants. Proteins identified were validated by western blot. Neurons were exposed to macrophage-conditioned media in presence or absence of antibodies against cathepsin B and interacting proteins. Apoptosis was measured using TUNEL labeling. Immunohistochemistry of post-mortem brain tissue samples from healthy, HIV-infected, and Alzheimer’s disease patients was performed to observe the ex vivo expression of the proteins identified. Results Nine proteins co-immunoprecipitated differentially with cathepsin B between uninfected and HIV-infected macrophages. Serum amyloid p component (SAPC) -cathepsin B interaction increased in HIV-infected macrophage supernatants, while matrix metalloprotease 9 (MMP-9) -cathepsin B interaction decreased. Pre-treatment of HIV-infected macrophage-conditioned media with antibodies against cathepsin B and SAPC decreased neuronal apoptosis. The addition of MMP-9 antibodies was not protective. SAPC was over-expressed in post-mortem brain tissue from HIV-positive neurocognitive impaired patients compared to HIV positive with normal cognition and healthy controls, while MMP-9 expression was similar in all tissues. Conclusions Inhibiting SAPC-cathepsin B interaction protects against HIV–induced neuronal death and may help to find alternative treatments for HIV-associated neurocognitive disorders. PMID:26208400

  9. Tubulin domains for the interaction of microtubule associated protein DMAP-85 from Drosophila melanogaster.

    PubMed

    Henríquez, J P; Cambiazo, V; Maccioni, R B

    1996-05-24

    The interaction of microtubule associated proteins (MAPs) with the microtubule system has been characterized in depth in neuronal cells from various mammalian species. These proteins interact with well-defined domains within the acidic tubulin carboxyl-terminal regulatory region. However, there is little information on the mechanisms of MAPs-tubulin interactions in nonmammalian systems. Recently, a novel tau-like protein designated as DMAP-85 has been identified in Drosophila melanogaster, and the regulation of its interactions with cytoskeletal elements was analyzed throughout different developmental stages of this organism. In this report, the topographic domains involved in the binding of DMAP-85 with tubulin heterodimer were investigated. Affinity chromatography of DMAP-85 in matrixes of taxol-stabilized microtubules showed the reversible interaction of DMAP-85 with domains on the microtubular surface. Co-sedimentation studies using the subtilisin-treated tubulin (S-tubulin) indicated the lack of association of DMAP-85 to this tubulin moiety. Moreover, studies on affinity chromatography of the purified 4 kDa C-terminal tubulin peptide bound to an affinity column, confirmed that DMAP-85 interacts directly with this regulatory domain on tubulin subunits. Further studies on sequential affinity chromatography using a calmodulin affinity column followed by the microtubule column confirmed the similarities in the interaction behaviour of DMAP-85 with that of tau. DMAP-85 associated to both calmodulin and the microtubular polymer. These studies support the idea that the carboxyl-terminal region on tubulin constitutes a common binding domain for most microtubule-interacting proteins.

  10. Charting the Landscape of Tandem BRCT Domain-Mediated Protein Interactions

    PubMed Central

    Woods, Nicholas T.; Mesquita, Rafael D.; Sweet, Michael; Carvalho, Marcelo A.; Li, Xueli; Liu, Yun; Nguyen, Huey; Thomas, C. Eric; Iversen, Edwin S.; Marsillac, Sylvia; Karchin, Rachel; Koomen, John; Monteiro, Alvaro N.A.

    2014-01-01

    Eukaryotic cells have evolved an intricate system to resolve DNA damage to prevent its transmission to daughter cells. This system, collectively known as the DNA damage response (DDR) network, includes a large number of proteins responsible for detection of DNA damage, promotion of repair, and coordination with cell cycle progression. Because defects in this network can lead to cancer, this network constitutes a barrier against tumorigenesis. The BRCT domain is a modular protein domain critical for relaying signals in the DDR. We performed a systematic analysis of protein-protein interactions involving tandem BRCT domains (tBRCT) in the DDR by combining literature curation, yeast two hybrid (Y2H) screens, and tandem affinity purification coupled to mass spectrometry (TAP-MS). We identified one previously unrecognized BRCT protein and generated human protein-protein interaction network for this type of modular domain. This study also reveals several novel components in DNA damage signaling such as COMMD1 and mTORC2. Additionally, integration of tBRCT domain interactions with DDR phosphoprotein studies and analysis of kinase-substrate interactions revealed signaling subnetworks that may aid in understanding the involvement of tBRCT in disease and DNA repair. PMID:22990118

  11. VND-INTERACTING2, a NAC domain transcription factor, negatively regulates xylem vessel formation in Arabidopsis.

    PubMed

    Yamaguchi, Masatoshi; Ohtani, Misato; Mitsuda, Nobutaka; Kubo, Minoru; Ohme-Takagi, Masaru; Fukuda, Hiroo; Demura, Taku

    2010-04-01

    The Arabidopsis thaliana NAC domain transcription factor VASCULAR-RELATED NAC-DOMAIN7 (VND7) acts as a master regulator of xylem vessel differentiation. To understand the mechanism by which VND7 regulates xylem vessel differentiation, we used a yeast two-hybrid system to screen for proteins that interact with VND7 and identified cDNAs encoding two NAC domain proteins, VND-INTERACTING1 (VNI1) and VNI2. Binding assays demonstrated that VNI2 effectively interacts with VND7 and the VND family proteins, VND1-5, as well as with other NAC domain proteins at lower affinity. VNI2 is expressed in both xylem and phloem cells in roots and inflorescence stems. The expression of VNI2 overlaps with that of VND7 in elongating vessel precursors in roots. VNI2 contains a predicted PEST motif and a C-terminally truncated VNI2 protein, which lacks part of the PEST motif, is more stable than full-length VNI2. Transient reporter assays showed that VNI2 is a transcriptional repressor and can repress the expression of vessel-specific genes regulated by VND7. Expression of C-terminally truncated VNI2 under the control of the VND7 promoter inhibited the normal development of xylem vessels in roots and aerial organs. These data suggest that VNI2 regulates xylem cell specification as a transcriptional repressor that interacts with VND proteins and possibly also with other NAC domain proteins.

  12. Interactions of U24 from Roseolovirus with WW domains: canonical vs noncanonical.

    PubMed

    Sang, Yurou; Zhang, Rui; Creagh, A Louise; Haynes, Charles A; Straus, Suzana K

    2017-06-01

    U24 is a C-terminal membrane-anchored protein found in both human herpes virus type 6 and 7 (HHV-6 and HHV-7), with an N-terminal segment that is rich in prolines (PPxY motif in both HHV-6A and 7; PxxP motif in HHV-6A). Previous work has shown that U24 interacts strongly with Nedd4 WW domains, in particular, hNedd4L-WW3*. It was also shown that this interaction depends strongly on the nature of the amino acids that are upstream from the PY motif in U24. In this contribution, data was obtained from pull-downs, isothermal titration calorimetry, and NMR to further determine what modulates U24:WW domain interactions. Specifically, 3 non-canonical WW domains from human Smad ubiquitination regulatory factor (Smurf), namely hSmurf2-WW2, hSmurf2-WW3, and a tandem construct hSmurf2-WW2 + 3, were studied. Overall, the interactions between U24 and these Smurf WW domains were found to be weaker than those in U24:Nedd4 WW domain pairs, suggesting that U24 function is tightly linked to specific E3 ubiqitin ligases.

  13. Molecular analysis of laminin N-terminal domains mediating self-interactions.

    PubMed

    Odenthal, Uwe; Haehn, Sebastian; Tunggal, Patrick; Merkl, Barbara; Schomburg, Dietmar; Frie, Christian; Paulsson, Mats; Smyth, Neil

    2004-10-22

    The ability of laminins to self-polymerize is crucial for the formation of basement membranes. Development of this polymerized network has profound effects upon tissue architecture as well as on the intracellular organization and differentiation of neighboring cells. The laminin N-terminal (LN) domains have been shown to mediate this interaction and studies using proteolytic fragments derived from laminin-1 led to the theory that network assembly depends on the formation of a heterotrimeric complex between LN domains derived from alpha, beta, and gamma chains in different laminin molecules with homologous interactions being insignificant. The laminin family consists of 15 known isoforms formed from five alpha, three beta, and three gamma chains, of which some are truncated and lack the N-terminal LN domain. To address whether the model of heterotrimeric complex formation is applicable to laminin isoforms other than laminin-1, eight LN domains found in the laminin protein family were recombinantly expressed and tested in three different assays for homologous and heterologous interactions. The results showed that the lack of homologous interactions is an exception, with such interactions being seen for LN domains derived from all alpha chains and from the beta2 and beta3 subunits. The gamma chain-derived LN domains showed a far more limited binding repertoire, particularly in the case of the gamma3 chain, which is found present in a range of non-basement membrane locations. Further, whereas the interactions depended upon Ca2+ ions, with EDTA reversibly abrogating binding, EDTA-induced conformational changes were not reversible. Together these results demonstrate that the assembly model proposed on the basis of laminin-1 may be a simplification, with the assembly of naturally occurring laminin networks being far more complex and highly dependent upon which laminin isoforms are present.

