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Sample records for pseudokinase domain target

  1. Pseudokinases: update on their functions and evaluation as new drug targets.

    PubMed

    Byrne, Dominic P; Foulkes, Daniel M; Eyers, Patrick A

    2017-01-01

    The pseudokinase complement of the human kinase superfamily consists of approximately 60 signaling proteins, which lacks one or more of the amino acids typically required to correctly align ATP and metal ions, and phosphorylate protein substrates. Recent studies in the pseudokinase field have begun to expose the biological relevance of pseudokinases, which are now thought to perform a diverse range of physiological roles and are connected to a multitude of human diseases, including cancer. In this review, we discuss how and why members of the 'pseudokinome' represent important new targets for drug discovery, and describe how knowledge of protein structure and function provides informative clues to help guide the rational chemical design or repurposing of inhibitors to target pseudokinases.

  2. Structure of the pseudokinase-kinase domains from protein kinase TYK2 reveals a mechanism for Janus kinase (JAK) autoinhibition.

    PubMed

    Lupardus, Patrick J; Ultsch, Mark; Wallweber, Heidi; Bir Kohli, Pawan; Johnson, Adam R; Eigenbrot, Charles

    2014-06-03

    Janus kinases (JAKs) are receptor-associated multidomain tyrosine kinases that act downstream of many cytokines and interferons. JAK kinase activity is regulated by the adjacent pseudokinase domain via an unknown mechanism. Here, we report the 2.8-Å structure of the two-domain pseudokinase-kinase module from the JAK family member TYK2 in its autoinhibited form. We find that the pseudokinase and kinase interact near the kinase active site and that most reported mutations in cancer-associated JAK alleles cluster in or near this interface. Mutation of residues near the TYK2 interface that are analogous to those in cancer-associated JAK alleles, including the V617F and "exon 12" JAK2 mutations, results in increased kinase activity in vitro. These data indicate that JAK pseudokinases are autoinhibitory domains that hold the kinase domain inactive until receptor dimerization stimulates transition to an active state.

  3. Tyrosine Kinase 2-mediated Signal Transduction in T Lymphocytes Is Blocked by Pharmacological Stabilization of Its Pseudokinase Domain*

    PubMed Central

    Tokarski, John S.; Zupa-Fernandez, Adriana; Tredup, Jeffrey A.; Pike, Kristen; Chang, ChiehYing; Xie, Dianlin; Cheng, Lihong; Pedicord, Donna; Muckelbauer, Jodi; Johnson, Stephen R.; Wu, Sophie; Edavettal, Suzanne C.; Hong, Yang; Witmer, Mark R.; Elkin, Lisa L.; Blat, Yuval; Pitts, William J.; Weinstein, David S.; Burke, James R.

    2015-01-01

    Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified that bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor but not Tyk2-independent signaling and transcriptional cellular assays, including stimulation through the receptors for IL-2 (JAK1- and JAK3-dependent) and thrombopoietin (JAK2-dependent), demonstrating the high functional selectivity of this approach. A crystal structure of the pseudokinase domain liganded with a representative example showed the compound bound to a site analogous to the ATP-binding site in catalytic kinases with features consistent with high ligand selectivity. The results support a model where the pseudokinase domain regulates activation of the catalytic domain by forming receptor-regulated inhibitory interactions. Tyk2 pseudokinase stabilizers, therefore, represent a novel approach to the design of potent and selective agents for the treatment of autoimmunity. PMID:25762719

  4. A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties.

    PubMed

    Murphy, James M; Zhang, Qingwei; Young, Samuel N; Reese, Michael L; Bailey, Fiona P; Eyers, Patrick A; Ungureanu, Daniela; Hammaren, Henrik; Silvennoinen, Olli; Varghese, Leila N; Chen, Kelan; Tripaydonis, Anne; Jura, Natalia; Fukuda, Koichi; Qin, Jun; Nimchuk, Zachary; Mudgett, Mary Beth; Elowe, Sabine; Gee, Christine L; Liu, Ling; Daly, Roger J; Manning, Gerard; Babon, Jeffrey J; Lucet, Isabelle S

    2014-01-15

    Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.

  5. The Toxoplasma pseudokinase ROP5 is an allosteric inhibitor of the immunity-related GTPases.

    PubMed

    Reese, Michael L; Shah, Niket; Boothroyd, John C

    2014-10-03

    The Red Queen hypothesis proposes that there is an evolutionary arms race between host and pathogen. One possible example of such a phenomenon could be the recently discovered interaction between host defense proteins known as immunity-related GTPases (IRGs) and a family of rhoptry pseudokinases (ROP5) expressed by the protozoan parasite, Toxoplasma gondii. Mouse IRGs are encoded by an extensive and rapidly evolving family of over 20 genes. Similarly, the ROP5 family is highly polymorphic and consists of 4-10 genes, depending on the strain of Toxoplasma. IRGs are known to be avidly bound and functionally inactivated by ROP5 proteins, but the molecular basis of this interaction/inactivation has not previously been known. Here we show that ROP5 uses a highly polymorphic surface to bind adjacent to the nucleotide-binding domain of an IRG and that this produces a profound allosteric change in the IRG structure. This has two dramatic effects: 1) it prevents oligomerization of the IRG, and 2) it alters the orientation of two threonine residues that are targeted by the Toxoplasma Ser/Thr kinases, ROP17 and ROP18. ROP5s are highly specific in the IRGs that they will bind, and the fact that it is the most highly polymorphic surface of ROP5 that binds the IRG strongly supports the notion that these two protein families are co-evolving in a way predicted by the Red Queen hypothesis.

  6. Dimeric structure of pseudokinase RNase L bound to 2-5A reveals a basis for interferon induced antiviral activity

    PubMed Central

    Huang, Hao; Zeqiraj, Elton; Dong, Beihua; Jha, Babal Kant; Duffy, Nicole; Orlicky, Stephen; Thevakumaran, Neroshan; Talukdar, Manisha; Pillon, Monica C.; Ceccarelli, Derek F.; Wan, Leo; Juang, Yu-Chi; Mao, Daniel Y.L.; Gaughan, Christina; Brinton, Margo A.; Perelygin, Andrey A.; Kourinov, Igor; Guarné, Alba; Silverman, Robert H.; Sicheri, Frank

    2014-01-01

    Summary RNase L is an ankyrin repeat domain containing dual endoribonuclease-pseudokinase that is activated by unusual 2′,5′-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here, we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small angle x-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the non-hydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain together with nucleotide binding, impose a rigid intertwined dimer configuration that is essential for RNase catalytic and anti-viral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold. PMID:24462203

  7. Targeting Discoidin Domain Receptors in Prostate Cancer

    DTIC Science & Technology

    2016-08-01

    1 AWARD NUMBER: W81XWH-15-1-0226 TITLE: Targeting Discoidin Domain Receptors in Prostate Cancer PRINCIPAL INVESTIGATOR: Dr. Rafael Fridman...4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER W81XWH-15-1-0226 Targeting Discoidin Domain Receptors in Prostate Cancer 5b. GRANT NUMBER W81XWH-15...DDRs in prostate cancer . During the first funding period, we conducted immunohistochemical studies by staining a 200 case Grade/Stage tissue

  8. Discoidin Domains as Emerging Therapeutic Targets.

    PubMed

    Villoutreix, Bruno O; Miteva, Maria A

    2016-08-01

    Discoidin (DS) domains are found in eukaryotic and prokaryotic extracellular and transmembrane multidomain proteins. These small domains play different functional roles and can interact with phospholipids, glycans, and proteins, including collagens. DS domain-containing proteins are often involved in cellular adhesion, migration, proliferation, and matrix-remodeling events, while some play a major role in blood coagulation. Mutations in DS domains have been associated with various disease conditions. This review provides an update on the structure, function, and modulation of the DS domains, with a special emphasis on two circulating blood coagulation cofactors, factor V and factor VIII, and the transmembrane neuropilin receptors that have been targeted for inhibition by biologics and small chemical compounds.

  9. A pseudokinase couples signaling pathways to enable asymmetric cell division in a bacterium

    PubMed Central

    Childers, W. S.; Shapiro, Lucy

    2014-01-01

    Bacteria face complex decisions when initiating developmental events such as sporulation, nodulation, virulence, and asymmetric cell division. These developmental decisions require global changes in genomic readout, and bacteria typically employ intricate (yet poorly understood) signaling networks that enable changes in cell function. The bacterium Caulobacter crescentus divides asymmetrically to yield two functionally distinct cells: a motile, chemotactic swarmer cell, and a sessile stalked cell with replication and division capabilities. Work from several Caulobacter labs has revealed that differentiation requires concerted regulation by several two-component system (TCS) signaling pathways that are differentially positioned at the poles of the predivisional cell (Figure 1). The strict unidirectional flow from histidine kinase (HK) to the response regulator (RR), observed in most studied TCS, is difficult to reconcile with the notion that information can be transmitted between two or more TCS signaling pathways. In this study, we uncovered a mechanism by which daughter cell fate, which is specified by the DivJ-DivK-PleC system and effectively encoded in the phosphorylation state of the single-domain RR DivK, is communicated to the CckA-ChpT-CtrA signaling pathway that regulates more than 100 genes for polar differentiation, replication initiation and cell division. Using structural biology and biochemical findings we proposed a mechanistic basis for TCS pathway coupling in which the DivL pseudokinase is repurposed as a sensor rather than participant in phosphotransduction.

  10. The Rhoptry Pseudokinase ROP54 Modulates Toxoplasma gondii Virulence and Host GBP2 Loading.

    PubMed

    Kim, Elliot W; Nadipuram, Santhosh M; Tetlow, Ashley L; Barshop, William D; Liu, Philip T; Wohlschlegel, James A; Bradley, Peter J

    2016-01-01

    Toxoplasma gondii uses unique secretory organelles called rhoptries to inject an array of effector proteins into the host cytoplasm that hijack host cell functions. We have discovered a novel rhoptry pseudokinase effector, ROP54, which is injected into the host cell upon invasion and traffics to the cytoplasmic face of the parasitophorous vacuole membrane (PVM). Disruption of ROP54 in a type II strain of T. gondii does not affect growth in vitro but results in a 100-fold decrease in virulence in vivo, suggesting that ROP54 modulates some aspect of the host immune response. We show that parasites lacking ROP54 are more susceptible to macrophage-dependent clearance, further suggesting that ROP54 is involved in evasion of innate immunity. To determine how ROP54 modulates parasite virulence, we examined the loading of two known innate immune effectors, immunity-related GTPase b6 (IRGb6) and guanylate binding protein 2 (GBP2), in wild-type and ∆rop54II mutant parasites. While no difference in IRGb6 loading was seen, we observed a substantial increase in GBP2 loading on the parasitophorous vacuole (PV) of ROP54-disrupted parasites. These results demonstrate that ROP54 is a novel rhoptry effector protein that promotes Toxoplasma infections by modulating GBP2 loading onto parasite-containing vacuoles. IMPORTANCE The interactions between intracellular microbes and their host cells can lead to the discovery of novel drug targets. During Toxoplasma infections, host cells express an array of immunity-related GTPases (IRGs) and guanylate binding proteins (GBPs) that load onto the parasite-containing vacuole to clear the parasite. To counter this mechanism, the parasite secretes effector proteins that traffic to the vacuole to disarm the immunity-related loading proteins and evade the immune response. While the interplay between host IRGs and Toxoplasma effector proteins is well understood, little is known about how Toxoplasma neutralizes the GBP response. We describe here a T

  11. The Rhoptry Pseudokinase ROP54 Modulates Toxoplasma gondii Virulence and Host GBP2 Loading

    PubMed Central

    Kim, Elliot W.; Nadipuram, Santhosh M.; Tetlow, Ashley L.; Barshop, William D.; Liu, Philip T.; Wohlschlegel, James A.

    2016-01-01

    ABSTRACT Toxoplasma gondii uses unique secretory organelles called rhoptries to inject an array of effector proteins into the host cytoplasm that hijack host cell functions. We have discovered a novel rhoptry pseudokinase effector, ROP54, which is injected into the host cell upon invasion and traffics to the cytoplasmic face of the parasitophorous vacuole membrane (PVM). Disruption of ROP54 in a type II strain of T. gondii does not affect growth in vitro but results in a 100-fold decrease in virulence in vivo, suggesting that ROP54 modulates some aspect of the host immune response. We show that parasites lacking ROP54 are more susceptible to macrophage-dependent clearance, further suggesting that ROP54 is involved in evasion of innate immunity. To determine how ROP54 modulates parasite virulence, we examined the loading of two known innate immune effectors, immunity-related GTPase b6 (IRGb6) and guanylate binding protein 2 (GBP2), in wild-type and ∆rop54II mutant parasites. While no difference in IRGb6 loading was seen, we observed a substantial increase in GBP2 loading on the parasitophorous vacuole (PV) of ROP54-disrupted parasites. These results demonstrate that ROP54 is a novel rhoptry effector protein that promotes Toxoplasma infections by modulating GBP2 loading onto parasite-containing vacuoles. IMPORTANCE The interactions between intracellular microbes and their host cells can lead to the discovery of novel drug targets. During Toxoplasma infections, host cells express an array of immunity-related GTPases (IRGs) and guanylate binding proteins (GBPs) that load onto the parasite-containing vacuole to clear the parasite. To counter this mechanism, the parasite secretes effector proteins that traffic to the vacuole to disarm the immunity-related loading proteins and evade the immune response. While the interplay between host IRGs and Toxoplasma effector proteins is well understood, little is known about how Toxoplasma neutralizes the GBP response. We describe

  12. Targeting of passenger protein domains to multiple intracellular membranes.

    PubMed Central

    Janiak, F; Glover, J R; Leber, B; Rachubinski, R A; Andrews, D W

    1994-01-01

    The role of passenger domains in protein targeting was examined by fusing previously characterized targeting motifs to different protein sequences. To compare the targeting requirements for a variety of subcellular compartments, targeting of the fusion proteins was examined for endoplasmic reticulum, mitochondria and peroxisomes in vitro and in yeast. Although most passenger domains were only partially passive to translocation, motif-dependent targeting via motifs positioned at either end of one passenger domain (gPA) was demonstrated for all of the subcellular compartments tested. The data presented extend earlier suggestions that translocation competence is an intrinsic property of the passenger protein. However, the properties that determine protein targeting are not mutually exclusive for the compartments tested. Therefore, although the primary determinant of specificity is the targeting motif, our results suggest that translocation competence of the targeted protein augments the fidelity of transport. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8198533

  13. Exogenous agents that target transmembrane domains of proteins.

    PubMed

    Yin, Hang

    2008-01-01

    Although membrane proteins account for approximately one third of all proteins encoded in the human genome, the functions and structures of their transmembrane domains are much less understood than the water-soluble regions. A major hurdle in studying these transmembrane domains is the lack of appropriate exogenous agents that can be used as specific probes. Despite the daunting challenges, major strides have recently been made in targeting the transmembrane domains of a variety of membrane proteins. High affinity and selectivity have been achieved in model biophysical systems, membranes of bacteria, and mammalian cells.