  14. The bacterial second messenger c-di-GMP: probing interactions with protein and RNA binding partners using cyclic dinucleotide analogs.

    PubMed

    Shanahan, Carly A; Strobel, Scott A

    2012-12-14

    The ability of bacteria to adapt to a changing environment is essential for their survival. One mechanism used to facilitate behavioral adaptations is the second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). c-di-GMP is widespread throughout the bacterial domain and plays a vital role in regulating the transition between the motile planktonic lifestyle and the sessile biofilm forming state. This second messenger also controls the virulence response of pathogenic organisms and is thought to be connected to quorum sensing, the process by which bacteria communicate with each other. The intracellular concentration of c-di-GMP is tightly regulated by the opposing enzymatic activities of diguanlyate cyclases and phosphodiesterases, which synthesize and degrade the second messenger, respectively. The change in the intracellular concentration of c-di-GMP is directly sensed by downstream targets of the second messenger, both protein and RNA, which induce the appropriate phenotypic response. This review will summarize our current state of knowledge of c-di-GMP signaling in bacteria with a focus on protein and RNA binding partners of the second messenger. Efforts towards the synthesis of c-di-GMP and its analogs are discussed as well as studies aimed at targeting these macromolecular effectors with chemically synthesized cyclic dinucleotide analogs.

  15. The bacterial second messenger c-di-GMP: Probing interactions with protein and RNA binding partners using cyclic dinucleotide analogs

    PubMed Central

    Shanahan, Carly A.; Strobel, Scott A.

    2013-01-01

    The ability of bacteria to adapt to a changing environment is essential for their survival. One mechanism used to facilitate behavioral adaptations is the second messenger signaling molecule bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). c-di-GMP is widespread throughout the bacterial domain and plays a vital role in regulating the transition between the motile planktonic lifestyle and the sessile biofilm forming state. This second messenger also controls the virulence response of pathogenic organisms and is thought to be connected to quorum sensing, the process by which bacteria communicate with each other. The intracellular concentration of c-di-GMP is tightly regulated by the opposing enzymatic activities of diguanlyate cyclases and phosphodiesterases, which synthesize and degrade the second messenger, respectively. The change in the intracellular concentration of c-di-GMP is directly sensed by downstream targets of the second messenger, both protein and RNA, which induce the appropriate phenotypic response. This review will summarize our current state of knowledge of c-di-GMP signaling in bacteria with a focus on protein and RNA binding partners of the second messenger. Efforts towards the synthesis of c-di-GMP and its analogs are discussed as well as studies aimed at targeting these macromolecular effectors with chemically synthesized cyclic dinucleotide analogs. PMID:23108253

  16. π and ρ mesons, and their diquark partners, from a contact interaction

    NASA Astrophysics Data System (ADS)

    Roberts, H. L. L.; Bashir, A.; Gutiérrez-Guerrero, L. X.; Roberts, C. D.; Wilson, D. J.

    2011-06-01

    We present a unified Dyson-Schwinger equation treatment of static and electromagnetic properties of pseudoscalar and vector mesons, and scalar and axial-vector diquark correlations, based upon a vector-vector contact interaction. A basic motivation for this paper is the need to document a comparison between the electromagnetic form factors of mesons and those diquarks that play a material role in nucleon structure. A notable result, therefore, is the large degree of similarity between related meson and diquark form factors. The simplicity of the interaction enables computation of the form factors at arbitrarily large spacelike Q2, which enables us to expose a zero in the ρ-meson electric form factor at zQρ≈√6mρ. Notably, rρzQρ≈rDzQD, where rρ and rD are, respectively, the electric radii of the ρ-meson and deuteron.

  17. Structures of the NLRP14 pyrin domain reveal a conformational switch mechanism regulating its molecular interactions

    SciTech Connect

    Eibl, Clarissa; Hessenberger, Manuel; Wenger, Julia; Brandstetter, Hans

    2014-07-01

    Pyrin domains (PYDs) recruit downstream effector molecules in NLR signalling. A specific charge-relay system suggests a the formation of a signalling complex involving a PYD dimer. The cytosolic tripartite NLR receptors serve as important signalling platforms in innate immunity. While the C-terminal domains act as sensor and activation modules, the N-terminal death-like domain, e.g. the CARD or pyrin domain, is thought to recruit downstream effector molecules by homotypic interactions. Such homotypic complexes have been determined for all members of the death-domain superfamily except for pyrin domains. Here, crystal structures of human NLRP14 pyrin-domain variants are reported. The wild-type protein as well as the clinical D86V mutant reveal an unexpected rearrangement of the C-terminal helix α6, resulting in an extended α5/6 stem-helix. This reordering mediates a novel symmetric pyrin-domain dimerization mode. The conformational switching is controlled by a charge-relay system with a drastic impact on protein stability. How the identified charge relay allows classification of NLRP receptors with respect to distinct recruitment mechanisms is discussed.

  18. Structural diversity in the RGS domain and its interaction with heterotrimeric G protein α-subunits

    PubMed Central

    Soundararajan, Meera; Willard, Francis S.; Kimple, Adam J.; Turnbull, Andrew P.; Ball, Linda J.; Schoch, Guillaume A.; Gileadi, Carina; Fedorov, Oleg Y.; Dowler, Elizabeth F.; Higman, Victoria A.; Hutsell, Stephanie Q.; Sundström, Michael; Doyle, Declan A.; Siderovski, David P.

    2008-01-01

    Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs—receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Gα when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Gα, RGS domain binding consequently accelerates Gα-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Gα substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Gα selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Gα complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Gα substrate, suggests that unique structural determinants specific to particular RGS protein/Gα pairings exist and could be used to achieve selective inhibition by small molecules. PMID:18434541

  19. Current-induced 360° domain wall motion with Dzyaloshinskii-Moriya interaction

    NASA Astrophysics Data System (ADS)

    Jin, Chendong; Zhang, Senfu; Zhu, Qiyuan; Liu, Xianyin; Chen, Shujun; Song, Chengkun; Wang, Jianbo; Liu, Qingfang

    2016-05-01

    By micromagnetic simulation, we investigated the effect of the Dzyaloshinskii-Moriya interaction (DMI) on the static and dynamic characteristics of a 360° domain wall. Simulation results show that both the energy and the size of a 360° domain wall decrease with the increase of DMI intensity. In the presence of DMI, the stable motion of a 360° domain wall can be either along the  +x direction or  -x direction depending on the sign of the DMI. For stable motion, the maximum velocity of a 360° domain wall is 19.87% larger than that without the DMI. Increasing the current density beyond the Walker threshold, conversion between the 360° domain wall state and the vortex state was observed. Further increasing the current density, the proliferation of 360° domain walls becomes possible. Moreover, the 360° domain wall becomes more flexible and easier to pass a notch by considering the DMI. These findings may offer guidance for the development of 360° domain wall-based racetrack memories.

  20. Distinct Interaction Modes of the Kinesin-13 Motor Domain with the Microtubule

    PubMed Central

    Chatterjee, Chandrima; Benoit, Matthieu P.M.H.; DePaoli, Vania; Diaz-Valencia, Juan D.; Asenjo, Ana B.; Gerfen, Gary J.; Sharp, David J.; Sosa, Hernando

    2016-01-01

    Kinesins-13s are members of the kinesin superfamily of motor proteins that depolymerize microtubules (MTs) and have no motile activity. Instead of generating unidirectional movement over the MT lattice, like most other kinesins, kinesins-13s undergo one-dimensional diffusion (ODD) and induce depolymerization at the MT ends. To understand the mechanism of ODD and the origin of the distinct kinesin-13 functionality, we used ensemble and single-molecule fluorescence polarization microscopy to analyze the behavior and conformation of Drosophila melanogaster kinesin-13 KLP10A protein constructs bound to the MT lattice. We found that KLP10A interacts with the MT in two coexisting modes: one in which the motor domain binds with a specific orientation to the MT lattice and another where the motor domain is very mobile and able to undergo ODD. By comparing the orientation and dynamic behavior of mutated and deletion constructs we conclude that 1) the Kinesin-13 class specific neck domain and loop-2 help orienting the motor domain relative to the MT. 2) During ODD the KLP10A motor-domain changes orientation rapidly (rocks or tumbles). 3) The motor domain alone is capable of undergoing ODD. 4) A second tubulin binding site in the KLP10A motor domain is not critical for ODD. 5) The neck domain is not the element preventing KLP10A from binding to the MT lattice like motile kinesins. PMID:27074684

  1. The transcription activation domains of Fos and Jun induce DNA bending through electrostatic interactions.

    PubMed Central

    Kerppola, T K; Curran, T

    1997-01-01

    Transcription factor-induced DNA bending is essential for the assembly of active transcription complexes at many promoters. However, most eukaryotic transcription regulatory proteins have modular DNA-binding and activation domains, which appeared to exclude DNA bending as a mechanism of transcription activation by these proteins. We show that the transcription activation domains of Fos and Jun induce DNA bending. In chimeric proteins, the transcription activation domains induce DNA bending independent of the DNA-binding domains. DNA bending by the chimeric proteins is directed diametrically away from the transcription activation domains. Therefore, the opposite directions of DNA bending by Fos and Jun are caused, in part, by the opposite locations of the transcription activation domains relative to the DNA-binding domains in these proteins. DNA bending is reduced in the presence of multivalent cations, indicating that electrostatic interactions contribute to DNA bending by Fos and Jun. Consequently, regions outside the minimal DNA-binding domain can influence DNA structure, and may thereby contribute to the architectural reorganization of the promoter region required for gene activation. PMID:9184234

  2. Definition of a spliceosome interaction domain in yeast Prp2 ATPase.

    PubMed

    Edwalds-Gilbert, Gretchen; Kim, Dong-Ho; Silverman, Edward; Lin, Ren-Jang

    2004-02-01

    The Saccharomyces cerevisiae splicing factor Prp2 is an RNA-dependent ATPase required before the first transesterification reaction in pre-mRNA splicing. Prp2 binds to the spliceosome in the absence of ATP and is released following ATP hydrolysis. It contains three domains: a unique N-terminal domain, a helicase domain that is highly conserved in the DExD/H protein family, and a C-terminal domain that is conserved in spliceosomal DEAH proteins Prp2, Prp16, Prp22, and Prp43. We examined the role of each domain of Prp2 by deletion mutagenesis. Whereas deletions of either the helicase or C-terminal domain are lethal, deletions in the N-terminal domain have no detectable effect on Prp2 activity. Overexpression of the C-terminal domain of Prp2 exacerbates the temperature-sensitive phenotype of a prp2(Ts) strain, suggesting that the C-domain interferes with the activity of the Prp2(Ts) protein. A genetic approach was then taken to study interactions between Prp2 and the spliceosome. Previously, we isolated dominant negative mutants in the helicase domain of Prp2 that inhibit the activity of wild-type Prp2 when the mutant protein is overexpressed. We mutagenized one prp2 release mutant gene and screened for loss of dominant negative function. Several weak binding mutants were isolated and mapped to the C terminus of Prp2, further indicating the importance of the C terminus in spliceosome binding. This study is the first to indicate that amino acid substitutions outside the helicase domain can abolish spliceosome contact and splicing activity of a spliceosomal DEAH protein.

  3. RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy*

    PubMed Central

    Erbil, Secil; Oral, Ozlem; Mitou, Geraldine; Kig, Cenk; Durmaz-Timucin, Emel; Guven-Maiorov, Emine; Gulacti, Ferah; Gokce, Gokcen; Dengjel, Jörn; Sezerman, Osman Ugur; Gozuacik, Devrim

    2016-01-01

    Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double- or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways. PMID:27325703

  4. Krit 1 interactions with microtubules and membranes are regulated by Rap1 and integrin cytoplasmic domain associated protein-1.

    PubMed

    Béraud-Dufour, Sophie; Gautier, Romain; Albiges-Rizo, Corinne; Chardin, Pierre; Faurobert, Eva

    2007-11-01

    The small G protein Rap1 regulates diverse cellular processes such as integrin activation, cell adhesion, cell-cell junction formation and cell polarity. It is crucial to identify Rap1 effectors to better understand the signalling pathways controlling these processes. Krev interaction trapped 1 (Krit1), a protein with FERM (band four-point-one/ezrin/radixin/moesin) domain, was identified as a Rap1 partner in a yeast two-hybrid screen, but this interaction was not confirmed in subsequent studies. As the evidence suggests a role for Krit1 in Rap1-dependent pathways, we readdressed this question. In the present study, we demonstrate by biochemical assays that Krit1 interacts with Rap1A, preferentially its GTP-bound form. We show that, like other FERM proteins, Krit1 adopts two conformations: a closed conformation in which its N-terminal NPAY motif interacts with its C-terminus and an opened conformation bound to integrin cytoplasmic domain associated protein (ICAP)-1, a negative regulator of focal adhesion assembly. We show that a ternary complex can form in vitro between Krit1, Rap1 and ICAP-1 and that Rap1 binds the Krit1 FERM domain in both closed and opened conformations. Unlike ICAP-1, Rap1 does not open Krit1. Using sedimentation assays, we show that Krit1 binds in vitro to microtubules through its N- and C-termini and that Rap1 and ICAP-1 inhibit Krit1 binding to microtubules. Consistently, YFP-Krit1 localizes on cyan fluorescent protein-labelled microtubules in baby hamster kidney cells and is delocalized from microtubules upon coexpression with activated Rap1V12. Finally, we show that Krit1 binds to phosphatidylinositol 4,5-P(2)-containing liposomes and that Rap1 enhances this binding. Based on these results, we propose a model in which Krit1 would be delivered by microtubules to the plasma membrane where it would be captured by Rap1 and ICAP-1.

  5. Systematic Analysis of Bacterial Effector-Postsynaptic Density 95/Disc Large/Zonula Occludens-1 (PDZ) Domain Interactions Demonstrates Shigella OspE Protein Promotes Protein Kinase C Activation via PDLIM Proteins*

    PubMed Central

    Yi, Chae-ryun; Allen, John E.; Russo, Brian; Lee, Soo Young; Heindl, Jason E.; Baxt, Leigh A.; Herrera, Bobby Brooke; Kahoud, Emily; MacBeath, Gavin; Goldberg, Marcia B.

    2014-01-01

    Diseases caused by many Gram-negative bacterial pathogens depend on the activities of bacterial effector proteins that are delivered into eukaryotic cells via specialized secretion systems. Effector protein function largely depends on specific subcellular targeting and specific interactions with cellular ligands. PDZ domains are common domains that serve to provide specificity in protein-protein interactions in eukaryotic systems. We show that putative PDZ-binding motifs are significantly enriched among effector proteins delivered into mammalian cells by certain bacterial pathogens. We use PDZ domain microarrays to identify candidate interaction partners of the Shigella flexneri effector proteins OspE1 and OspE2, which contain putative PDZ-binding motifs. We demonstrate in vitro and in cells that OspE proteins interact with PDLIM7, a member of the PDLIM family of proteins, which contain a PDZ domain and one or more LIM domains, protein interaction domains that participate in a wide variety of functions, including activation of isoforms of protein kinase C (PKC). We demonstrate that activation of PKC during S. flexneri infection is attenuated in the absence of PDLIM7 or OspE proteins and that the OspE PDZ-binding motif is required for wild-type levels of PKC activation. These results are consistent with a model in which binding of OspE to PDLIM7 during infection regulates the activity of PKC isoforms that bind to the PDLIM7 LIM domain. PMID:25124035

  6. RNA polymerase II conserved protein domains as platforms for protein-protein interactions

    PubMed Central

    García-López, M Carmen

    2011-01-01

    RNA polymerase II establishes many protein-protein interactions with transcriptional regulators to coordinate gene expression, but little is known about protein domains involved in the contact with them. We use a new approach to look for conserved regions of the RNA pol II of S. cerevisiae located at the surface of the structure of the complex, hypothesizing that they might be involved in the interaction with transcriptional regulators. We defined five different conserved domains and demonstrate that all of them make contact with transcriptional regulators. PMID:21922063

  7. Human Foamy Virus Capsid Formation Requires an Interaction Domain in the N Terminus of Gag

    PubMed Central

    Tobaly-Tapiero, Joelle; Bittoun, Patricia; Giron, Marie-Lou; Neves, Manuel; Koken, Marcel; Saïb, Ali; de Thé, Hugues

    2001-01-01

    Retroviral Gag expression is sufficient for capsid assembly, which occurs through interaction between distinct Gag domains. Human foamy virus (HFV) capsids assemble within the cytoplasm, although their budding, which mainly occurs in the endoplasmic reticulum, requires the presence of homologous Env. Yet little is known about the molecular basis of HFV Gag precursor assembly. Using fusions between HFV Gag and a nuclear reporter protein, we have identified a strong interaction domain in the N terminus of HFV Gag which is predicted to contain a conserved coiled-coil motif. Deletion within this region in an HFV provirus abolishes viral production through inhibition of capsid assembly. PMID:11287585

  8. The expanded octarepeat domain selectively binds prions and disrupts homomeric prion protein interactions.

    PubMed

    Leliveld, Sirik Rutger; Dame, Remus Thei; Wuite, Gijs J L; Stitz, Lothar; Korth, Carsten

    2006-02-10

    Insertion of additional octarepeats into the prion protein gene has been genetically linked to familial Creutzfeldt Jakob disease and hence to de novo generation of infectious prions. The pivotal event during prion formation is the conversion of the normal prion protein (PrPC) into the pathogenic conformer PrPSc, which subsequently induces further conversion in an autocatalytic manner. Apparently, an expanded octarepeat domain directs folding of PrP toward the PrPSc conformation and initiates a self-replicating conversion process. Here, based on three main observations, we have provided a model on how altered molecular interactions between wild-type and mutant PrP set the stage for familial Creutzfeldt Jakob disease with octarepeat insertions. First, we showed that wild-type octarepeat domains interact in a copper-dependent and reversible manner, a "copper switch." This interaction becomes irreversible upon domain expansion, possibly reflecting a loss of function. Second, expanded octarepeat domains of increasing length gradually form homogenous globular multimers of 11-21 nm in the absence of copper ions when expressed as soluble glutathione S-transferase fusion proteins. Third, octarepeat domain expansion causes a gain of function with at least 10 repeats selectively binding PrPSc in a denaturant-resistant complex in the absence of copper ions. Thus, the combination of both a loss and gain of function profoundly influences homomeric interaction behavior of PrP with an expanded octarepeat domain. A multimeric cluster of prion proteins carrying expanded octarepeat domains may therefore capture and incorporate spontaneously arising short-lived PrPSc-like conformers, thereby providing a matrix for their conversion.