  14. The crystal structure of PknI from Mycobacterium tuberculosis shows an inactive, pseudokinase-like conformation.

    PubMed

    Lisa, María-Natalia; Wagner, Tristan; Alexandre, Matthieu; Barilone, Nathalie; Raynal, Bertrand; Alzari, Pedro M; Bellinzoni, Marco

    2017-02-01

    Eukaryotic-like Ser/Thr protein kinases (ePKs) have been identified in many bacterial species, where they are known to mediate signalling mechanisms that share several features with their eukaryotic counterparts. In Mycobacterium tuberculosis, PknI is one of the 11 predicted ePKs and it has been related to bacterial virulence. In order to better understand the molecular basis of its role in mycobacterial signalling, we solved the crystal structure of the PknI cytoplasmic domain. We found that even though PknI possesses most conserved elements characteristic of Hanks-type kinases, it is degraded in several motifs that are essential for the ePKs catalytic activity. Most notably, PknI presents a remarkably short activation segment lacking a peptide-substrate binding site. Consistent with this observation and similar to earlier findings for eukaryotic pseudokinases, no kinase activity was detected for the catalytic domain of PknI, against different substrates and in various experimental conditions. Based on these results, we conclude that PknI may rely on unconventional mechanism(s) for kinase activity and/or it could play alternative role(s) in mycobacterial signalling.

  15. Limited Proteolysis Combined with Stable Isotope Labeling Reveals Conformational Changes in Protein (Pseudo)kinases upon Binding Small Molecules.

    PubMed

    Di Michele, Michela; Stes, Elisabeth; Vandermarliere, Elien; Arora, Rohit; Astorga-Wells, Juan; Vandenbussche, Jonathan; van Heerde, Erika; Zubarev, Roman; Bonnet, Pascal; Linders, Joannes T M; Jacoby, Edgar; Brehmer, Dirk; Martens, Lennart; Gevaert, Kris

    2015-10-02

    Likely due to conformational rearrangements, small molecule inhibitors may stabilize the active conformation of protein kinases and paradoxically promote tumorigenesis. We combined limited proteolysis with stable isotope labeling MS to monitor protein conformational changes upon binding of small molecules. Applying this method to the human serine/threonine kinase B-Raf, frequently mutated in cancer, we found that binding of ATP or its nonhydrolyzable analogue AMP-PNP, but not ADP, stabilized the structure of both B-Raf(WT) and B-Raf(V600E). The ATP-competitive type I B-Raf inhibitor vemurafenib and the type II inhibitor sorafenib stabilized the kinase domain (KD) but had distinct effects on the Ras-binding domain. Stabilization of the B-Raf(WT) KD was confirmed by hydrogen/deuterium exchange MS and molecular dynamics simulations. Our results are further supported by cellular assays in which we assessed cell viability and phosphorylation profiles in cells expressing B-Raf(WT) or B-Raf(V600E) in response to vemurafenib or sorafenib. Our data indicate that an overall stabilization of the B-Raf structure by specific inhibitors activates MAPK signaling and increases cell survival, helping to explain clinical treatment failure. We also applied our method to monitor conformational changes upon nucleotide binding of the pseudokinase KSR1, which holds high potential for inhibition in human diseases.

  16. The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2.

    PubMed

    Sklodowski, Kamil; Riedelsberger, Janin; Raddatz, Natalia; Riadi, Gonzalo; Caballero, Julio; Chérel, Isabelle; Schulze, Waltraud; Graf, Alexander; Dreyer, Ingo

    2017-03-16

    The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H(+)-ATPase-energized K(+) uptake. Moreover, through reversible post-translational modifications it can also function as an open, K(+)-selective channel, which taps a 'potassium battery', providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.

  17. The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2

    PubMed Central

    Sklodowski, Kamil; Riedelsberger, Janin; Raddatz, Natalia; Riadi, Gonzalo; Caballero, Julio; Chérel, Isabelle; Schulze, Waltraud; Graf, Alexander; Dreyer, Ingo

    2017-01-01

    The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a ‘potassium battery’, providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals. PMID:28300158

  18. The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2

    NASA Astrophysics Data System (ADS)

    Sklodowski, Kamil; Riedelsberger, Janin; Raddatz, Natalia; Riadi, Gonzalo; Caballero, Julio; Chérel, Isabelle; Schulze, Waltraud; Graf, Alexander; Dreyer, Ingo

    2017-03-01

    The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a ‘potassium battery’, providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.

  19. Superdiffusive motion of membrane-targeting C2 domains

    NASA Astrophysics Data System (ADS)

    Campagnola, Grace; Nepal, Kanti; Schroder, Bryce W.; Peersen, Olve B.; Krapf, Diego

    2015-12-01

    Membrane-targeting domains play crucial roles in the recruitment of signalling molecules to the plasma membrane. For most peripheral proteins, the protein-to-membrane interaction is transient. After proteins dissociate from the membrane they have been observed to rebind following brief excursions in the bulk solution. Such membrane hops can have broad implications for the efficiency of reactions on membranes. We study the diffusion of membrane-targeting C2 domains using single-molecule tracking in supported lipid bilayers. The ensemble-averaged mean square displacement (MSD) exhibits superdiffusive behaviour. However, traditional time-averaged MSD analysis of individual trajectories remains linear and does not reveal superdiffusion. Our observations are explained in terms of bulk excursions that introduce jumps with a heavy-tail distribution. These hopping events allow proteins to explore large areas in a short time. The experimental results are shown to be consistent with analytical models of bulk-mediated diffusion and numerical simulations.

  20. Superdiffusive motion of membrane-targeting C2 domains

    PubMed Central

    Campagnola, Grace; Nepal, Kanti; Schroder, Bryce W.; Peersen, Olve B.; Krapf, Diego

    2015-01-01

    Membrane-targeting domains play crucial roles in the recruitment of signalling molecules to the plasma membrane. For most peripheral proteins, the protein-to-membrane interaction is transient. After proteins dissociate from the membrane they have been observed to rebind following brief excursions in the bulk solution. Such membrane hops can have broad implications for the efficiency of reactions on membranes. We study the diffusion of membrane-targeting C2 domains using single-molecule tracking in supported lipid bilayers. The ensemble-averaged mean square displacement (MSD) exhibits superdiffusive behaviour. However, traditional time-averaged MSD analysis of individual trajectories remains linear and does not reveal superdiffusion. Our observations are explained in terms of bulk excursions that introduce jumps with a heavy-tail distribution. These hopping events allow proteins to explore large areas in a short time. The experimental results are shown to be consistent with analytical models of bulk-mediated diffusion and numerical simulations. PMID:26639944

  1. Radar target imaging by time-domain inverse scattering

    NASA Astrophysics Data System (ADS)

    Morag, M.

    1981-03-01

    This thesis describes the study and development of a workable inverse scattering method for imaging and identification of radar targets. The space-time integral approach is used for iterative target shape reconstruction. Following an overview of transient electromagnetics, the integral equation is applied for thin-wire transient response computation. The analytical time domain integral equation is derived and solved numerically, for general conducting bodies of revolution. Finally the algorithm for an inverse scattering computer solution is derived and tested under simulation of physical environments.

  2. Structural Basis for Endosomal Targeting by the Bro1 Domain

    PubMed Central

    Kim, Jaewon; Sitaraman, Sujatha; Hierro, Aitor; Beach, Bridgette M.; Odorizzi, Greg; Hurley, James H.

    2010-01-01

    Summary Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs. PMID:15935782

  3. Structural basis for endosomal targeting by the Bro1 domain.

    PubMed

    Kim, Jaewon; Sitaraman, Sujatha; Hierro, Aitor; Beach, Bridgette M; Odorizzi, Greg; Hurley, James H

    2005-06-01

    Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs.

  4. Adenoviral targeting using genetically incorporated camelid single variable domains

    PubMed Central

    Kaliberov, Sergey A.; Kaliberova, Lyudmila N.; Buggio, Maurizio; Tremblay, Jacqueline M.; Shoemaker, Charles B.; Curiel, David T.

    2014-01-01

    The unique ability of human adenovirus serotype 5 (Ad5) to accomplish efficient transduction has allowed the use of Ad5-based vectors for a range of gene therapy applications. Several strategies have been developed to alter tropism of Ad vectors to achieve a cell-specific gene delivery by employing fiber modifications via genetic incorporation of targeting motifs. In this study we have explored the utility of novel anti-human carcinoembryonic antigen (hCEA) single variable domains derived from heavy chain (VHH) camelid family of antibodies to achieve targeted gene transfer. To obtain anti-CEA VHHs we produced a VHH-display library from peripheral blood lymphocytes RNA of alpacas at the peak of immune response to the hCEA antigen. We genetically incorporated an anti-hCEA VHH into a de-knobbed Ad5 fiber-fibritin chimera and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of target cells. We report that the anti-hCEA VHH employed in this study retains antigen recognition functionality and provides specificity for gene transfer of capsid-modified Ad5 vectors. These studies clearly demonstrated the feasibility of retargeting of Ad5-based gene transfer using VHHs. PMID:24933423

  5. Adenoviral targeting using genetically incorporated camelid single variable domains.

    PubMed

    Kaliberov, Sergey A; Kaliberova, Lyudmila N; Buggio, Maurizio; Tremblay, Jacqueline M; Shoemaker, Charles B; Curiel, David T

    2014-08-01

    The unique ability of human adenovirus serotype 5 (Ad5) to accomplish efficient transduction has allowed the use of Ad5-based vectors for a range of gene therapy applications. Several strategies have been developed to alter tropism of Ad vectors to achieve a cell-specific gene delivery by using fiber modifications via genetic incorporation of targeting motifs. In this study, we have explored the utility of novel anti-human carcinoembryonic antigen (hCEA) single variable domains derived from heavy chain (VHH) camelid family of antibodies to achieve targeted gene transfer. To obtain anti-CEA VHHs, we produced a VHH-display library from peripheral blood lymphocytes RNA of alpacas at the peak of immune response to the hCEA antigen (Ag). We genetically incorporated an anti-hCEA VHH into a de-knobbed Ad5 fiber-fibritin chimera and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of target cells. We report that the anti-hCEA VHH used in this study retains Ag recognition functionality and provides specificity for gene transfer of capsid-modified Ad5 vectors. These studies clearly demonstrated the feasibility of retargeting of Ad5-based gene transfer using VHHs.

  6. The pseudokinase CaMKv is required for the activity-dependent maintenance of dendritic spines

    PubMed Central

    Liang, Zhuoyi; Zhan, Yi; Shen, Yang; Wong, Catherine C. L.; Yates, John R.; Plattner, Florian; Lai, Kwok-On; Ip, Nancy Y.

    2016-01-01

    Dendritic spine stabilization depends on afferent synaptic input and requires changes in actin cytoskeleton dynamics and protein synthesis. However, the underlying molecular mechanism remains unclear. Here we report the identification of ‘calmodulin kinase-like vesicle-associated' (CaMKv), a pseudokinase of the CaMK family with unknown function, as a synaptic protein crucial for dendritic spine maintenance. CaMKv mRNA localizes at dendrites, and its protein synthesis is regulated by neuronal activity. CaMKv function is inhibited upon phosphorylation by cyclin-dependent kinase 5 (Cdk5) at Thr345. Furthermore, CaMKv knockdown in mouse hippocampal CA1 pyramidal neurons impairs synaptic transmission and plasticity in vivo, resulting in hyperactivity and spatial memory impairment. These findings collectively indicate that the precise regulation of CaMKv through activity-dependent synthesis and post-translational phosphorylation is critical for dendritic spine maintenance, revealing an unusual signalling pathway in the regulation of synaptic transmission and brain function that involves a pseudokinase. PMID:27796283

  7. Fourier domain target transformation analysis in the thermal infrared

    NASA Technical Reports Server (NTRS)

    Anderson, D. L.

    1993-01-01

    Remote sensing uses of principal component analysis (PCA) of multispectral images include band selection and optimal color selection for display of information content. PCA has also been used for quantitative determination of mineral types and abundances given end member spectra. The preliminary results of the investigation of target transformation PCA (TTPCA) in the fourier domain to both identify end member spectra in an unknown spectrum, and to then calculate the relative concentrations of these selected end members are presented. Identification of endmember spectra in an unknown sample has previously been performed through bandmatching, expert systems, and binary classifiers. Both bandmatching and expert system techniques require the analyst to select bands or combinations of bands unique to each endmember. Thermal infrared mineral spectra have broad spectral features which vary subtly with composition. This makes identification of unique features difficult. Alternatively, whole spectra can be used in the classification process, in which case there is not need for an expert to identify unique spectra. Use of binary classifiers on whole spectra to identify endmember components has met with some success. These techniques can be used, along with a least squares fit approach on the endmembers identified, to derive compositional information. An alternative to the approach outlined above usese target transformation in conjunction with PCA to both identify and quantify the composition of unknown spectra. Preprocessing of the library and unknown spectra into the fourier domain, and using only a specific number of the components, allows for significant data volume reduction while maintaining a linear relationship in a Beer's Law sense. The approach taken here is to iteratively calculate concentrations, reducing the number of endmember components until only non-negative concentrations remain.

  8. Discoidin Domain Receptors: Potential Actors and Targets in Cancer

    PubMed Central

    Rammal, Hassan; Saby, Charles; Magnien, Kevin; Van-Gulick, Laurence; Garnotel, Roselyne; Buache, Emilie; El Btaouri, Hassan; Jeannesson, Pierre; Morjani, Hamid

    2016-01-01

    The extracellular matrix critically controls cancer cell behavior by inducing several signaling pathways through cell membrane receptors. Besides conferring structural properties to tissues around the tumor, the extracellular matrix is able to regulate cell proliferation, survival, migration, and invasion. Among these receptors, the integrins family constitutes a major class of receptors that mediate cell interactions with extracellular matrix components. Twenty years ago, a new class of extracellular matrix receptors has been discovered. These tyrosine kinase receptors are the two discoidin domain receptors DDR1 and DDR2. DDR1 was first identified in the Dictyostelium discoideum and was shown to mediate cell aggregation. DDR2 shares highly conserved sequences with DDR1. Both receptors are activated upon binding to collagen, one of the most abundant proteins in extracellular matrix. While DDR2 can only be activated by fibrillar collagen, particularly types I and III, DDR1 is mostly activated by type I and IV collagens. In contrast with classical growth factor tyrosine kinase receptors which display a rapid and transient activation, DDR1 and DDR2 are unique in that they exhibit delayed and sustained receptor phosphorylation upon binding to collagen. Recent studies have reported differential expression and mutations of DDR1 and DDR2 in several cancer types and indicate clearly that these receptors have to be taken into account as new players in the different aspects of tumor progression, from non-malignant to highly malignant and invasive stages. This review will discuss the current knowledge on the role of DDR1 and DDR2 in malignant transformation, cell proliferation, epithelial to mesenchymal transition, migratory, and invasive processes, and finally the modulation of the response to chemotherapy. These new insights suggest that DDR1 and DDR2 are new potential targets in cancer therapy. PMID:27014069

  9. Discoidin Domain Receptors: Potential Actors and Targets in Cancer.

    PubMed

    Rammal, Hassan; Saby, Charles; Magnien, Kevin; Van-Gulick, Laurence; Garnotel, Roselyne; Buache, Emilie; El Btaouri, Hassan; Jeannesson, Pierre; Morjani, Hamid

    2016-01-01

    The extracellular matrix critically controls cancer cell behavior by inducing several signaling pathways through cell membrane receptors. Besides conferring structural properties to tissues around the tumor, the extracellular matrix is able to regulate cell proliferation, survival, migration, and invasion. Among these receptors, the integrins family constitutes a major class of receptors that mediate cell interactions with extracellular matrix components. Twenty years ago, a new class of extracellular matrix receptors has been discovered. These tyrosine kinase receptors are the two discoidin domain receptors DDR1 and DDR2. DDR1 was first identified in the Dictyostelium discoideum and was shown to mediate cell aggregation. DDR2 shares highly conserved sequences with DDR1. Both receptors are activated upon binding to collagen, one of the most abundant proteins in extracellular matrix. While DDR2 can only be activated by fibrillar collagen, particularly types I and III, DDR1 is mostly activated by type I and IV collagens. In contrast with classical growth factor tyrosine kinase receptors which display a rapid and transient activation, DDR1 and DDR2 are unique in that they exhibit delayed and sustained receptor phosphorylation upon binding to collagen. Recent studies have reported differential expression and mutations of DDR1 and DDR2 in several cancer types and indicate clearly that these receptors have to be taken into account as new players in the different aspects of tumor progression, from non-malignant to highly malignant and invasive stages. This review will discuss the current knowledge on the role of DDR1 and DDR2 in malignant transformation, cell proliferation, epithelial to mesenchymal transition, migratory, and invasive processes, and finally the modulation of the response to chemotherapy. These new insights suggest that DDR1 and DDR2 are new potential targets in cancer therapy.

  10. Knowledge-based approach for generating target system specifications from a domain model

    NASA Technical Reports Server (NTRS)

    Gomaa, Hassan; Kerschberg, Larry; Sugumaran, Vijayan

    1992-01-01

    Several institutions in industry and academia are pursuing research efforts in domain modeling to address unresolved issues in software reuse. To demonstrate the concepts of domain modeling and software reuse, a prototype software engineering environment is being developed at George Mason University to support the creation of domain models and the generation of target system specifications. This prototype environment, which is application domain independent, consists of an integrated set of commercial off-the-shelf software tools and custom-developed software tools. This paper describes the knowledge-based tool that was developed as part of the environment to generate target system specifications from a domain model.

  11. Structures and target recognition modes of PDZ domains: recurring themes and emerging pictures.

    PubMed

    Ye, Fei; Zhang, Mingjie

    2013-10-01

    PDZ domains are highly abundant protein-protein interaction modules and are often found in multidomain scaffold proteins. PDZ-domain-containing scaffold proteins regulate multiple biological processes, including trafficking and clustering receptors and ion channels at defined membrane regions, organizing and targeting signalling complexes at specific cellular compartments, interfacing cytoskeletal structures with membranes, and maintaining various cellular structures. PDZ domains, each with ~90-amino-acid residues folding into a highly similar structure, are best known to bind to short C-terminal tail peptides of their target proteins. A series of recent studies have revealed that, in addition to the canonical target-binding mode, many PDZ-target interactions involve amino acid residues beyond the regular PDZ domain fold, which we refer to as extensions. Such extension sequences often form an integral structural and functional unit with the attached PDZ domain, which is defined as a PDZ supramodule. Correspondingly, PDZ-domain-binding sequences from target proteins are frequently found to require extension sequences beyond canonical short C-terminal tail peptides. Formation of PDZ supramodules not only affords necessary binding specificities and affinities demanded by physiological functions of PDZ domain targets, but also provides regulatory switches to be built in the PDZ-target interactions. At the 20th anniversary of the discovery of PDZ domain proteins, we try to summarize structural features and target-binding properties of such PDZ supramodules emerging from studies in recent years.

  12. Unsupervised Spatial Event Detection in Targeted Domains with Applications to Civil Unrest Modeling

    PubMed Central

    Zhao, Liang; Chen, Feng; Dai, Jing; Hua, Ting; Lu, Chang-Tien; Ramakrishnan, Naren

    2014-01-01

    Twitter has become a popular data source as a surrogate for monitoring and detecting events. Targeted domains such as crime, election, and social unrest require the creation of algorithms capable of detecting events pertinent to these domains. Due to the unstructured language, short-length messages, dynamics, and heterogeneity typical of Twitter data streams, it is technically difficult and labor-intensive to develop and maintain supervised learning systems. We present a novel unsupervised approach for detecting spatial events in targeted domains and illustrate this approach using one specific domain, viz. civil unrest modeling. Given a targeted domain, we propose a dynamic query expansion algorithm to iteratively expand domain-related terms, and generate a tweet homogeneous graph. An anomaly identification method is utilized to detect spatial events over this graph by jointly maximizing local modularity and spatial scan statistics. Extensive experiments conducted in 10 Latin American countries demonstrate the effectiveness of the proposed approach. PMID:25350136

  13. Using llama derived single domain antibodies to target botulinum neurotoxins

    NASA Astrophysics Data System (ADS)

    Swain, Marla D.; Anderson, George P.; Bernstein, Rachael D.; Liu, Jinny L.; Goldman, Ellen R.

    2010-04-01

    Llama serum contains both conventional IgG as well as unique forms of antibody that contain only heavy chains where antigen binding is mediated through a single variable domain. These variable domains can be expressed recombinantly and are referred to as single domain antibodies (sdAb). SdAb are among the smallest known naturally derived antigen binding fragments, possess good solubility, thermal stability, and can refold after heat and chemical denaturation. Llamas were immunized with either BoNT A or B toxoid and phage display libraries prepared. Single domain antibodies (sdAb) that were able to detect botulinum neurotoxin (BoNT) serotypes A and B were selected from their respective libraries. Here, the binders obtained by panning the BoNT B library on either BoNT B toxoid or BoNT B complex toxoid coated plates or BoNT B toxin coupled microspheres are described. Using these panning methods, we selected for binders that showed specificity for BoNT B. Phage displayed binders were screened, moved to a protein expression vector and soluble sdAb was produced. Using a Luminex flow cytometer binders were evaluated in direct binding assays. We have exploited the unique properties of sdAb and used them as biological recognition elements in immuno-based sensors that can detect BoNT B.

  14. Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains.

    PubMed

    Shi, Junwei; Wang, Eric; Milazzo, Joseph P; Wang, Zihua; Kinney, Justin B; Vakoc, Christopher R

    2015-06-01

    CRISPR-Cas9 genome editing technology holds great promise for discovering therapeutic targets in cancer and other diseases. Current screening strategies target CRISPR-Cas9-induced mutations to the 5' exons of candidate genes, but this approach often produces in-frame variants that retain functionality, which can obscure even strong genetic dependencies. Here we overcome this limitation by targeting CRISPR-Cas9 mutagenesis to exons encoding functional protein domains. This generates a higher proportion of null mutations and substantially increases the potency of negative selection. We also show that the magnitude of negative selection can be used to infer the functional importance of individual protein domains of interest. A screen of 192 chromatin regulatory domains in murine acute myeloid leukemia cells identifies six known drug targets and 19 additional dependencies. A broader application of this approach may allow comprehensive identification of protein domains that sustain cancer cells and are suitable for drug targeting.

  15. Positive Regulation of Interleukin-2 Expression by a Pseudokinase, Tribbles 1, in Activated T Cells.

    PubMed

    Miyajima, Chiharu; Itoh, Yuka; Inoue, Yasumichi; Hayashi, Hidetoshi

    2015-01-01

    Tribbles 1 (TRB1), a member of the Tribbles family, is a pseudokinase that is conserved among species and implicated in various human diseases including leukemia, cardiovascular diseases, and metabolic disorders. However, the role of TRB1 in the immune response is not understood. To evaluate this role, we examined regulation of TRB1 expression and the function of TRB1 in interleukin-2 (IL-2) induction in Jurkat cells, a human acute T cell leukemia cell line. We found that TRB1 was strongly induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin in these cells. IL-2 expression was induced in Jurkat cells activated by PMA and ionomycin; however, knockdown of TRB1 resulted in decreased induction of IL-2. TRB1 null Jurkat cells established using the CRISPR/Cas9 system also showed reduction of IL-2 expression on PMA/ionomycin stimulation. TRB1 knockdown also markedly inhibited IL-2 promoter activation. To determine the mechanism of the stimulatory effect on IL-2 induction, we focused on histone deacetylases (HDACs), and found that HDAC1 preferentially interacts with TRB1. TRB1 suppressed the interaction of HDAC1 with nuclear factor of activated T cells 2 (NFAT2), which is a crucial transcription factor for IL-2 induction. These results indicate that TRB1 is a positive regulator of IL-2 induction in activated T cells.

  16. The pseudokinase MLKL mediates programmed hepatocellular necrosis independently of RIPK3 during hepatitis

    PubMed Central

    Günther, Claudia; He, Gui-Wei; Kremer, Andreas E.; Murphy, James M.; Petrie, Emma J.; Amann, Kerstin; Linkermann, Andreas; Poremba, Christopher; Schleicher, Ulrike; Dewitz, Christin; Neurath, Markus F.; Becker, Christoph; Wirtz, Stefan

    2016-01-01

    Although necrosis and necroinflammation are central features of many liver diseases, the role of programmed necrosis in the context of inflammation-dependent hepatocellular death remains to be fully determined. Here, we have demonstrated that the pseudokinase mixed lineage kinase domain–like protein (MLKL), which plays a key role in the execution of receptor-interacting protein (RIP) kinase–dependent necroptosis, is upregulated and activated in human autoimmune hepatitis and in a murine model of inflammation-dependent hepatitis. Using genetic and pharmacologic approaches, we determined that hepatocellular necrosis in experimental hepatitis is driven by an MLKL-dependent pathway that occurs independently of RIPK3. Moreover, we have provided evidence that the cytotoxic activity of the proinflammatory cytokine IFN-γ in hepatic inflammation is strongly connected to induction of MLKL expression via activation of the transcription factor STAT1. In summary, our results reveal a pathway for MLKL-dependent programmed necrosis that is executed in the absence of RIPK3 and potentially drives the pathogenesis of severe liver diseases. PMID:27756058

  17. The Replication Focus Targeting Sequence (RFTS) Domain Is a DNA-competitive Inhibitor of Dnmt1

    SciTech Connect

    Syeda, Farisa; Fagan, Rebecca L.; Wean, Matthew; Avvakumov, George V.; Walker, John R.; Xue, Sheng; Dhe-Paganon, Sirano; Brenner, Charles

    2015-11-30

    Dnmt1 (DNA methyltransferase 1) is the principal enzyme responsible for maintenance of cytosine methylation at CpG dinucleotides in the mammalian genome. The N-terminal replication focus targeting sequence (RFTS) domain of Dnmt1 has been implicated in subcellular localization, protein association, and catalytic function. However, progress in understanding its function has been limited by the lack of assays for and a structure of this domain. Here, we show that the naked DNA- and polynucleosome-binding activities of Dnmt1 are inhibited by the RFTS domain, which functions by virtue of binding the catalytic domain to the exclusion of DNA. Kinetic analysis with a fluorogenic DNA substrate established the RFTS domain as a 600-fold inhibitor of Dnmt1 enzymatic activity. The crystal structure of the RFTS domain reveals a novel fold and supports a mechanism in which an RFTS-targeted Dnmt1-binding protein, such as Uhrf1, may activate Dnmt1 for DNA binding.