  9. A systematic, family-wide investigation reveals that ~30% of mammalian PDZ domains engage in PDZ-PDZ interactions

    PubMed Central

    Chang, Bryan H.; Gujral, Taranjit S.; Karp, Ethan S.; BuKhalid, Raghida; Grantcharova, Viara P.; MacBeath, Gavin

    2012-01-01

    Summary PDZ domains are independently folded modules that typically mediate protein-protein interactions by binding to the C-termini of their target proteins. In a few instances, however, PDZ domains have been reported to dimerize with other PDZ domains. To investigate this noncanonical binding mode further, we used protein microarrays comprising virtually every mouse PDZ domain to systematically query all possible PDZ-PDZ pairs. We then used fluorescence polarization to retest and quantify novel interactions and co-affinity purification to test biophysically validated interactions in the context of their full-length proteins. Overall, we discovered 37 PDZ-PDZ interactions involving 46 PDZ domains (~30% of all PDZ domains tested), revealing that dimerization is a more frequently used binding mode than was previously appreciated. This suggests that many PDZ domains evolved to form multiprotein complexes by simultaneously interacting with more than one ligand. PMID:21944753

  10. Accommodation of structural rearrangements in the huntingtin-interacting protein 1 coiled-coil domain

    SciTech Connect

    Wilbur, Jeremy D.; Hwang, Peter K.; Brodsky, Frances M.; Fletterick, Robert J.

    2010-03-01

    Variable packing interaction related to the conformational flexibility within the huntingtin-interacting protein 1 coiled coil domain. Huntingtin-interacting protein 1 (HIP1) is an important link between the actin cytoskeleton and clathrin-mediated endocytosis machinery. HIP1 has also been implicated in the pathogenesis of Huntington’s disease. The binding of HIP1 to actin is regulated through an interaction with clathrin light chain. Clathrin light chain binds to a flexible coiled-coil domain in HIP1 and induces a compact state that is refractory to actin binding. To understand the mechanism of this conformational regulation, a high-resolution crystal structure of a stable fragment from the HIP1 coiled-coil domain was determined. The flexibility of the HIP1 coiled-coil region was evident from its variation from a previously determined structure of a similar region. A hydrogen-bond network and changes in coiled-coil monomer interaction suggest that the HIP1 coiled-coil domain is uniquely suited to allow conformational flexibility.

  11. The basic amino acids in the coiled-coil domain of CIN85 regulate its interaction with c-Cbl and phosphatidic acid during epidermal growth factor receptor (EGFR) endocytosis.

    PubMed

    Zheng, Xiudan; Zhang, Jing; Liao, Kan

    2014-07-08

    During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain. Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation. As an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation.

  12. The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N′ and Regulates Light-Dependent Cell Death1[OPEN

    PubMed Central

    Sekine, Ken-Taro; Wallon, Thérèse; Sugiwaka, Yuji; Kobayashi, Kappei

    2016-01-01

    One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N′, which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N′ results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N′ is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins. PMID:26951433

  13. Does communication partner training improve the conversation skills of speech-language pathology students when interacting with people with aphasia?

    PubMed

    Finch, Emma; Cameron, Ashley; Fleming, Jennifer; Lethlean, Jennifer; Hudson, Kyla; McPhail, Steven

    2017-07-01

    Aphasia is a common consequence of stroke. Despite receiving specialised training in communication, speech-language pathology students may lack confidence when communicating with People with Aphasia (PWA). This paper reports data from secondary outcome measures from a randomised controlled trial. The aim of the current study was to examine the effects of communication partner training on the communication skills of speech-language pathology students during conversations with PWA. Thirty-eight speech-language pathology students were randomly allocated to trained and untrained groups. The first group received a lecture about communication strategies for communicating with PWA then participated in a conversation with PWA (Trained group), while the second group of students participated in a conversation with the PWA without receiving the lecture (Untrained group). The conversations between the groups were analysed according to the Measure of skill in Supported Conversation (MSC) scales, Measure of Participation in Conversation (MPC) scales, types of strategies used in conversation, and the occurrence and repair of conversation breakdowns. The trained group received significantly higher MSC Revealing Competence scores, used significantly more props, and introduced significantly more new ideas into the conversation than the untrained group. The trained group also used more gesture and writing to facilitate the conversation, however, the difference was not significant. There was no significant difference between the groups according to MSC Acknowledging Competence scores, MPC Interaction or Transaction scores, or in the number of interruptions, minor or major conversation breakdowns, or in the success of strategies initiated to repair the conversation breakdowns. Speech-language pathology students may benefit from participation in communication partner training programs. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. The dipole moment of the electron carrier adrenodoxin is not critical for redox partner interaction and electron transfer.

    PubMed

    Hannemann, Frank; Guyot, Arnaud; Zöllner, Andy; Müller, Jürgen J; Heinemann, Udo; Bernhardt, Rita

    2009-07-01

    Dipole moments of proteins arise from helical dipoles, hydrogen bond networks and charged groups at the protein surface. High protein dipole moments were suggested to contribute to the electrostatic steering between redox partners in electron transport chains of respiration, photosynthesis and steroid biosynthesis, although so far experimental evidence for this hypothesis was missing. In order to probe this assumption, we changed the dipole moment of the electron transfer protein adrenodoxin and investigated the influence of this on protein-protein interactions and electron transfer. In bovine adrenodoxin, the [2Fe-2S] ferredoxin of the adrenal glands, a dipole moment of 803 Debye was calculated for a full-length adrenodoxin model based on the Adx(4-108) and the wild type adrenodoxin crystal structures. Large distances and asymmetric distribution of the charged residues in the molecule mainly determine the observed high value. In order to analyse the influence of the resulting inhomogeneous electric field on the biological function of this electron carrier the molecular dipole moment was systematically changed. Five recombinant adrenodoxin mutants with successively reduced dipole moment (from 600 to 200 Debye) were analysed for their redox properties, their binding affinities to the redox partner proteins and for their function during electron transfer-dependent steroid hydroxylation. None of the mutants, not even the quadruple mutant K6E/K22Q/K24Q/K98E with a dipole moment reduced by about 70% showed significant changes in the protein function as compared with the unmodified adrenodoxin demonstrating that neither the formation of the transient complex nor the biological activity of the electron transfer chain of the endocrine glands was affected. This is the first experimental evidence that the high dipole moment observed in electron transfer proteins is not involved in electrostatic steering among the proteins in the redox chain.

  15. NMR Solution Structure, Stability, and Interaction of the Recombinant Bovine Fibrinogen αC-Domain Fragment†

    PubMed Central

    Burton, Robert A.; Tsurupa, Galina; Hantgan, Roy R.; Tjandra, Nico; Medved, Leonid

    2008-01-01

    According to the current hypothesis, in fibrinogen, the COOH-terminal portions of two Aα chains are folded into compact αC-domains that interact intramolecularly with each other and with the central region of the molecule; in fibrin, the αC-domains switch to an intermolecular interaction resulting in αC polymers. In agreement, our recent NMR study identified within the bovine fibrinogen Aα374-538 αC-domain fragment an ordered compact structure including a β-hairpin restricted at the base by a 423–453 disulfide linkage. To establish the complete structure of the αC-domain and to further test the hypothesis, we expressed a shorter αC-fragment, Aα406-483, and performed detailed analysis of its structure, stability, and interactions. NMR experiments on the Aα406-483 fragment identified a second loose β-hairpin formed by residues 459–476, yielding a structure consisting of an intrinsically unstable mixed parallel/anti-parallel β-sheet. Size-exclusion chromatography and sedimentation velocity experiments revealed that the Aα406-483 fragment forms soluble oligomers whose fraction increases with increasing concentration. This was confirmed by sedimentation equilibrium analysis, which also revealed that the addition of each monomer to an assembling αC oligomer substantially increases its stabilizing free energy. In agreement, unfolding experiments monitored by CD established that oligomerization of Aα406-483 results in increased thermal stability. Altogether, these experiments establish the complete NMR solution structure of the Aα406-483 αC-domain fragment, provide direct evidence for the intra- and intermolecular interactions between the αC-domains, and confirm that these interactions are thermodynamically driven. PMID:17590019

  16. {pi} and {rho} mesons, and their diquark partners, from a contact interaction

    SciTech Connect

    Roberts, H. L. L.; Bashir, A.; Gutierrez-Guerrero, L. X.; Roberts, C. D.; Wilson, D. J.