  18. Expanding the Targeting Process into the Space Domain

    DTIC Science & Technology

    2008-06-01

    estimated to average 1 hour per response, including the time for reviewing instruction, searching existing data sources, gathering and maintaining...planning and operations. The process is a continuous method by which information is converted into intelligence and made available to users...Targeting personnel and organizations consume intelligence produced by various agencies and organizations. Actionable and predictive intelligence applies to

  19. Defining the Domain Arrangement of the Mammalian Target of Rapamycin Complex Component Rictor Protein

    PubMed Central

    Zhou, Ping; Zhang, Ning; Nussinov, Ruth

    2015-01-01

    Abstract Mammalian target of rapamycin (mTOR) complexes play a pivotal role in the cell. Raptor and Rictor proteins interact with mTOR to form two distinct complexes, mTORC1 and mTORC2, respectively. While the domain structure of Raptor is known, current bioinformatics tools failed to classify the domains in Rictor. Here we focus on identifying specific domains in Rictor by searching for conserved regions. We scanned the pdb structural database and constructed three protein domain datasets. Next we carried out multiple pairwise sequence alignments of the proteins in the domain dataset. By analyzing the z-scores of Rictor sequence similarity to protein sequences in the dataset, we assigned the structural and functional domains of Rictor. We found that, like Raptor, Rictor also has HEAT and WD40 domains, which could be the common motif binding to mTORC. Rictor may also have pleckstrin homology domains, which mediate cellular localization and transmit signals to downstream targets, as well as a domain that is homologous to 50S protein L17 and human 39S protein L17. This putative ribosome binding domain could mediate mTORC2–ribosome interaction. PMID:26176550

  20. Ligands located within a cholesterol domain enhance gene delivery to the target tissue

    PubMed Central

    Xu, Long; Betker, Jamie; Yin, Hao; Anchordoquy, Thomas J.

    2012-01-01

    Targeted gene delivery provides enormous potential for clinical treatment of many incurable diseases. Liposomes formulated with targeting ligands have been tested extensively both in vitro and in vivo, and many studies have strived to identify more efficacious ligands. However, the environment of the ligand within the delivery vehicle is generally not considered, and this study assesses the effect of ligand micoenvironment by utilizing a lipoplex possessing a cholesterol domain. Our recent work has shown that the presence of the targeting ligand within the cholesterol domain promotes more productive transfection in cultured cells. In the present study, lipoplexes having the identical lipid composition were formulated with different conjugates of the folate ligand such that the ligand was included in, or excluded from, the cholesterol domain. The effect of locating the ligand within the cholesterol domain was then tested in a xenograft tumor model in mice. Lipoplexes that included the ligand within the cholesterol domain showed significantly higher luciferase expression and plasmid accumulation in tumors as compared to lipoplexes in which the ligand was excluded from the domain. These results demonstrate that the microenvironment of the ligand can affect gene delivery to tumors, and show that ligand-mediated delivery can be enhanced by locating targeting ligands within a cholesterol domain. PMID:22440429

  1. Coupling of tandem Smad ubiquitination regulatory factor (Smurf) WW domains modulates target specificity.

    PubMed

    Chong, P Andrew; Lin, Hong; Wrana, Jeffrey L; Forman-Kay, Julie D

    2010-10-26

    Smad ubiquitination regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase that participates in degradation of TGF-β receptors and other targets. Smurf2 WW domains recognize PPXY (PY) motifs on ubiquitin ligase target proteins or on adapters, such as Smad7, that bind to E3 target proteins. We previously demonstrated that the isolated WW3 domain of Smurf2, but not the WW2 domain, can directly bind to a Smad7 PY motif. We show here that the WW2 augments this interaction by binding to the WW3 and making auxiliary contacts with the PY motif and a novel E/D-S/T-P motif, which is N-terminal to all Smad PY motifs. The WW2 likely enhances the selectivity of Smurf2 for the Smad proteins. NMR titrations confirm that Smad1 and Smad2 are bound by Smurf2 with the same coupled WW domain arrangement used to bind Smad7. The analogous WW domains in the short isoform of Smurf1 recognize the Smad7 PY peptide using the same coupled mechanism. However, a longer Smurf1 isoform, which has an additional 26 residues in the inter-WW domain linker, is only partially able to use the coupled WW domain binding mechanism. The longer linker results in a decrease in affinity for the Smad7 peptide. Interdomain coupling of WW domains enhances selectivity and enables the tuning of interactions by isoform switching.

  2. Versatile TPR domains accommodate different modes of target protein recognition and function.

    PubMed

    Allan, Rudi Kenneth; Ratajczak, Thomas

    2011-07-01

    The tetratricopeptide repeat (TPR) motif is one of many repeat motifs that form structural domains in proteins that can act as interaction scaffolds in the formation of multi-protein complexes involved in numerous cellular processes such as transcription, the cell cycle, protein translocation, protein degradation and host defence against invading pathogens. The crystal structures of many TPR domain-containing proteins have been determined, showing TPR motifs as two anti-parallel α-helices packed in tandem arrays to form a structure with an amphipathic groove which can bind a target peptide. This is however not the only mode of target recognition by TPR domains, with short amino acid insertions and alternative TPR motif conformations also shown to contribute to protein interactions, highlighting diversity in TPR domains and the versatility of this structure in mediating biological events.

  3. 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.

  4. Chloroplast targeting factor AKR2 evolved from an ankyrin repeat domain coincidentally binds two chloroplast lipids

    PubMed Central

    Kim, Dae Heon; Park, Mi-Jeong; Gwon, Gwang Hyeon; Silkov, Antonina; Xu, Zheng-Yi; Yang, Eun Chan; Song, Seohyeon; Song, Kyungyoung; Kim, Younghyun; Yoon, Hwan Su; Honig, Barry; Cho, Wonhwa; Cho, Yunje; Hwang, Inhwan

    2014-01-01

    SUMMARY In organellogenesis of the chloroplast from endosymbiotic cyanobacterium, the establishment of protein targeting mechanisms to the chloroplast should have been pivotal. However, it is still mysterious how these mechanisms were established and how they work in plant cells. Here, we show that AKR2A, the cytosolic targeting factor for chloroplast outer membrane (COM) proteins, evolved from the ankyrin repeat domain (ARD) of the host cell by stepwise extensions of its N-terminal domain, and two lipids monogalactosyldiacylglycerol (MGDG) and phosphatidylglycerol (PG) of the endosymbiont were selected to function as the AKR2A receptor. Structural analysis, molecular modeling and mutational analysis of the ARD identified two adjacent sites for coincidental and synergistic binding of MGDG and PG. Based on these findings, we propose that the targeting mechanism of COM proteins was established using components from both the endosymbiont and host cell through a modification of the protein-protein interacting ARD into a lipid binding domain. PMID:25203210

  5. Crystallization of the Focal Adhesion Kinase Targeting (FAT) Domain in a Primitive Orthorhombic Space Group

    SciTech Connect

    Magis,A.; Bailey, K.; Kurenova, E.; Hernandez Prada, J.; Cance, W.; Ostrov, D.

    2008-01-01

    X-ray diffraction data from the targeting (FAT) domain of focal adhesion kinase (FAK) were collected from a single crystal that diffracted to 1.99 Angstroms resolution and reduced to the primitive orthorhombic lattice. A single molecule was predicted to be present in the asymmetric unit based on the Matthews coefficient. The data were phased using molecular-replacement methods using an existing model of the FAK FAT domain. All structures of human focal adhesion kinase FAT domains solved to date have been solved in a C-centered orthorhombic space group.

  6. Generation and isolation of target-specific single-domain antibodies from shark immune repertoires.

    PubMed

    Müller, Mischa Roland; O'Dwyer, Ronan; Kovaleva, Marina; Rudkin, Fiona; Dooley, Helen; Barelle, Caroline Jane

    2012-01-01

    The drive to exploit novel targets and biological pathways has lead to the expansion of classical antibody research into innovative fragment adaptations and novel scaffolds. The hope being that alternative or cryptic epitopes may be targeted, tissue inaccessibility may be overcome, and easier engineering options will facilitate multivalent, multi-targeting approaches. To this end, we have been isolating shark single domains to gain a greater understanding of their potential as therapeutic agents. Their unique shape, small size, inherent stability, and simple molecular architecture make them attractive candidates from a drug discovery perspective. Here we describe protocols to capture the immune repertoire of an immunized shark species and to build and select via phage-display target-specific IgNAR variable domains (VNARs).

  7. A Proteomic Approach to Identify Phosphorylation-Dependent Targets of BRCT Domains

    DTIC Science & Technology

    2007-03-01

    AD_________________ Award Number: W81XWH-05-1-0233 TITLE: A Proteomic Approach to...COVERED (From - To) 1 Mar 2006 – 28 Feb 2007 5a. CONTRACT NUMBER A Proteomic Approach to Identify Phosphorylation-Dependent Targets of BRCT...this award) BRCT domain, peptide library, OPAL, peptide array, proteomics , genome wide, signal transduction pathways, androgen receptor 16. SECURITY

  8. GRP1 pleckstrin homology domain: activation parameters and novel search mechanism for rare target lipid.

    PubMed

    Corbin, John A; Dirkx, Ronald A; Falke, Joseph J

    2004-12-28

    Pleckstrin homology (PH) domains play a central role in a wide array of signaling pathways by binding second messenger lipids of the phosphatidylinositol phosphate (PIP) lipid family. A given type of PIP lipid is formed in a specific cellular membrane where it is generally a minor component of the bulk lipid mixture. For example, the signaling lipid PI(3,4,5)P(3) (or PIP(3)) is generated primarily in the inner leaflet of the plasma membrane where it is believed to never exceed 0.02% of the bulk lipid. The present study focuses on the PH domain of the general receptor for phosphoinositides, isoform 1 (GRP1), which regulates the actin cytoskeleton in response to PIP(3) signals at the plasma membrane surface. The study systematically analyzes both the equilibrium and kinetic features of GRP1-PH domain binding to its PIP lipid target on a bilayer surface. Equilibrium binding measurements utilizing protein-to-membrane fluorescence resonance energy transfer (FRET) to detect GRP1-PH domain docking to membrane-bound PIP lipids confirm specific binding to PIP(3). A novel FRET competitive binding measurement developed to quantitate docking affinity yields a K(D) of 50 +/- 10 nM for GRP1-PH domain binding to membrane-bound PIP(3) in a physiological lipid mixture approximating the composition of the plasma membrane inner leaflet. This observed K(D) lies in a suitable range for regulation by physiological PIP(3) signals. Interestingly, the affinity of the interaction decreases at least 12-fold when the background anionic lipids phosphatidylserine (PS) and phosphatidylinositol (PI) are removed from the lipid mixture. Stopped-flow kinetic studies using protein-to-membrane FRET to monitor association and dissociation time courses reveal that this affinity decrease arises from a corresponding decrease in the on-rate for GRP1-PH domain docking with little or no change in the off-rate for domain dissociation from membrane-bound PIP(3). Overall, these findings indicate that the PH

  9. Characterization of the membrane-targeting C1 domain in Pasteurella multocida toxin.

    PubMed

    Kamitani, Shigeki; Kitadokoro, Kengo; Miyazawa, Masayuki; Toshima, Hirono; Fukui, Aya; Abe, Hiroyuki; Miyake, Masami; Horiguchi, Yasuhiko

    2010-08-13

    Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some forms of pasteurellosis. The toxin activates G(q)- and G(12/13)-dependent pathways through the deamidation of a glutamine residue in the alpha-subunit of heterotrimeric GTPases. We recently reported the crystal structure of the C terminus (residues 575-1285) of PMT (C-PMT), which is composed of three domains (C1, C2, and C3), and that the C1 domain is involved in the localization of C-PMT to the plasma membrane, and the C3 domain possesses a cysteine protease-like catalytic triad. In this study, we analyzed the membrane-targeting function of the C1 domain in detail. The C1 domain consists of seven helices of which the first four (residues 590-670), showing structural similarity to the N terminus of Clostridium difficile toxin B, were found to be involved in the recruitment of C-PMT to the plasma membrane. C-PMT lacking these helices (C-PMT DeltaC1(4H)) neither localized to the plasma membrane nor stimulated the G(q/12/13)-dependent signaling pathways. When the membrane-targeting property was complemented by a peptide tag with an N-myristoylation motif, C-PMT DeltaC1(4H) recovered the PMT activity. Direct binding between the C1 domain and liposomes containing phospholipids was evidenced by surface plasmon resonance analyses. These results indicate that the C1 domain of C-PMT functions as a targeting signal for the plasma membrane.

  10. A conserved polybasic domain mediates plasma membrane targeting of Lgl and its regulation by hypoxia

    PubMed Central

    Dong, Wei; Zhang, Xuejing; Liu, Weijie; Chen, Yi-jiun; Huang, Juan; Austin, Erin; Celotto, Alicia M.; Jiang, Wendy Z.; Palladino, Michael J.; Jiang, Yu; Hammond, Gerald R.V.

    2015-01-01

    Lethal giant larvae (Lgl) plays essential and conserved functions in regulating both cell polarity and tumorigenesis in Drosophila melanogaster and vertebrates. It is well recognized that plasma membrane (PM) or cell cortex localization is crucial for Lgl function in vivo, but its membrane-targeting mechanisms remain poorly understood. Here, we discovered that hypoxia acutely and reversibly inhibits Lgl PM targeting through a posttranslational mechanism that is independent of the well-characterized atypical protein kinase C (aPKC) or Aurora kinase–mediated phosphorylations. Instead, we identified an evolutionarily conserved polybasic (PB) domain that targets Lgl to the PM via electrostatic binding to membrane phosphatidylinositol phosphates. Such PB domain–mediated PM targeting is inhibited by hypoxia, which reduces inositol phospholipid levels on the PM through adenosine triphosphate depletion. Moreover, Lgl PB domain contains all the identified phosphorylation sites of aPKC and Aurora kinases, providing a molecular mechanism by which phosphorylations neutralize the positive charges on the PB domain to inhibit Lgl PM targeting. PMID:26483556

  11. Neutralization of Clostridium difficile Toxin A with Single-domain Antibodies Targeting the Cell Receptor Binding Domain*

    PubMed Central

    Hussack, Greg; Arbabi-Ghahroudi, Mehdi; van Faassen, Henk; Songer, J. Glenn; Ng, Kenneth K.-S.; MacKenzie, Roger; Tanha, Jamshid

    2011-01-01

    Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two high molecular weight exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease and are targets for C. difficile-associated disease therapy. Here, recombinant single-domain antibody fragments (VHHs), which specifically target the cell receptor binding domains of TcdA or TcdB, were isolated from an immune llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8), all shown to recognize conformational epitopes, were potent neutralizers of the cytopathic effects of toxin A on fibroblast cells in an in vitro assay. The neutralizing potency was further enhanced when VHHs were administered in paired or triplet combinations at the same overall VHH concentration, suggesting recognition of nonoverlapping TcdA epitopes. Biacore epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Further binding assays revealed TcdA-specific VHHs neutralized toxin A by binding to sites other than the carbohydrate binding pocket of the toxin. With favorable characteristics such as high production yield, potent toxin neutralization, and intrinsic stability, these VHHs are attractive systemic therapeutics but are more so as oral therapeutics in the destabilizing environment of the gastrointestinal tract. PMID:21216961

  12. Targeted ablation of the WW domain-containing oxidoreductase tumor suppressor leads to impaired steroidogenesis.

    PubMed

    Aqeilan, Rami I; Hagan, John P; de Bruin, Alain; Rawahneh, Maysoon; Salah, Zaidoun; Gaudio, Eugenio; Siddiqui, Hasan; Volinia, Stefano; Alder, Hansjuerg; Lian, Jane B; Stein, Gary S; Croce, Carlo M

    2009-03-01

    The WW domain-containing oxidoreductase (WWOX) gene encodes a 46-kDa tumor suppressor. The Wwox protein contains two N-terminal WW domains that interact with several transcriptional activators containing proline-tyrosine motifs and a central short-chain dehydrogenase/reductase domain that has been suggested to play a role in steroid metabolism. Recently, we have shown that targeted deletion of the Wwox gene in mice leads to postnatal lethality and defects in bone growth. Here, we report that Wwox-deficient mice display impaired steroidogenesis. Mutant homozygous mice are born with gonadal abnormalities, including failure of Leydig cell development in testis and reduced theca cell proliferation in ovary. Furthermore, Wwox(-/-) mice displayed impaired gene expression of key steroidogenesis enzymes. Affymetrix microarray gene analysis revealed differentially expressed related genes in steroidogenesis in knockout mice testis and ovary as compared with control mice. These results demonstrate the essential requirement for the Wwox tumor suppressor in proper steroidogenesis.

  13. Analysis of the Protein Domain and Domain Architecture Content in Fungi and Its Application in the Search of New Antifungal Targets

    PubMed Central

    Barrera, Alejandro; Alastruey-Izquierdo, Ana; Martín, María J.; Cuesta, Isabel; Vizcaíno, Juan Antonio

    2014-01-01

    Over the past several years fungal infections have shown an increasing incidence in the susceptible population, and caused high mortality rates. In parallel, multi-resistant fungi are emerging in human infections. Therefore, the identification of new potential antifungal targets is a priority. The first task of this study was to analyse the protein domain and domain architecture content of the 137 fungal proteomes (corresponding to 111 species) available in UniProtKB (UniProt KnowledgeBase) by January 2013. The resulting list of core and exclusive domain and domain architectures is provided in this paper. It delineates the different levels of fungal taxonomic classification: phylum, subphylum, order, genus and species. The analysis highlighted Aspergillus as the most diverse genus in terms of exclusive domain content. In addition, we also investigated which domains could be considered promiscuous in the different organisms. As an application of this analysis, we explored three different ways to detect potential targets for antifungal drugs. First, we compared the domain and domain architecture content of the human and fungal proteomes, and identified those domains and domain architectures only present in fungi. Secondly, we looked for information regarding fungal pathways in public repositories, where proteins containing promiscuous domains could be involved. Three pathways were identified as a result: lovastatin biosynthesis, xylan degradation and biosynthesis of siroheme. Finally, we classified a subset of the studied fungi in five groups depending on their occurrence in clinical samples. We then looked for exclusive domains in the groups that were more relevant clinically and determined which of them had the potential to bind small molecules. Overall, this study provides a comprehensive analysis of the available fungal proteomes and shows three approaches that can be used as a first step in the detection of new antifungal targets. PMID:25033262

  14. Identification of Novel Molecular Targets for Pleckstrin Homology (PH) Domains Found in Oncogenes Implicated in Breast Cancer

    DTIC Science & Technology

    2005-03-01

    cannot account for the specific subcellular localization of several PH domains - particularly in the case of the Golgi-targeted OSBP PH domain, it...vivo) and subcellular localization , and we been shown to bind phosphoinositides only weakly and modeled the electrostatic properties for all 33 PH do...Oshl p and inositide binding properties with subcellular localization Osh2p PH domains also drove robust Ras recruitment, of isolated yeast PH domains

  15. Epigenetic and genetic deregulation in cancer target distinct signaling pathway domains

    PubMed Central

    Gao, Yang; Teschendorff, Andrew E.

    2017-01-01

    Cancer is characterized by both genetic and epigenetic alterations. While cancer driver mutations and copy-number alterations have been studied at a systems-level, relatively little is known about the systems-level patterns exhibited by their epigenetic counterparts. Here we perform a pan-cancer wide systems-level analysis, mapping candidate cancer-driver DNA methylation (DNAm) alterations onto a human interactome. We demonstrate that functional DNAm alterations in cancer tend to map to nodes of lower connectivity and inter-connectivity, compared to the corresponding alterations at the genomic level. We find that epigenetic alterations are relatively over-represented in extracellular and transmembrane signaling domains, whereas cancer genes undergoing amplification or deletion tend to be enriched within the intracellular domain. A pan-cancer wide meta-analysis identifies WNT and chemokine signaling, as two key pathways where epigenetic deregulation preferentially targets extracellular components. We further pinpoint specific chemokine ligands/receptors whose epigenetic deregulation associates with key epigenetic enzymes, representing potential targets for epigenetic therapy. Our results suggest that epigenetic deregulation in cancer not only targets tissue-specific transcription factors, but also modulates signaling within the extra-cellular domain, providing novel system-level insight into the potential distinctive role of genetic and epigenetic alterations in cancer. PMID:27899617

  16. Molecular Pathways: The Necrosome-A Target for Cancer Therapy.

    PubMed

    Seifert, Lena; Miller, George

    2017-03-01

    Necroptosis is a caspase-8-independent cell death that requires coactivation of receptor-interacting protein 1 (RIP1) and receptor-interacting protein 3 (RIP3) kinases. The necrosome is a complex consisting of RIP1, RIP3, and Fas-associated protein with death domain leading to activation of the pseudokinase mixed lineage kinase like followed by a rapid plasma membrane rupture and inflammatory response through the release of damage-associated molecular patterns and cytokines. The necrosome has been shown to be relevant in multiple tumor types, including pancreatic adenocarcinoma, melanoma, and several hematologic malignancies. Preclinical data suggest that targeting this complex can have differential impact on tumor progression and that the effect of necroptosis on oncogenesis is cell-type and context dependent. The emerging data suggest that targeting the necrosome may lead to immunogenic reprogramming in the tumor microenvironment in multiple tumors and that combining therapies targeting the necrosome with either conventional chemotherapy or immunotherapy may have beneficial effects. Thus, understanding the interplay of necroptotic cell death, transformed cells, and the immune system may enable the development of novel therapeutic approaches. Clin Cancer Res; 23(5); 1132-6. ©2016 AACR.

  17. Evaluation of Waveform Structure Features on Time Domain Target Recognition under Cross Polarization

    NASA Astrophysics Data System (ADS)

    Selver, M. A.; Seçmen, M.; Zoral, E. Y.

    2016-08-01

    Classification of aircraft targets from scattered electromagnetic waves is a challenging application, which suffers from aspect angle dependency. In order to eliminate the adverse effects of aspect angle, various strategies were developed including the techniques that rely on extraction of several features and design of suitable classification systems to process them. Recently, a hierarchical method, which uses features that take advantage of waveform structure of the scattered signals, is introduced and shown to have effective results. However, this approach has been applied to the special cases that consider only a single planar component of electric field that cause no-cross polarization at the observation point. In this study, two small scale aircraft models, Boeing-747 and DC-10, are selected as the targets and various polarizations are used to analyse the cross-polarization effects on system performance of the aforementioned method. The results reveal the advantages and the shortcomings of using waveform structures in time-domain target identification.

  18. The TCF C-clamp DNA binding domain expands the Wnt transcriptome via alternative target recognition

    PubMed Central

    Hoverter, Nate P.; Zeller, Michael D.; McQuade, Miriam M.; Garibaldi, Angela; Busch, Anke; Selwan, Elizabeth M.; Hertel, Klemens J.; Baldi, Pierre; Waterman, Marian L.

    2014-01-01

    LEF/TCFs direct the final step in Wnt/β-catenin signalling by recruiting β-catenin to genes for activation of transcription. Ancient, non-vertebrate TCFs contain two DNA binding domains, a High Mobility Group box for recognition of the Wnt Response Element (WRE; 5′-CTTTGWWS-3′) and the C-clamp domain for recognition of the GC-rich Helper motif (5′-RCCGCC-3′). Two vertebrate TCFs (TCF-1/TCF7 and TCF-4/TCF7L2) use the C-clamp as an alternatively spliced domain to regulate cell-cycle progression, but how the C-clamp influences TCF binding and activity genome-wide is not known. Here, we used a doxycycline inducible system with ChIP-seq to assess how the C-clamp influences human TCF1 binding genome-wide. Metabolic pulse-labeling of nascent RNA with 4′Thiouridine was used with RNA-seq to connect binding to the Wnt transcriptome. We find that the C-clamp enables targeting to a greater number of gene loci for stronger occupancy and transcription regulation. The C-clamp uses Helper sites concurrently with WREs for gene targeting, but it also targets TCF1 to sites that do not have readily identifiable canonical WREs. The coupled ChIP-seq/4′Thiouridine-seq analysis identified new Wnt target genes, including additional regulators of cell proliferation. Thus, C-clamp containing isoforms of TCFs are potent transcriptional regulators with an expanded transcriptome directed by C-clamp-Helper site interactions. PMID:25414359

  19. Crystal Structures of Free and Ligand-Bound Focal Adhesion Targeting Domain of Pyk2

    SciTech Connect

    Lulo, J.; Yuzawa, S; Schlessinger, J

    2009-01-01

    Focal adhesion targeting (FAT) domains target the non-receptor tyrosine kinases FAK and Pyk2 to cellular focal adhesion areas, where the signaling molecule paxillin is also located. Here, we report the crystal structures of the Pyk2 FAT domain alone or in complex with paxillin LD4 peptides. The overall structure of Pyk2-FAT is an antiparallel four-helix bundle with an up-down, up-down, right-handed topology. In the LD4-bound FAT complex, two paxillin LD4 peptides interact with two opposite sides of Pyk2-FAT, at the surfaces of the a1a4 and a2a3 helices of each FAT molecule. We also demonstrate that, while paxillin is phosphorylated by Pyk2, complex formation between Pyk2 and paxillin does not depend on Pyk2 tyrosine kinase activity. These experiments reveal the structural basis underlying the selectivity of paxillin LD4 binding to the Pyk2 FAT domain and provide insights about the molecular details which influence the different behavior of these two closely-related kinases.

  20. Membrane domain formation—a key factor for targeted intracellular drug delivery

    PubMed Central

    Popov-Čeleketić, Dušan; van Bergen en Henegouwen, Paul M. P.