    2011-06-15

    We present a unified Dyson-Schwinger equation treatment of static and electromagnetic properties of pseudoscalar and vector mesons, and scalar and axial-vector diquark correlations, based upon a vector-vector contact interaction. A basic motivation for this paper is the need to document a comparison between the electromagnetic form factors of mesons and those diquarks that play a material role in nucleon structure. A notable result, therefore, is the large degree of similarity between related meson and diquark form factors. The simplicity of the interaction enables computation of the form factors at arbitrarily large spacelike Q{sup 2}, which enables us to expose a zero in the {rho}-meson electric form factor at z{sub Q}{sup {rho}}{approx_equal}{radical}6m{sub {rho}}. Notably, r{sub {rho}}zQ{sup {rho}}{approx_equal}r{sub D}z{sub Q}{sup D}, where r{sub {rho}} and r{sub D} are, respectively, the electric radii of the {rho}-meson and deuteron.

  17. Dual regulatory switch through interactions of Tcf7l2/Tcf4 with stage-specific partners propels oligodendroglial maturation

    PubMed Central

    Zhao, Chuntao; Deng, Yaqi; Liu, Lei; Yu, Kun; Zhang, Liguo; Wang, Haibo; He, Xuelian; Wang, Jincheng; Lu, Changqing; Wu, Laiman N; Weng, Qinjie; Mao, Meng; Li, Jianrong; van Es, Johan H; Xin, Mei; Parry, Lee; Goldman, Steven A; Clevers, Hans; Lu, Q. Richard

    2016-01-01

    Constitutive activation of Wnt/β-catenin inhibits oligodendrocyte myelination. Tcf7l2/Tcf4, a β-catenin transcriptional partner, is required for oligodendrocyte differentiation. How Tcf7l2 modifies β-catenin signalling and controls myelination remains elusive. Here we define a stage-specific Tcf7l2-regulated transcriptional circuitry in initiating and sustaining oligodendrocyte differentiation. Multistage genome occupancy analyses reveal that Tcf7l2 serially cooperates with distinct co-regulators to control oligodendrocyte lineage progression. At the differentiation onset, Tcf7l2 interacts with a transcriptional co-repressor Kaiso/Zbtb33 to block β-catenin signalling. During oligodendrocyte maturation, Tcf7l2 recruits and cooperates with Sox10 to promote myelination. In that context, Tcf7l2 directly activates cholesterol biosynthesis genes and cholesterol supplementation partially rescues oligodendrocyte differentiation defects in Tcf712 mutants. Together, we identify stage-specific co-regulators Kaiso and Sox10 that sequentially interact with Tcf7l2 to coordinate the switch at the transitions of differentiation initiation and maturation during oligodendrocyte development, and point to a previously unrecognized role of Tcf7l2 in control of cholesterol biosynthesis for CNS myelinogenesis. PMID:26955760

  18. Small heterodimer partner-interacting leucine zipper protein inhibits adipogenesis by regulating peroxisome proliferator-activated receptor γ activity.

    PubMed

    Jang, Hoon; Kim, Hyoung-Joo; Kim, Dong-Hwan; Park, Jae-Kyung; Sun, Wu-Sheng; Hwang, Seongsoo; Oh, Keon-Bong; Jang, Won-Gu; Lee, Jeong-Woong

    2015-07-01

    Adipocytes play a critical role in energy balance. Growth of fat tissue is achieved via an increase in adipocyte mass and the formation of newly differentiated adipocytes from precursor cells. Understanding the cellular and molecular mechanisms of adipocyte differentiation is crucial for the study of obesity- and fat-related diseases. The present study was designed to study whether small heterodimer partner-interacting leucine zipper protein (SMILE), a novel co-repressor, could regulate differentiation of adipocyte in 3T3-L1 cells. Treatment of endoplasmic stress inducers, thapsigargin and tunicamycin, inhibited adipocyte differentiation, stimulated Smile mRNA expression, and repressed the expression of adiponectin (Adipoq) in 3T3-L1 pre-adipocyte. Overexpression of SMILE in 3T3-L1 cells decreased the expression of the mRNA encoding Adipoq, a major marker of adipocytes, significantly. Furthermore, knockdown of SMILE recovered the thapsigargin-mediated repression of Adipoq transcription. Co-immunoprecipitation experiments revealed that SMILE interacted physically with PPARγ in 3T3-L1 cells. In addition, chromatin immunoprecipitation experiments revealed that SMILE suppressed the binding affinity of PPARγ for the Adipoq promoter. We demonstrate that SMILE controls adipocyte differentiation by regulating the transactivity of peroxisome proliferator-activated receptor γ (PPARγ). These findings demonstrate that SMILE represses adipocyte differentiation by regulating PPARγ transactivity; hence, SMILE is a potential regulator of PPARγ-related diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Cloning and characterization of GRIPE, a novel interacting partner of the transcription factor E12 in developing mouse forebrain.

    PubMed

    Heng, Julian Ik Tsen; Tan, Seong-Seng

    2002-11-08

    The helix-loop-helix (HLH) family of transcription factors are key contributors to a wide array of developmental processes, including neurogenesis and hematopoiesis. These factors are thought to exert their regulatory influences by binding to cognate promoter-DNA sequences as dimers. Although studies in mice have convincingly demonstrated that neurogenic HLH proteins such as NeuroD are intimately involved in neuronal fate determination, the role of the ubiquitously expressed HLH protein, E12, in mammalian neurogenesis remains ambiguous. To address this, a yeast two-hybrid interaction screen was employed to identify dimerization partners to E12. Screening of an embryonic day 11.5 forebrain library resulted in the cloning of GRIPE, a novel GAP-related interacting protein to E12. GRIPE binds to the HLH region of E12 and may require E12 for nuclear import. Furthermore, GRIPE may negatively regulate E12-dependent target gene transcription. High levels of GRIPE and E12 mRNA were coincidentally detected during embryogenesis, but only GRIPE mRNA levels remained high in adult brain, particularly in neurons of the cortex and hippocampus. These observations were recapitulated through an in vitro model of neurogenesis. Taken together, these results indicate that GRIPE is a novel protein dimerization of which with E12 has important consequences for cells undergoing neuronal differentiation.

  20. Bacterial interactomes: Interacting protein partners share similar function and are validated in independent assays more frequently than previously reported.

    DOE PAGES

    Shatsky, Maxim; Allen, Simon; Gold, Barbara; ...

    2016-05-01

    Numerous affinity purification – mass-spectrometry (AP-MS) and yeast two hybrid (Y2H) screens have each defined thousands of pairwise protein-protein interactions (PPIs), most between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial Y2H and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli. Compared to the nine published interactomes, our two networks are smaller; are much less highly connected; have significantly lower false discovery rates; and are much moremore » enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays. Lastly, our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.« less

  1. Bacterial interactomes: Interacting protein partners share similar function and are validated in independent assays more frequently than previously reported.

    SciTech Connect

    Shatsky, Maxim; Allen, Simon; Gold, Barbara; Liu, Nancy L.; Juba, Thomas R.; Elias, Dwayne A; Reveco, Sonia A.; Prathapam, Ramadevi; He, Jennifer; Yang, Wenhong; Szakal, Evelin D.; Liu, Haichuan; Singer, Mary E.; Geller, Jil T.; Lam, Bonita R.; Saini, Avneesh; Trotter, Valentine V.; Hall, Steven C.; Fisher, Susan J.; Brenner, Steven E.; Chhabra, Swapnil; Hazen, Terry C.; Wall, Judy D.; Witkowska, Ewa; Biggin, Mark D.; Chandonia, John-Marc; Butland, Gareth

    2016-05-01

    Numerous affinity purification – mass-spectrometry (AP-MS) and yeast two hybrid (Y2H) screens have each defined thousands of pairwise protein-protein interactions (PPIs), most between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial Y2H and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli. Compared to the nine published interactomes, our two networks are smaller; are much less highly connected; have significantly lower false discovery rates; and are much more enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays. Lastly, our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.