    2014-01-01

    Protein molecules, toxins and viruses internalize into the cell via receptor-mediated endocytosis (RME) using specific proteins and lipids in the plasma membrane. The plasma membrane is a barrier for many pharmaceutical agents to enter into the cytoplasm of target cells. In the case of cancer cells, tissue-specific biomarkers in the plasma membrane, like cancer-specific growth factor receptors, could be excellent candidates for RME-dependent drug delivery. Recent data suggest that agent binding to these receptors at the cell surface, resulting in membrane domain formation by receptor clustering, can be used for the initiation of RME. As a result, these pharmaceutical agents are internalized into the cells and follow different routes until they reach their final intracellular targets like lysosomes or Golgi. We propose that clustering induced formation of plasma membrane microdomains enriched in receptors, sphingolipids, and inositol lipids, leads to membrane bending which functions as the onset of RME. In this review we will focus on the role of domain formation in RME and discuss potential applications for targeted intracellular drug delivery. PMID:25520666

  1. Novel Kinase Inhibitors Targeting the PH Domain of AKT for Preventing and Treating Cancer | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute's Medical Oncology Branch is seeking statements of capability or interest from parties interested in licensing and co-development collaborative research to further develop, evaluate, or commercialize novel kinase inhibitors targeting the PH domain of AKT.

  2. Proposal of Dual Inhibitor Targeting ATPase Domains of Topoisomerase II and Heat Shock Protein 90

    PubMed Central

    Jun, Kyu-Yeon; Kwon, Youngjoo

    2016-01-01

    There is a conserved ATPase domain in topoisomerase II (topo II) and heat shock protein 90 (Hsp90) which belong to the GHKL (gyrase, Hsp90, histidine kinase, and MutL) family. The inhibitors that target each of topo II and Hsp90 are intensively studied as anti-cancer drugs since they play very important roles in cell proliferation and survival. Therefore the development of dual targeting anti-cancer drugs for topo II and Hsp90 is suggested to be a promising area. The topo II and Hsp90 inhibitors, known to bind to their ATP binding site, were searched. All the inhibitors investigated were docked to both topo II and Hsp90. Four candidate compounds as possible dual inhibitors were selected by analyzing the molecular docking study. The pharmacophore model of dual inhibitors for topo II and Hsp90 were generated and the design of novel dual inhibitor was proposed. PMID:27582553

  3. GRP1 PH Domain, Like AKT1 PH Domain, Possesses a Sentry Glutamate Residue Essential for Specific Targeting to Plasma Membrane PI(3,4,5)P3

    PubMed Central

    Pilling, Carissa; Landgraf, Kyle E.; Falke, Joseph J.

    2011-01-01

    During the appearance of the signaling lipid PI(3,4,5)P3, an important subset of pleckstrin homology (PH) domains target signaling proteins to the plasma membrane. To ensure proper pathway regulation, such PI(3,4,5)P3-specific PH domains must exclude the more prevalant, constitutive plasma membrane lipid PI(4,5)P2 and bind the rare PI(3,4,5)P3 target lipid with sufficiently high affinity. Our previous study of the E17K mutant of protein kinase B (AKT1) PH domain, together with evidence from Carpten et al (1), revealed that the native AKT1 E17 residue serves as a sentry glutamate that excludes PI(4,5)P2, thereby playing an essential role in specific PI(3,4,5)P3 targeting (2). The sentry glutamate hypothesis proposes that an analogous sentry glutamate residue is a widespread feature of PI(3,4,5)P3-specific PH domains, and that charge reversal mutation at the sentry glutamate position will yield both increased PI(4,5)P2 affinity and constitutive plasma membrane targeting. To test this hypothesis the present study investigates the E345 residue, a putative sentry glutamate, of General Receptor for Phosphoinositides 1 (GRP1) PH domain. The results show that incorporation of the E345K charge reversal mutation into GRP1 PH domain enhances PI(4,5)P2 affinity 8-fold and yields constitutive plasma membrane targeting in cells, reminiscent of the effects of the E17K mutation in AKT1 PH domain. Hydrolysis of plasma membrane PI(4,5)P2 releases E345K GRP1 PH domain into the cytoplasm and the efficiency of this release increases when target Arf6 binding is disrupted. Overall, the findings provide strong support for the sentry glutamate hypothesis and suggest that the GRP1 E345K mutation will be linked to changes in cell physiology and human pathologies, as demonstrated for AKT1 E17K (1, 3). Analysis of available PH domain structures suggests that a lone glutamate residue (or, in some cases an aspartate) is a common, perhaps ubiquitous, feature of PI(3,4,5)P3-specific binding

  4. DNA damage targets PKC{eta} to the nuclear membrane via its C1b domain

    SciTech Connect

    Tamarkin, Ana; Zurgil, Udi; Braiman, Alex; Hai, Naama; Krasnitsky, Ella; Maissel, Adva; Ben-Ari, Assaf; Yankelovich, Liat; Livneh, Etta

    2011-06-10

    Translocation to cellular membranes is one of the hallmarks of PKC activation, occurring as a result of the generation of lipid secondary messengers in target membrane compartments. The activation-induced translocation of PKCs and binding to membranes is largely directed by their regulatory domains. We have previously reported that PKC{eta}, a member of the novel subfamily and an epithelial specific isoform, is localized at the cytoplasm and ER/Golgi and is translocated to the plasma membrane and the nuclear envelope upon short-term activation by PMA. Here we show that PKC{eta} is shuttling between the cytoplasm and the nucleus and that upon etoposide induced DNA damage is tethered at the nuclear envelope. Although PKC{eta} expression and its phosphorylation on the hydrophobic motif (Ser675) are increased by etoposide, this phosphorylation is not required for its accumulation at the nuclear envelope. Moreover, we demonstrate that the C1b domain is sufficient for translocation to the nuclear envelope. We further show that, similar to full-length PKC{eta}, the C1b domain could also confer protection against etoposide-induced cell death. Our studies demonstrate translocation of PKC{eta} to the nuclear envelope, and suggest that its spatial regulation could be important for its cellular functions including effects on cell death.

  5. Mammalian Pseudokinase TRIB3 in Normal Physiology and Disease: Charting the Progress in Old and New Avenues.

    PubMed

    Örd, Tiit; Örd, Tõnis

    2017-04-06

    Tribbles homolog 3 (TRIB3) is a mammalian gene that is upregulated in response to several types of cell death-inducing cellular stress. The TRIB3 protein is a pseudokinase, a protein kinase-like scaffold with impaired catalytic activity. However, research has revealed it to be prolific at forming protein-protein interactions. By binding to and regulating the activity of several key proteins, including the protein kinase Akt and transcription factors ATF4, CHOP and NF-κB, TRIB3 is at a junction of several signaling pathways. This review begins by providing insights into the characteristic protein structure and gene expression regulation mechanisms of TRIB3. Further, the diverse reported molecular roles of TRIB3 as a regulator of cell death, stress responses, inflammation, cell differentiation, protein degradation and other processes are discussed. Special attention is devoted to the involvement of TRIB3 in the pathogenesis of cancer and type 2 diabetes, two fields where TRIB3 has generated particular interest, as well as considerable debate, from a biomedical standpoint. Throughout, emphasis is placed on results obtained from animal models with altered TRIB3 expression (Trib3 knockout or overexpression mice), in order to provide insight into the contributions of TRIB3 to physiology and disease at the organism level.

  6. CdiA Effectors Use Modular Receptor-Binding Domains To Recognize Target Bacteria

    PubMed Central

    Ruhe, Zachary C.; Nguyen, Josephine Y.; Xiong, Jing; Koskiniemi, Sanna; Beck, Christina M.; Perkins, Basil R.; Low, David A.

    2017-01-01

    ABSTRACT Contact-dependent growth inhibition (CDI) systems encode CdiA effectors, which bind to specific receptors on neighboring bacteria and deliver C-terminal toxin domains to suppress target cell growth. Two classes of CdiA effectors that bind distinct cell surface receptors have been identified, but the molecular basis of receptor specificity is not understood. Alignment of BamA-specific CdiAEC93 from Escherichia coli EC93 and OmpC-specific CdiAEC536 from E. coli 536 suggests that the receptor-binding domain resides within a central region that varies between the two effectors. In support of this hypothesis, we find that CdiAEC93 fragments containing residues Arg1358 to Phe1646 bind specifically to purified BamA. Moreover, chimeric CdiAEC93 that carries the corresponding sequence from CdiAEC536 is endowed with OmpC-binding activity, demonstrating that this region dictates receptor specificity. A survey of E. coli CdiA proteins reveals two additional effector classes, which presumably recognize distinct receptors. Using a genetic approach, we identify the outer membrane nucleoside transporter Tsx as the receptor for a third class of CdiA effectors. Thus, CDI systems exploit multiple outer membrane proteins to identify and engage target cells. These results underscore the modularity of CdiA proteins and suggest that novel effectors can be constructed through genetic recombination to interchange different receptor-binding domains and toxic payloads. PMID:28351921

  7. The axon-dendrite targeting of Kv3 (Shaw) channels is determined by a targeting motif that associates with the T1 domain and ankyrin G.

    PubMed

    Xu, Mingxuan; Cao, Ruifeng; Xiao, Rui; Zhu, Michael X; Gu, Chen

    2007-12-19

    Kv3 (Shaw) channels regulate rapid spiking, transmitter release and dendritic integration of many central neurons. Crucial to functional diversity are the complex targeting patterns of channel proteins. However, the targeting mechanisms are not known. Here we report that the axon-dendrite targeting of Kv3.1 is controlled by a conditional interaction of a C-terminal axonal targeting motif (ATM) with the N-terminal T1 domain and adaptor protein ankyrin G. In cultured hippocampal neurons, although the two splice variants of Kv3.1, Kv3.1a and Kv3.1b, are differentially targeted to the somatodendritic and axonal membrane, respectively, the lysine-rich ATM is surprisingly common for both splice variants. The ATM not only directly binds to the T1 domain in a Zn2+-dependent manner, but also associates with the ankyrin-repeat domain of ankyrin G. However, the full-length channel proteins of Kv3.1b display stronger association to ankyrin G than those of Kv3.1a, suggesting that the unique splice domain at Kv3.1b C terminus influences ATM binding to T1 and ankyrin G. Because ankyrin G mainly resides at the axon initial segment, we propose that it may function as a barrier for axon-dendrite targeting of Kv3.1 channels. In support of this idea, disrupting ankyrin G function either by over-expressing a dominant-negative mutant or by siRNA knockdown decreases polarized axon-dendrite targeting of both Kv3.1a and Kv3.1b. We conclude that the conditional ATM masked by the T1 domain in Kv3.1a is exposed by the splice domain in Kv3.1b, and is subsequently recognized by ankyrin G to target Kv3.1b into the axon.

  8. DNA-binding domain fusions enhance the targeting range and precision of Cas9

    PubMed Central

    Bolukbasi, Mehmet Fatih; Gupta, Ankit; Oikemus, Sarah; Derr, Alan G.; Garber, Manuel; Brodsky, Michael H.; Zhu, Lihua Julie; Wolfe, Scot A.

    2015-01-01

    The CRISPR-Cas9 system is commonly employed in biomedical research; however, the precision of Cas9 is sub-optimal for gene therapy applications that involve editing a large population of cells. Variations on the standard Cas9 system have yielded improvements in the precision of targeted DNA cleavage, but often restrict the range of targetable sequences. It remains unclear whether these variants can limit lesions to a single site within the human genome over a large cohort of treated cells. Here, we demonstrate that fusing a programmable DNA-binding domain (pDBD) to Cas9 combined with the attenuation of Cas9’s inherent DNA binding affinity produces a Cas9-pDBD chimera with dramatically improved precision and increased targeting range. Because the specificity and affinity of this framework is easily tuned, Cas9-pDBDs provide a flexible system that can be tailored to achieve extremely precise genome editing at nearly any genomic locus – characteristics that are ideal for gene therapy applications. PMID:26480473

  9. Effects of gender-related domain violations and sexual orientation on perceptions of male and female targets: an analogue study.

    PubMed

    Blashill, Aaron J; Powlishta, Kimberly K

    2012-10-01

    The current study examined factors that influenced heterosexual male and female raters' evaluations of male and female targets who were gay or heterosexual, and who displayed varying gender roles (i.e., typical vs. atypical) in multiple domains (i.e., activities, traits, and appearance). Participants were 305 undergraduate students from a private, midwestern Jesuit institution who read vignettes describing one of 24 target types and then rated the target on possession of positive and negative characteristics, psychological adjustment, and on measures reflecting the participants' anticipated behavior toward or comfort with the target. Results showed that gender atypical appearance and activity attributes (but not traits) were viewed more negatively than their gender typical counterparts. It was also found that male participants in particular viewed gay male targets as less desirable than lesbian and heterosexual male targets. These findings suggest a nuanced approach for understanding sexual prejudice, which incorporates a complex relationship among sex, gender, sexual orientation, and domain of gendered attributes.

  10. Single-Domain Antibodies Targeting Neuraminidase Protect against an H5N1 Influenza Virus Challenge

    PubMed Central

    Cardoso, Francisco Miguel; Ibañez, Lorena Itatí; Van den Hoecke, Silvie; De Baets, Sarah; Smet, Anouk; Roose, Kenny; Schepens, Bert; Descamps, Francis J.; Fiers, Walter; Muyldermans, Serge

    2014-01-01

    ABSTRACT Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We isolated a set of H5N1 NA-specific single-domain antibodies (N1-VHHm) and evaluated their in vitro and in vivo antiviral potential. Two of them inhibited the NA activity and in vitro replication of clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two methods. First, we made N1-VHHb by genetically joining two N1-VHHm moieties with a flexible linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm moiety with the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1 of both bivalent N1-VHHb formats was 30- to 240-fold higher than that of their monovalent counterparts, with 50% inhibitory concentrations in the low nanomolar range. Moreover, single-dose prophylactic treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal challenge with H5N1 virus, including an oseltamivir-resistant H5N1 variant. Surprisingly, an N1-VHH-Fc fusion without in vitro NA-inhibitory or antiviral activity also protected mice against an H5N1 challenge. Virus escape selection experiments indicated that one amino acid residue close to the catalytic site is required for N1-VHHm binding. We conclude that single-domain antibodies directed against influenza virus NA protect against H5N1 virus infection, and when engineered with a conventional Fc domain, they can do so in the absence of detectable NA-inhibitory activity. IMPORTANCE Highly pathogenic H5N1 viruses are a zoonotic threat. Outbreaks of avian influenza caused by these viruses occur in many parts of the world and are associated with tremendous economic loss, and these viruses can cause very severe disease in humans. In such cases, small-molecule inhibitors of the viral NA are among the few treatment options for patients. However, treatment with such drugs often results in the emergence of resistant viruses

  11. Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1.

    PubMed

    Nuttall, Stewart D; Humberstone, Karen S; Krishnan, Usha V; Carmichael, Jennifer A; Doughty, Larissa; Hattarki, Meghan; Coley, Andrew M; Casey, Joanne L; Anders, Robin F; Foley, Michael; Irving, Robert A; Hudson, Peter J

    2004-04-01

    The new antigen receptor (IgNAR) is an antibody unique to sharks and consists of a disulphide-bonded dimer of two protein chains, each containing a single variable and five constant domains. The individual variable (V(NAR)) domains bind antigen independently, and are candidates for the smallest antibody-based immune recognition units. We have previously produced a library of V(NAR) domains with extensive variability in the CDR1 and CDR3 loops displayed on the surface of bacteriophage. Now, to test the efficacy of this library, and further explore the dynamics of V(NAR) antigen binding we have performed selection experiments against an infectious disease target, the malarial Apical Membrane Antigen-1 (AMA1) from Plasmodium falciparum. Two related V(NAR) clones were selected, characterized by long (16- and 18-residue) CDR3 loops. These recombinant V(NAR)s could be harvested at yields approaching 5mg/L of monomeric protein from the E. coli periplasm, and bound AMA1 with nanomolar affinities (K(D)= approximately 2 x 10(-7) M). One clone, designated 12Y-2, was affinity-matured by error prone PCR, resulting in several variants with mutations mapping to the CDR1 and CDR3 loops. The best of these variants showed approximately 10-fold enhanced affinity over 12Y-2 and was Plasmodium falciparum strain-specific. Importantly, we demonstrated that this monovalent V(NAR) co-localized with rabbit anti-AMA1 antisera on the surface of malarial parasites and thus may have utility in diagnostic applications.

  12. 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

  13. Proposed docking interface between peptidoglycan and the target recognition domain of zoocin A

    SciTech Connect

    Chen, Yinghua; Simmonds, Robin S.; Timkovich, Russell

    2013-11-15

    Highlights: •Peptidoglycan added to zoocin rTRD perturbs NMR resonances around W115. •Simulations predict docking to a shallow surface groove near W115. •The docking interface is similar to mammalian antibody–antigen sites. •EDTA binds to a distinct surface site. -- Abstract: A docking model is proposed for the target recognition domain of the lytic exoenzyme zoocin A with the peptidoglycan on the outer cell surface of sensitive bacterial strains. Solubilized fragments from such peptidoglycans perturb specific backbone and side chain amide resonances in the recombinant form of the domain designated rTRD as detected in two-dimensional {sup 1}H–{sup 15}N correlation NMR spectra. The affected residues comprise a shallow surface cleft on the protein surface near W115, N53, N117, and Q105 among others, which interacts with the peptide portion of the peptidoglycan. Calculations with AutoDock Vina provide models of the docking interface. There is approximate homology between the rTDR-peptidoglycan docking site and the antigen binding site of Fab antibodies with the immunoglobin fold. EDTA was also found to bind to rTRD, but at a site distinct from the proposed peptidoglycan docking site.

  14. Receptor-binding domain as a target for developing SARS vaccines.

    PubMed

    Zhu, Xiaojie; Liu, Qi; Du, Lanying; Lu, Lu; Jiang, Shibo

    2013-08-01

    A decade ago, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a global pandemic with a mortality rate of 10%. Reports of recent outbreaks of a SARS-like disease caused by Middle East respiratory syndrome coronavirus (MERS-CoV) have raised serious concerns of a possible reemergence of SARS-CoV, either by laboratory escape or the presence of a natural reservoir. Therefore, the development of effective and safe SARS vaccines is still needed. Based on our previous studies, we believe that the receptor-binding domain (RBD) in the S1 subunit of the SARS-CoV spike (S) protein is the most important target for developing a SARS vaccine. In particular, RBD of S protein contains the critical neutralizing domain (CND), which is able to induce highly potent neutralizing antibody response and cross-protection against divergent SARS-CoV strains. Furthermore, a RBD-based subunit vaccine is expected to be safer than other vaccines that may induce Th2-type immunopathology. This review will discuss key advances in the development of RBD-based SARS vaccines and the possibility of using a similar strategy to develop vaccines against MERS-CoV.

  15. Modulators of Hepatic Lipoprotein Metabolism Identified in a Search for Small-Molecule Inducers of Tribbles Pseudokinase 1 Expression

    PubMed Central

    Nagiec, Marek M.; Skepner, Adam P.; Negri, Joseph; Eichhorn, Michelle; Kuperwasser, Nicolas; Comer, Eamon; Muncipinto, Giovanni; Subramanian, Aravind; Clish, Clary; Musunuru, Kiran; Duvall, Jeremy R.; Foley, Michael; Perez, Jose R.; Palmer, Michelle A. J.

    2015-01-01

    Recent genome wide association studies have linked tribbles pseudokinase 1 (TRIB1) to the risk of coronary artery disease (CAD). Based on the observations that increased expression of TRIB1 reduces secretion of VLDL and is associated with lower plasma levels of LDL cholesterol and triglycerides, higher plasma levels of HDL cholesterol and reduced risk for myocardial infarction, we carried out a high throughput phenotypic screen based on quantitative RT-PCR assay to identify compounds that induce TRIB1 expression in human HepG2 hepatoma cells. In a screen of a collection of diversity-oriented synthesis (DOS)-derived compounds, we identified a series of benzofuran-based compounds that upregulate TRIB1 expression and phenocopy the effects of TRIB1 cDNA overexpression, as they inhibit triglyceride synthesis and apoB secretion in cells. In addition, the compounds downregulate expression of MTTP and APOC3, key components of the lipoprotein assembly pathway. However, CRISPR-Cas9 induced chromosomal disruption of the TRIB1 locus in HepG2 cells, while confirming its regulatory role in lipoprotein metabolism, demonstrated that the effects of benzofurans persist in TRIB1-null cells indicating that TRIB1 is sufficient but not necessary to transmit the effects of the drug. Remarkably, active benzofurans, as well as natural products capable of TRIB1 upregulation, also modulate hepatic cell cholesterol metabolism by elevating the expression of LDLR transcript and LDL receptor protein, while reducing the levels of PCSK9 transcript and secreted PCSK9 protein and stimulating LDL uptake. The effects of benzofurans are not masked by cholesterol depletion and are independent of the SREBP-2 regulatory circuit, indicating that these compounds represent a novel class of chemically tractable small-molecule modulators that shift cellular lipoprotein metabolism in HepG2 cells from lipogenesis to scavenging. PMID:25811180

  16. Molecular functions of the TLE tetramerization domain in Wnt target gene repression

    PubMed Central

    Chodaparambil, Jayanth V; Pate, Kira T; Hepler, Margretta R D; Tsai, Becky P; Muthurajan, Uma M; Luger, Karolin; Waterman, Marian L; Weis, William I

    2014-01-01

    Wnt signaling activates target genes by promoting association of the co-activator β-catenin with TCF/LEF transcription factors. In the absence of β-catenin, target genes are silenced by TCF-mediated recruitment of TLE/Groucho proteins, but the molecular basis for TLE/TCF-dependent repression is unclear. We describe the unusual three-dimensional structure of the N-terminal Q domain of TLE1 that mediates tetramerization and binds to TCFs. We find that differences in repression potential of TCF/LEFs correlates with their affinities for TLE-Q, rather than direct competition between β-catenin and TLE for TCFs as part of an activation–repression switch. Structure-based mutation of the TLE tetramer interface shows that dimers cannot mediate repression, even though they bind to TCFs with the same affinity as tetramers. Furthermore, the TLE Q tetramer, not the dimer, binds to chromatin, specifically to K20 methylated histone H4 tails, suggesting that the TCF/TLE tetramer complex promotes structural transitions of chromatin to mediate repression. PMID:24596249

  17. Design and implementation of random noise radar with spectral-domain correlation for moving target detection

    NASA Astrophysics Data System (ADS)

    Kim, Jeong Phill; Jeong, Chi Hyun; Kim, Cheol Hoo

    2011-06-01

    A correlation processing algorithm in the spectral domain is proposed for detecting moving targets with random noise radar. AD converted reference and Rx signals are passed through FFT block, and they are multiplied after the reference signal is complex conjugated. Now inverse FFT yields the sub-correlation results, and range and velocity information can be accurately extracted by an additional FFT processing. In this design procedure, specific considerations have to be made for correlation length, averaging number, and number of sub-correlation data for Doppler processing. The proposed algorithm was verified by Simulink (Mathworks) simulation, and its logic was implemented with Xilinx FPGA device (Vertex5 series) by System Generator block sets (Xilinx) in the Simulink environment. A CW X-band random-FM noise radar prototype with an instantaneous bandwidth of 100 MHz was designed and implemented, and laboratory and field tests were conducted to detect moving targets, and the observed results showed the validity of the proposed algorithm and the operation of implemented FPGA logics.

  18. ETS-domain transcription factor Elk-1 mediates neuronal survival: SMN as a potential target.

    PubMed

    Demir, Ozlem; Aysit, Nese; Onder, Zeynep; Turkel, Nezaket; Ozturk, Gurkan; Sharrocks, Andrew D; Kurnaz, Isil Aksan

    2011-06-01

    Elk-1 belongs to the ternary complex factors (TCFs) subfamily of the ETS domain proteins, and plays a critical role in the expression of immediate-early genes (IEGs) upon mitogen stimulation and activation of the mitogen-activated protein kinase (MAPK) cascade. The association of TCFs with serum response elements (SREs) on IEG promoters has been widely studied and a role for Elk-1 in promoting cell cycle entry has been determined. However, the presence of the ETS domain transcription factor Elk-1 in axons and dendrites of post-mitotic adult brain neurons has implications for an alternative function for Elk-1 in neurons other than controlling proliferation. In this study, possible alternative roles for Elk-1 in neurons were investigated, and it was demonstrated that blocking TCF-mediated transactivation in neuronal cells leads to apoptosis through a caspase-dependent mechanism. Indeed RNAi-mediated depletion of endogenous Elk-1 results in increased caspase activity. Conversely, overexpression of either Elk-1 or Elk-VP16 fusion proteins was shown to rescue PC12 cells from chemically-induced apoptosis, and that higher levels of endogenous Elk-1 correlated with longer survival of DRGs in culture. It was shown that Elk-1 regulated the Mcl-1 gene expression required for survival, and that RNAi-mediated degradation of endogenous Elk-1 resulted in elimination of the mcl-1 message. We have further identified the survival-of-motor neuron-1 (SMN1) gene as a novel target of Elk-1, and show that the ets motifs in the SMN1 promoter are involved in this regulation.

  19. ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation

    SciTech Connect

    Shi, Fumin; Telesco, Shannon E.; Liu, Yingting; Radhakrishnan, Ravi; Lemmon, Mark A.

    2010-06-21

    ErbB3/HER3 is one of four members of the human epidermal growth factor receptor (EGFR/HER) or ErbB receptor tyrosine kinase family. ErbB3 binds neuregulins via its extracellular region and signals primarily by heterodimerizing with ErbB2/HER2/Neu. A recently appreciated role for ErbB3 in resistance of tumor cells to EGFR/ErbB2-targeted therapeutics has made it a focus of attention. However, efforts to inactivate ErbB3 therapeutically in parallel with other ErbB receptors are challenging because its intracellular kinase domain is thought to be an inactive pseudokinase that lacks several key conserved (and catalytically important) residues - including the catalytic base aspartate. We report here that, despite these sequence alterations, ErbB3 retains sufficient kinase activity to robustly trans-autophosphorylate its intracellular region - although it is substantially less active than EGFR and does not phosphorylate exogenous peptides. The ErbB3 kinase domain binds ATP with a K{sub d} of approximately 1.1 {micro}M. We describe a crystal structure of ErbB3 kinase bound to an ATP analogue, which resembles the inactive EGFR and ErbB4 kinase domains (but with a shortened {alpha}C-helix). Whereas mutations that destabilize this configuration activate EGFR and ErbB4 (and promote EGFR-dependent lung cancers), a similar mutation conversely inactivates ErbB3. Using quantum mechanics/molecular mechanics simulations, we delineate a reaction pathway for ErbB3-catalyzed phosphoryl transfer that does not require the conserved catalytic base and can be catalyzed by the 'inactive-like'configuration observed crystallographically. These findings suggest that ErbB3 kinase activity within receptor dimers may be crucial for signaling and could represent an important therapeutic target.

  20. Mutations in the linker domain affect phospho-STAT3 function and suggest targets for interrupting STAT3 activity.

    PubMed

    Mertens, Claudia; Haripal, Bhagwattie; Klinge, Sebastian; Darnell, James E

    2015-12-01

    Crystallography of the cores of phosphotyrosine-activated dimers of STAT1 (132-713) and STAT3 (127-722) bound to a similar double-stranded deoxyoligonucleotide established the domain structure of the STATs and the structural basis for activation through tyrosine phosphorylation and dimerization. We reported earlier that mutants in the linker domain of STAT1 that connect the DNA-binding domain and SH2 domain can prevent transcriptional activation. Because of the pervasive importance of persistently activated STAT3 in many human cancers and the difficulty of finding useful drug candidates aimed at disrupting the pY interchange in active STAT3 dimers, we have examined effects of an array of mutants in the STAT3 linker domain. We have found several STAT3 linker domain mutants to have profound effects of inhibiting STAT3 transcriptional activation. From these results, we propose (i) there is definite functional interaction of the linker both with the DNA binding domain and with the SH2 domain, and (ii) these putative contacts provide potential new targets for small molecule-induced pSTAT3 inhibition.