  2. Tctex-1, a Novel Interaction Partner of Rab3D, Is Required for Osteoclastic Bone Resorption ▿

    PubMed Central

    Pavlos, Nathan J.; Cheng, Tak Sum; Qin, An; Ng, Pei Ying; Feng, Hao-Tian; Ang, Estabelle S. M.; Carrello, Amerigo; Sung, Ching-Hwa; Jahn, Reinhard; Zheng, Ming-Hao; Xu, Jiake

    2011-01-01

    Vesicular transport along microtubules must be strictly regulated to sustain the unique structural and functional polarization of bone-resorbing osteoclasts. However, the molecular mechanisms bridging these vesicle-microtubule interactions remain largely obscure. Rab3D, a member of the Rab3 subfamily (Rab3A/B/C/D) of small exocytotic GTPases, represents a core component of the osteoclastic vesicle transport machinery. Here, we identify a new Rab3D-interacting partner, Tctex-1, a light chain of the cytoplasmic dynein microtubule motor complex, by a yeast two-hybrid screen. We demonstrate that Tctex-1 binds specifically to Rab3D in a GTP-dependent manner and co-occupies Rab3D-bearing vesicles in bone-resorbing osteoclasts. Furthermore, we provide evidence that Tctex-1 and Rab3D intimately associate with the dynein motor complex and microtubules in osteoclasts. Finally, targeted disruption of Tctex-1 by RNA interference significantly impairs bone resorption capacity and mislocalizes Rab3D vesicles in osteoclasts, attesting to the notion that components of the Rab3D-trafficking pathway contribute to the maintenance of osteoclastic resorptive function. PMID:21262767

  3. Bacterial interactomes: Interacting protein partners share similar function and are validated in independent assays more frequently than previously reported.

    SciTech Connect

    Shatsky, Maxim; Allen, Simon; Gold, Barbara; Liu, Nancy L.; Juba, Thomas R.; Elias, Dwayne A; Reveco, Sonia A.; Prathapam, Ramadevi; He, Jennifer; Yang, Wenhong; Szakal, Evelin D.; Liu, Haichuan; Singer, Mary E.; Geller, Jil T.; Lam, Bonita R.; Saini, Avneesh; Trotter, Valentine V.; Hall, Steven C.; Fisher, Susan J.; Brenner, Steven E.; Chhabra, Swapnil; Hazen, Terry C.; Wall, Judy D.; Witkowska, Ewa; Biggin, Mark D.; Chandonia, John-Marc; Butland, Gareth

    2016-05-01

    Numerous affinity purification – mass-spectrometry (AP-MS) and yeast two hybrid (Y2H) screens have each defined thousands of pairwise protein-protein interactions (PPIs), most between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial Y2H and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli. Compared to the nine published interactomes, our two networks are smaller; are much less highly connected; have significantly lower false discovery rates; and are much more enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays. Lastly, our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.

  4. Biochemical and functional significance of F-BAR domain proteins interaction with WASP/N-WASP.

    PubMed

    Chen, Yolande; Aardema, Jorie; Corey, Seth J

    2013-04-01

    The Bin-Amphiphysin-Rvs (BAR) domain family of proteins includes groups which promote positive (classical BAR, N-BAR, and F-BAR) and negative (I-BAR) membrane deformation. Of these groups, the F-BAR subfamily is the most diverse in its biochemical properties. F-BAR domain proteins dimerize to form a tight scaffold about the membrane. The F-BAR domain provides a banana-shaped, alpha-helical structure that senses membrane curvature. Different types of F-BAR domain proteins contain tyrosine kinase or GTPase activities; some interact with phosphatases and RhoGTPases. Most possess an SH3 domain that facilitates the recruitment and activation of WASP/N-WASP. Thus, F-BAR domain proteins affect remodeling of both membrane and the actin cytoskeleton. The purpose of this review is to highlight the role of F-BAR proteins in coupling WASP/N-WASP to cytoskeletal remodeling. A role for F-BAR/WASP interaction in human diseases affecting nervous, blood, and neoplastic tissues is discussed.

  5. The hemopexin domain of MMP3 is responsible for mammary epithelial invasion and morphogenesis through extracellular interaction with HSP90β

    PubMed Central

    Correia, Ana Luísa; Mori, Hidetoshi; Chen, Emily I.; Schmitt, Fernando C.; Bissell, Mina J.

    2013-01-01

    Matrix metalloproteinases (MMPs) are crucial mediators in sculpting tissue architecture and are required for many physiological and pathological processes. MMP3 has been shown to regulate branching morphogenesis in the mammary gland. Ectopic expression of proteolytically active MMP3 in mouse mammary epithelia triggers supernumerary lateral branching and, eventually, tumors. Using a three-dimensional collagen-I (Col-1) gel assay that simulates epithelial invasion and branching, we show that it is the hemopexin domain that directs these processes. Using three different engineered constructs containing a variation on MMP3 structural domains, we confirmed the importance of the hemopexin domain also in primary organoids of the mammary gland. A proteomic screen of MMP3-binding partners surprisingly revealed that the intracellular chaperone heat-shock protein 90 β (HSP90β) is present extracellularly, and its interaction with the hemopexin domain of MMP3 is critical for invasion. Blocking of HSP90β with inhibitory antibodies added to the medium abolished invasion and branching. These findings shift the focus from the proteolytic activity of MMP3 as the central player to its hemopexin domain and add a new dimension to HSP90β's functions by revealing a hitherto undescribed mechanism of MMP3 regulation. Our data also may shed light on the failure of strategies to use MMP inhibitors in cancer treatment and other related disorders. PMID:23592797

  6. Using compound similarity and functional domain composition for prediction of drug-target interaction networks.

    PubMed

    Chen, Lei; He, Zhi-Song; Huang, Tao; Cai, Yu-Dong

    2010-11-01

    Study of interactions between drugs and target proteins is an essential step in genomic drug discovery. It is very hard to determine the compound-protein interactions or drug-target interactions by experiment alone. As supplementary, effective prediction model using machine learning or data mining methods can provide much help. In this study, a prediction method based on Nearest Neighbor Algorithm and a novel metric, which was obtained by combining compound similarity and functional domain composition, was proposed. The target proteins were divided into the following groups: enzymes, ion channels, G protein-coupled receptors, and nuclear receptors. As a result, four predictors with the optimal parameters were established. The overall prediction accuracies, evaluated by jackknife cross-validation test, for four groups of target proteins are 90.23%, 94.74%, 97.80%, and 97.51%, respectively, indicating that compound similarity and functional domain composition are very effective to predict drug-target interaction networks.

  7. Extracting sets of chemical substructures and protein domains governing drug-target interactions.

    PubMed

    Yamanishi, Yoshihiro; Pauwels, Edouard; Saigo, Hiroto; Stoven, Véronique

    2011-05-23

    The identification of rules governing molecular recognition between drug chemical substructures and protein functional sites is a challenging issue at many stages of the drug development process. In this paper we develop a novel method to extract sets of drug chemical substructures and protein domains that govern drug-target interactions on a genome-wide scale. This is made possible using sparse canonical correspondence analysis (SCCA) for analyzing drug substructure profiles and protein domain profiles simultaneously. The method does not depend on the availability of protein 3D structures. From a data set of known drug-target interactions including enzymes, ion channels, G protein-coupled receptors, and nuclear receptors, we extract a set of chemical substructures shared by drugs able to bind to a set of protein domains. These two sets of extracted chemical substructures and protein domains form components that can be further exploited in a drug discovery process. This approach successfully clusters protein domains that may be evolutionary unrelated but that bind a common set of chemical substructures. As shown in several examples, it can also be very helpful for predicting new protein-ligand interactions and addressing the problem of ligand specificity. The proposed method constitutes a contribution to the recent field of chemogenomics that aims to connect the chemical space with the biological space.

  8. Structure and function of the interacting domains of Spire and Fmn-family formins

    SciTech Connect

    Vizcarra, Christina L.; Kreutz, Barry; Rodal, Avital A.; Toms, Angela V.; Lu, Jun; Zheng, Wei; Quinlan, Margot E.; Eck, Michael J.

    2012-07-11

    Evidence for cooperation between actin nucleators is growing. The WH2-containing nucleator Spire and the formin Cappuccino interact directly, and both are essential for assembly of an actin mesh during Drosophila oogenesis. Their interaction requires the kinase noncatalytic C-lobe domain (KIND) domain of Spire and the C-terminal tail of the formin. Here we describe the crystal structure of the KIND domain of human Spir1 alone and in complex with the tail of Fmn2, a mammalian ortholog of Cappuccino. The KIND domain is structurally similar to the C-lobe of protein kinases. The Fmn2 tail is coordinated in an acidic cleft at the base of the domain that appears to have evolved via deletion of a helix from the canonical kinase fold. Our functional analysis of Cappuccino reveals an unexpected requirement for its tail in actin assembly. In addition, we find that the KIND/tail interaction blocks nucleation by Cappuccino and promotes its displacement from filament barbed ends providing insight into possible modes of cooperation between Spire and Cappuccino.

  9. Mother–Infant and Extra-Dyadic Interactions with a New Social Partner: Developmental Trajectories of Early Social Abilities during Play

    PubMed Central

    Fadda, Roberta; Lucarelli, Loredana

    2017-01-01

    Mother–infant interactions during feeding and play are pivotal experiences in the development of infants’ early social abilities (Stern, 1985, 1995; Biringen, 2000). Stern indicated distinctive characteristics of mother–infant interactions, respectively, during feeding and play, suggesting to evaluate both to better describe the complexity of such early affective and social experiences (Stern, 1996). Moreover, during the first years of life, infants acquire cognitive and social skills that allow them to interact with new social partners in extra-dyadic interactions. However, the relations between mother–child interactions and infants’ social skills in extra-dyadic interactions are still unknown. We investigated longitudinally the relations between mother–child interactions during feeding and play and child’s pre-verbal communicative abilities in extra-dyadic interactions during play. 20 dyads were evaluated at T1 (infants aged between 9–22 months) and 6 months later, at T2. The interdyadic differences in mother–infant interactions during feeding and play were evaluated, respectively, with the “Feeding Scale” (Chatoor et al., 1997) and with the “Play Scale” (Chatoor, 2006) and the socio-communicative abilities of children with a new social partner during play were evaluated with the “Early Social Communication Scales” (Mundy et al., 2003). We distinguished the dyads into two categories: dyads with functional interactions (high dyadic reciprocity, low dyadic conflict) and dyads with dysfunctional interactions (lower dyadic reciprocity, higher dyadic conflict). At T1, infants belonging to dyads with dysfunctional interactions were significantly lower in “Initiating Joint Attention” and in “Responding to Joint Attention” in interaction with a new social partner compared to the infants belonging to dyads with functional interactions. At T2, infants belonging to dyads with dysfunctional interactions were significantly lower in

  10. Dzyaloshinskii-Moriya interaction induced domain wall depinning anomaly in ferromagnetic nanowire

    NASA Astrophysics Data System (ADS)

    Kheng Teoh, Han; Goolaup, Sarjoosing; Siang Lew, Wen

    2017-01-01

    Magnetic domain wall positional manipulation is usually through the introduction of potential trap. In this work, we show that the presence of interfacial Dzyaloshinkii-Moriya interaction leads to a different static depinning field for Néel domain walls with the same handedness in a notched magnetic nanowire. The difference in static depinning field is due to the Néel domain wall spin orientation. The spin orientation leads to different torques being exerted on the localized magnetic moments. This inherently imposes a spin orientation dependent diode-like behavior for domain walls in a notched nanowire. An equation which relates the difference in static depinning field to the notch geometry is derived. Micromagnetic simulation with varying damping constant reveals the influence of damping constant on the strength of depinning anomaly.

  11. Domain walls in finite-width nanowires with interfacial Dzyaloshinskii-Moriya interaction

    NASA Astrophysics Data System (ADS)

    DeJong, M. D.; Livesey, K. L.

    2017-02-01

    It is widely known that the interfacial Dzyaloshinskii-Moriya interaction (DMI) may stabilize Néel walls rather than Bloch walls in magnetic thin films. When the DMI is weak, it results in a "tilted" Bloch wall. However, for most applications, domain walls are in nanowires rather than thin films. Here we present a semianalytic two-parameter calculation for the static domain wall in a nanowire of finite width and thickness, with DMI. The DMI strength that is needed to force a Néel wall is smaller in nanowires than in films due to demagnetizing energy. Even nanowires that are hundreds of nanometers wide may have different domain wall solutions than thin films and so their finite size must be considered. The impact of this result on current experiments is briefly discussed. We extend the model to show that applying a weak magnetic field allows the domain wall type to be tuned.

  12. Structures of the NLRP14 pyrin domain reveal a conformational switch mechanism regulating its molecular interactions

    PubMed Central

    Eibl, Clarissa; Hessenberger, Manuel; Wenger, Julia; Brandstetter, Hans

    2014-01-01

    The cytosolic tripartite NLR receptors serve as important signalling platforms in innate immunity. While the C-terminal domains act as sensor and activation modules, the N-terminal death-like domain, e.g. the CARD or pyrin domain, is thought to recruit downstream effector molecules by homotypic interactions. Such homotypic complexes have been determined for all members of the death-domain superfamily except for pyrin domains. Here, crystal structures of human NLRP14 pyrin-domain variants are reported. The wild-type protein as well as the clinical D86V mutant reveal an unexpected rearrangement of the C-terminal helix α6, resulting in an extended α5/6 stem-helix. This reordering mediates a novel symmetric pyrin-domain dimerization mode. The conformational switching is controlled by a charge-relay system with a drastic impact on protein stability. How the identified charge relay allows classification of NLRP receptors with respect to distinct recruitment mechanisms is discussed. PMID:25004977

  13. Determination of interfacial Dzyaloshinskii-Moriya exchange interaction from static domain size imaging

    NASA Astrophysics Data System (ADS)

    Agrawal, Parnika; Lemesh, Ivan; Schlotter, Sarah; Beach, Geoffrey

    Dzyaloshinskii-Moriya interaction (DMI) has been identified [1-2] as a necessary ingredient for the formation of chiral spin structures such as skyrmions and Néel domain walls in perpendicularly magnetized thin films. Various simulation and experimental studies have tried to quantify DMI from domain wall and skyrmion [3-4] motion with applied currents and magnetic fields. Here, a means to quantify DMI in multilayer films using only static magnetic characterizations is proposed. Static domain structure is observed using magnetic force microscopy (MFM) in multilayer stacks of [Pt(2.5-7.5nm)/CoFeB(0.8nm)/MgO(1.5nm)]15 where the thickness tpt of the Pt layer is systematically varied from 2.5 nm to 7.5 nm. A variation of domain size from ~300 nm to ~70 nm is seen in the labyrinthine demagnetized state as tpt is decreased. It is shown that the domain size as a function of tpt can be well-fitted analytically by a model in which the domain wall energy is the sole free parameter. Additional measurements of magnetic anisotropy of the film reveal the significant contribution of interfacial DMI (~1.4 mJ/m2) to the domain wall energy.

  14. Frequency- and Time-Domain Methods in Soil-Structure Interaction Analysis

    SciTech Connect

    Bolisetti, Chandrakanth; Whittaker, Andrew S.; Coleman, Justin L.

    2015-06-01

    Soil-structure interaction (SSI) analysis in the nuclear industry is currently performed using linear codes that function in the frequency domain. There is a consensus that these frequency-domain codes give reasonably accurate results for low-intensity ground motions that result in almost linear response. For higher intensity ground motions, which may result in nonlinear response in the soil, structure or at the vicinity of the foundation, the adequacy of frequency-domain codes is unproven. Nonlinear analysis, which is only possible in the time domain, is theoretically more appropriate in such cases. These methods are available but are rarely used due to the large computational requirements and a lack of experience with analysts and regulators. This paper presents an assessment of the linear frequency-domain code, SASSI, which is widely used in the nuclear industry, and the time-domain commercial finite-element code, LS-DYNA, for SSI analysis. The assessment involves benchmarking the SSI analysis procedure in LS-DYNA against SASSI for linearly elastic models. After affirming that SASSI and LS-DYNA result in almost identical responses for these models, they are used to perform nonlinear SSI analyses of two structures founded on soft soil. An examination of the results shows that, in spite of using identical material properties, the predictions of frequency- and time-domain codes are significantly different in the presence of nonlinear behavior such as gapping and sliding of the foundation.

  15. Tandem Domains with Tuned Interactions Are a Powerful Biological Design Principle.

    PubMed

    Nussinov, Ruth; Tsai, Chung-Jung

    2015-01-01

    Allosteric effects of mutations, ligand binding, or post-translational modifications on protein function occur through changes to the protein's shape, or conformation. In a cell, there are many copies of the same protein, all experiencing these perturbations in a dynamic fashion and fluctuating through different conformations and activity states. According to the "conformational selection and population shift" theory, ligand binding selects a particular conformation. This perturbs the ensemble and induces a population shift. In a new PLOS Biology paper, Melacini and colleagues describe a novel model of protein regulation, the "Double-Conformational Selection Model", which demonstrates how two tandem ligand-binding domains interact to regulate protein function. Here we explain how tandem domains with tuned interactions-but not single domains-can provide a blueprint for sensitive activation sensors within a narrow window of ligand concentration, thereby promoting signaling control.

  16. Importance of the REM (Ras exchange) domain for membrane interactions by RasGRP3.

    PubMed

    Czikora, Agnes; Kedei, Noemi; Kalish, Heather; Blumberg, Peter M

    2017-09-11

    RasGRP comprises a family of guanine nucleotide exchange factors, regulating the dissociation of GDP from Ras GTPases to enhance the formation of the active GTP-bound form. RasGRP1 possesses REM (Ras exchange), GEF (catalytic), EF-hand, C1, SuPT (suppressor of PT), and PT (plasma membrane-targeting) domains, among which the C1 domain drives membrane localization in response to diacylglycerol or phorbol ester and the PT domain recognizes phosphoinositides. The homologous family member RasGRP3 shows less plasma membrane localization. The objective of this study was to explore the role of the different domains of RasGRP3 in membrane translocation in response to phorbol esters. The full-length RasGRP3 shows limited translocation to the plasma membrane in response to PMA, even when the basic hydrophobic cluster in the PT domain, reported to be critical for RasGRP1 translocation to endogenous activators, is mutated to resemble that of RasGRP1. Moreover, exchange of the C-termini (SuPT-PT domain) of the two proteins had little effect on their plasma membrane translocation. On the other hand, while the C1 domain of RasGRP3 alone showed partial plasma membrane translocation, truncated RasGRP3 constructs, which contain the PT domain and are missing the REM, showed stronger translocation, indicating that the REM of RasGRP3 was a suppressor of its membrane interaction. The REM of RasGRP1 failed to show comparable suppression of RasGRP3 translocation. The marked differences between RasGRP3 and RasGRP1 in membrane interaction necessarily will contribute to their different behavior in cells and are relevant to the design of selective ligands as potential therapeutic agents. Published by Elsevier B.V.

  17. Complexity Theory: Inclusion of the Affective Domain in an Interactive Learning Model for Instructional Design.

    ERIC Educational Resources Information Center

    Tennyson, Robert D.; Nielsen, Milt

    1998-01-01

    Proposes an interactive learning model to serve as a psychological foundation for the field of instructional design theory. Topics include complexity theory, cognitive models of learning, cognitive and affective domains, sensory receptors component (memory) and the effects of external stimuli, affects component, cognitive strategies, and knowledge…

  18. Interactions among Domain-Specific Expectancies, Values, and Gender: Predictors of Test Anxiety during Early Adolescence

    ERIC Educational Resources Information Center

    Selkirk, Laura C.; Bouchey, Heather A.; Eccles, Jacquelynne S.

    2011-01-01

    This research focuses on the interaction between students' domain-specific expectancies and values as a predictor of test anxiety. A subsample of adolescents from the MSALT dataset are used in the current study; students complete measures during the spring of sixth grade and again during the spring of seventh grade. Overall, findings provide…

  19. A novel DFP tripeptide motif interacts with the coagulation factor XI apple 2 domain

    PubMed Central

    Wong, Szu S.; Østergaard, Søren; Hall, Gareth; Li, Chan; Williams, Philip M.; Stennicke, Henning

    2016-01-01

    Factor XI (FXI) is the zymogen of FXIa, which cleaves FIX in the intrinsic pathway of coagulation. FXI is known to exist as a dimer and interacts with multiple proteins via its 4 apple domains in the “saucer section” of the enzyme; however, to date, no complex crystal structure has been described. To investigate protein interactions of FXI, a large random peptide library consisting of 106 to 107 peptides was screened for FXI binding, which identified a series of FXI binding motifs containing the signature Asp-Phe-Pro (DFP) tripeptide. Motifs containing this core tripeptide were found in diverse proteins, including the known ligand high-molecular-weight kininogen (HK), as well as the extracellular matrix proteins laminin and collagen V. To define the binding site on FXI, we determined the crystal structure of FXI in complex with the HK-derived peptide NPISDFPDT. This revealed the location of the DFP peptide bound to the FXI apple 2 domain, and central to the interaction, the DFP phenylalanine side-chain inserts into a major hydrophobic pocket in the apple 2 domain and the isoleucine occupies a flanking minor pocket. Two further structures of FXI in complex with the laminin-derived peptide EFPDFP and a DFP peptide from the random screen demonstrated binding in the same pocket, although in a slightly different conformation, thus revealing some flexibility in the molecular interactions of the FXI apple 2 domain. PMID:27006387

  20. Using siRNA to define functional interactions between melanopsin and multiple G Protein partners.

    PubMed

    Hughes, Steven; Jagannath, Aarti; Hickey, Doron; Gatti, Silvia; Wood, Matthew; Peirson, Stuart N; Foster, Russell G; Hankins, Mark W

    2015-01-01

    Melanopsin expressing photosensitive retinal ganglion cells (pRGCs) represent a third class of ocular photoreceptors and mediate a range of non-image forming responses to light. Melanopsin is a G protein coupled receptor (GPCR) and existing data suggest that it employs a membrane bound signalling cascade involving Gnaq/11 type G proteins. However, to date the precise identity of the Gα subunits involved in melanopsin phototransduction remains poorly defined. Here we show that Gnaq, Gna11 and Gna14 are highly co-expressed in pRGCs of the mouse retina. Furthermore, using RNAi based gene silencing we show that melanopsin can signal via Gnaq, Gna11 or Gna14 in vitro, and demonstrate that multiple members of the Gnaq/11 subfamily, including Gna14 and at least Gnaq or Gna11, can participate in melanopsin phototransduction in vivo and contribute to the pupillary light responses of mice lacking rod and cone photoreceptors. This diversity of G protein interactions suggests additional complexity in the melanopsin phototransduction cascade and may provide a basis for generating the diversity of light responses observed from pRGC subtypes.

  1. The role of an E-box element: multiple frunctions and interacting partners.

    PubMed

    Seitz, Stefanie B; Voytsekh, Olga; Mohan, Karthik M; Mittag, Maria

    2010-09-01

    Circadian clocks can be entrained by light-dark or temperature cycles. In the green alga Chlamydomonas reinhardtii, 12h changes in temperature between 18°C and 28°C synchronize its clock. Both subunits of the circadian RNA-binding protein CHLAMY1, named C1 and C3, are able to integrate temperature information. C1 gets hyper-phosphorylated in cells grown at 18°C and the level of C3 is up-regulated at this temperature. In the long period mutant per1, where temperature entrainment is disturbed, the temperature-dependent regulation of C1 and C3 is altered. Up-regulation of C3 at the low temperature is mediated predominantly by an E-box element situated in its promoter region. This cis-acting element is also relevant for circadian expression of c3 as well as of its up-regulation in cells, where C1 is overexpressed. Among the few identified factors interacting with the E-box region, C3 is also present, suggesting that it feedbacks on its own transcription.

  2. Identification of novel PTEN-binding partners: PTEN interaction with fatty acid binding protein FABP4.

    PubMed

    Gorbenko, O; Panayotou, G; Zhyvoloup, A; Volkova, D; Gout, I; Filonenko, V

    2010-04-01

    PTEN is a tumor suppressor with dual protein and lipid-phosphatase activity, which is frequently deleted or mutated in many human advanced cancers. Recent studies have also demonstrated that PTEN is a promising target in type II diabetes and obesity treatment. Using C-terminal PTEN sequence in pEG202-NLS as bait, yeast two-hybrid screening on Mouse Embryo, Colon Cancer, and HeLa cDNA libraries was carried out. Isolated positive clones were validated by mating assay and identified through automated DNA sequencing and BLAST database searches. Sequence analysis revealed a number of PTEN-binding proteins linking this phosphatase to a number of different signaling cascades, suggesting that PTEN may perform other functions besides tumor-suppressing activity in different cell types. In particular, the interplay between PTEN function and adipocyte-specific fatty-acid-binding protein FABP4 is of notable interest. The demonstrable tautology of PTEN to FABP4 suggested a role for this phosphatase in the regulation of lipid metabolism and adipocyte differentiation. This interaction was further studied using coimmunoprecipitation and gel-filtration assays. Finally, based on Biacore assay, we have calculated the K(D) of PTEN-FABP4 complex, which is around 2.8 microM.

  3. 14-3-3 and its binding partners are regulators of protein–protein interactions during spermatogenesis

    PubMed Central

    Sun, Shengyi; Wong, Elissa W P; Li, Michelle W M; Lee, Will M; Cheng, C Yan

    2009-01-01

    During spermatogenesis, spermiation takes place at the adluminal edge of the seminiferous epithelium at stage VIII of the epithelial cycle during which fully developed spermatids (i.e. spermatozoa) detach from the epithelium in adult rat testes. This event coincides with the migration of preleptotene/leptotene spermatocytes across the blood–testis barrier from the basal to the apical (or adluminal) compartment. At stage XIV of the epithelial cycle, Pachytene spermatocytes (diploid, 2n) differentiate into diplotene spermatocytes (tetraploid, 4n) in the apical compartment of the epithelium, which begin meiosis I to be followed by meiosis II to form spermatids (haploid, 1n) at stage XIVof the epithelial cycle. These spermatids, in turn, undergo extensive morphological changes and traverse the seminiferous epithelium until they differentiate into elongated spermatids. Thus, there are extensive changes at the Sertoli–Sertoli and Sertoli–germ cell interface via protein ‘coupling’ and ‘uncoupling’ between cell adhesion protein complexes, as well as changes in interactions between integral membrane proteins and their peripheral adaptors, regulatory protein kinases and phosphatases, and the cytoskeletal proteins. These precisely coordinated protein–protein interactions affect cell adhesion and cell movement. In this review, we focus on the 14-3-3 protein family, whose members have different binding partners in the seminiferous epithelium. Recent studies have illustrated that 14-3-3 affects protein–protein interactions in the seminiferous epithelium, and regulates cell adhesion possibly via its effects on intracellular protein trafficking and cell-polarity proteins. This review provides a summary on the latest findings regarding the role of 14-3-3 family of proteins and their potential implications on spermatogenesis. We also highlight research areas that deserve attentions by investigators. PMID:19366886

  4. GIPC, a PDZ domain containing protein, interacts specifically with the C terminus