  1. Mutations in the linker domain affect phospho-STAT3 function and suggest targets for interrupting STAT3 activity

    PubMed Central

    Mertens, Claudia; Haripal, Bhagwattie; Klinge, Sebastian; Darnell, James E.

    2015-01-01

    Crystallography of the cores of phosphotyrosine-activated dimers of STAT1 (132–713) and STAT3 (127–722) bound to a similar double-stranded deoxyoligonucleotide established the domain structure of the STATs and the structural basis for activation through tyrosine phosphorylation and dimerization. We reported earlier that mutants in the linker domain of STAT1 that connect the DNA-binding domain and SH2 domain can prevent transcriptional activation. Because of the pervasive importance of persistently activated STAT3 in many human cancers and the difficulty of finding useful drug candidates aimed at disrupting the pY interchange in active STAT3 dimers, we have examined effects of an array of mutants in the STAT3 linker domain. We have found several STAT3 linker domain mutants to have profound effects of inhibiting STAT3 transcriptional activation. From these results, we propose (i) there is definite functional interaction of the linker both with the DNA binding domain and with the SH2 domain, and (ii) these putative contacts provide potential new targets for small molecule-induced pSTAT3 inhibition. PMID:26553978

  2. Structural basis of focal adhesion targeting domain-mediated signaling in cardiac hypertrophy.

    PubMed

    Mohanty, Pallavi; Bhatnagar, Sonika

    2017-02-01

    The focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK) exists in monomeric closed (c) or arm exchanged (ae) dimeric state. FAT interaction with Grb2 necessitates an intermediate open (o) state that interacts with Grb2 and activates signaling pathways leading to pathological cardiac hypertrophy. Targeted molecular dynamics (TMD) simulation was carried out in order to capture the structure of the intermediate formed by opening of Helix1 (H1) from monomeric cFAT leading to the formation of monomeric aeFAT. During TMD, H1 separated from the four helices bundle of cFAT, completely unfolded and performed a full turn before folding back to a helix inclined at an acute angle to the helical bundle in aeFAT. The entire transition can be described in six distinct intermediate structural stages. The most significant correlation of H1 motion was observed with Loop3 (L3) and is the likely reason for the complete disruption of the FAT interaction with paxillin during the transition. High-affinity analogs of the paxillin LD4 region can be a promising strategy to drive the equilibrium towards cFAT, thus antagonizing FAT-Grb2 association. During transition, the overall shift in orientation of all the four helices rejects paxillin binding and approves Grb2 association. Exposure and β-turn conformation of the YENV motif (residues 925-928) in oFAT-facilitated phosphorylation and Grb2 binding. Docking, MD simulation and conservation analysis of oFAT-Grb2 complex provided insight into the structural determinants of binding and specificity. Our work provides a structural basis for pharmacological modulation of dynamic conformational changes and interactions of FAT.

  3. DEDD, a novel death effector domain-containing protein, targeted to the nucleolus.

    PubMed Central

    Stegh, A H; Schickling, O; Ehret, A; Scaffidi, C; Peterhänsel, C; Hofmann, T G; Grummt, I; Krammer, P H; Peter, M E

    1998-01-01

    The CD95 signaling pathway comprises proteins that contain one or two death effector domains (DED), such as FADD/Mort1 or caspase-8. Here we describe a novel 37 kDa protein, DEDD, that contains an N-terminal DED. DEDD is highly conserved between human and mouse (98. 7% identity) and is ubiquitously expressed. Overexpression of DEDD in 293T cells induced weak apoptosis, mainly through its DED by which it interacts with FADD and caspase-8. Endogenous DEDD was found in the cytoplasm and translocated into the nucleus upon stimulation of CD95. Immunocytological studies revealed that overexpressed DEDD directly translocated into the nucleus, where it co-localizes in the nucleolus with UBF, a basal factor required for RNA polymerase I transcription. Consistent with its nuclear localization, DEDD contains two nuclear localization signals and the C-terminal part shares sequence homology with histones. Recombinant DEDD binds to both DNA and reconstituted mononucleosomes and inhibits transcription in a reconstituted in vitro system. The results suggest that DEDD is a final target of a chain of events by which the CD95-induced apoptotic signal is transferred into the nucleolus to shut off cellular biosynthetic activities. PMID:9774341

  4. Evidence that ethanol acts on a target in Loop 2 of the extracellular domain of alpha1 glycine receptors.

    PubMed

    Crawford, Daniel K; Trudell, James R; Bertaccini, Edward J; Li, Kaixun; Davies, Daryl L; Alkana, Ronald L

    2007-09-01

    Considerable evidence indicates that ethanol acts on specific residues in the transmembrane domains of glycine receptors (GlyRs). In this study, we tested the hypothesis that the extracellular domain is also a target for ethanol action by investigating the effect of cysteine substitutions at positions 52 (extracellular domain) and 267 (transmembrane domain) on responses to n-alcohols and propyl methanethiosulfonate (PMTS) in alpha1GlyRs expressed in Xenopus oocytes. In support of the hypothesis: (i) The A52C mutation changed ethanol sensitivity compared to WT GlyRs; (ii) PMTS produced irreversible alcohol-like potentiation in A52C GlyRs; and (iii) PMTS binding reduced the n-chain alcohol cutoff in A52C GlyRs. Further studies used PMTS binding to cysteines at positions 52 or 267 to block ethanol action at one site in order to determine its effect at other site(s). In these situations, ethanol caused negative modulation when acting at position 52 and positive modulation when acting at position 267. Collectively, these findings parallel the evidence that established the TM domain as a target for ethanol, suggest that positions 52 and 267 are part of the same alcohol pocket and indicate that the net effect of ethanol on GlyR function reflects the summation of its positive and negative modulatory effects on different targets.

  5. A sting in the tail: the N-terminal domain of the androgen receptor as a drug target

    PubMed Central

    Monaghan, Amy E; McEwan, Iain J

    2016-01-01

    The role of androgen receptor (AR) in the initiation and progression of prostate cancer (PCa) is well established. Competitive inhibition of the AR ligand-binding domain (LBD) has been the staple of antiandrogen therapies employed to combat the disease in recent years. However, their efficacy has often been limited by the emergence of resistance, mediated through point mutations, and receptor truncations. As a result, the prognosis for patients with malignant castrate resistant disease remains poor. The amino-terminal domain (NTD) of the AR has been shown to be critical for AR function. Its modular activation function (AF-1) is important for both gene regulation and participation in protein-protein interactions. However, due to the intrinsically disordered structure of the domain, its potential as a candidate for therapeutic intervention has been dismissed in the past. The recent emergence of the small molecule EPI-001 has provided evidence that AR-NTD can be targeted therapeutically, independent of the LBD. Targeting of AR-NTD has the potential to disrupt multiple intermolecular interactions between AR and its coregulatory binding partners, in addition to intramolecular cross-talk between the domains of the AR. Therapeutics targeting these protein-protein interactions or NTD directly should also have efficacy against emerging AR splice variants which may play a role in PCa progression. This review will discuss the role of intrinsic disorder in AR function and illustrate how emerging therapies might target NTD in PCa. PMID:27212126

  6. Structure-based Discovery of Novel Small Molecule Wnt Signaling Inhibitors by Targeting the Cysteine-rich Domain of Frizzled*

    PubMed Central

    Lee, Ho-Jin; Bao, Ju; Miller, Ami; Zhang, Chi; Wu, Jibo; Baday, Yiressy C.; Guibao, Cristina; Li, Lin; Wu, Dianqing; Zheng, Jie J.

    2015-01-01

    Frizzled is the earliest discovered glycosylated Wnt protein receptor and is critical for the initiation of Wnt signaling. Antagonizing Frizzled is effective in inhibiting the growth of multiple tumor types. The extracellular N terminus of Frizzled contains a conserved cysteine-rich domain that directly interacts with Wnt ligands. Structure-based virtual screening and cell-based assays were used to identify five small molecules that can inhibit canonical Wnt signaling and have low IC50 values in the micromolar range. NMR experiments confirmed that these compounds specifically bind to the Wnt binding site on the Frizzled8 cysteine-rich domain with submicromolar dissociation constants. Our study confirms the feasibility of targeting the Frizzled cysteine-rich domain as an effective way of regulating canonical Wnt signaling. These small molecules can be further optimized into more potent therapeutic agents for regulating abnormal Wnt signaling by targeting Frizzled. PMID:26504084

  7. miRNA863-3p sequentially targets negative immune regulator ARLPKs and positive regulator SERRATE upon bacterial infection

    PubMed Central

    Niu, Dongdong; Lii, Yifan E.; Chellappan, Padmanabhan; Lei, Lei; Peralta, Karl; Jiang, Chunhao; Guo, Jianhua; Coaker, Gitta; Jin, Hailing

    2016-01-01

    Plant small RNAs play important roles in gene regulation during pathogen infection. Here we show that miR863-3p is induced by the bacterial pathogen Pseudomonas syringae carrying various effectors. Early during infection, miR863-3p silences two negative regulators of plant defence, atypical receptor-like pseudokinase1 (ARLPK1) and ARLPK2, both lacking extracellular domains and kinase activity, through mRNA degradation to promote immunity. ARLPK1 associates with, and may function through another negative immune regulator ARLPK1-interacting receptor-like kinase 1 (AKIK1), an active kinase with an extracellular domain. Later during infection, miR863-3p silences SERRATE, which is essential for miRNA accumulation and positively regulates defence, through translational inhibition. This results in decreased miR863-3p levels, thus forming a negative feedback loop to attenuate immune responses after successful defence. This is an example of a miRNA that sequentially targets both negative and positive regulators of immunity through two modes of action to fine-tune the timing and amplitude of defence responses. PMID:27108563

  8. PTB domain-directed substrate targeting in a tyrosine kinase from the unicellular choanoflagellate Monosiga brevicollis.

    PubMed

    Prieto-Echagüe, Victoria; Chan, Perry M; Craddock, Barbara P; Manser, Edward; Miller, W Todd

    2011-04-26

    Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.

  9. DDR2 (discoidin domain receptor 2) suppresses osteoclastogenesis and is a potential therapeutic target in osteoporosis.

    PubMed

    Zhang, Yan; Su, Jin; Wu, Shufang; Teng, Yue; Yin, Zhanhai; Guo, Yan; Li, Jing; Li, Kun; Yao, Libo; Li, Xu

    2015-03-24

    Osteoporosis is a disease characterized by abnormal skeletal fragility due to an imbalance in bone homeostasis. Finding therapeutic targets that promote bone formation is valuable for treating osteoporosis. Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase that promotes the differentiation of bone-depositing cells, osteoblasts, and bone formation. We investigated the role of DDR2 in osteoclastogenesis, the formation of cells responsible for bone resorption. We found that, although differentiated osteoclasts had catalytically active DDR2, its abundance was decreased. Overexpression of DDR2 inhibited the expression of osteoclastic markers, osteoclast maturation, and osteoclast-mediated bone resorption in a culture model of bone matrix. Conversely, knocking down Ddr2 through RNA interference accelerated osteoclast differentiation and bone resorption in mice. Further, we identified the co-receptor Neuropilin-1 (Nrp1) as a DDR2-interacting protein. DDR2 facilitated the binding of Nrp1 and the co-receptor PlexinA1 by forming a DDR2-Nrp1-PlexinA1 complex, which blocked PlexinA1-mediated stimulation of osteoclastogenesis. DDR2 prevented PlexinA1 from interacting with the receptor TREM2 and the adaptor DAP12. Moreover, knocking down Nrp1 rescued the expression of osteoclastic markers observed in DDR2-overexpressing cells, whereas ectopic expression of Nrp1 in DDR2-silenced cells inhibited the induction of osteoclastogenesis. Nrp1 enhanced the function of DDR2 in promoting osteoblast differentiation and bone formation in mice. In an ovariectomized mouse model of osteoporosis, adenovirus-mediated delivery of DDR2 to the femur bone alleviated osteopenic phenotypes. Our results revealed that DDR2 functions as an inhibitor of osteoclastogenesis, as well as a promoter of osteoblastogenesis, suggesting that enhancing DDR2 may be therapeutic for patients with osteoporosis.

  10. Targeting domain-III hinging of dengue envelope (DENV-2) protein by MD simulations, docking and free energy calculations.

    PubMed

    Dubey, Kshatresh Dutta; Tiwari, Gargi; Ojha, Rajendra Prasad

    2017-04-01

    The entry of the dengue virus is mediated by the conformational change in the envelope protein due to change in the endosomal pH. The structural study reveals that domain-III of the dengue envelope protein (DENV) shows the largest shift in its position during the entry of the virus. Therefore, targeting the hinge region of the domain-III may block the conformational changes in the DENV. In the present work, we have targeted the domain I/III hinge region using four known ligands used for the dengue envelope protein (serotype-2) and have intended to explore the specificity of one ligand R1 (5-(3-chlorophenyl)-N-(2-phenyl-2H-benzo[d][1,2,3]triazol-6-yl)furan-2-carboxamide) that succeeded the dengue inhibition by the molecular dynamics (MD) simulations in conjunction of the molecular docking and the binding free energy calculations. The residue interactions map shows Lys 296 of domain-III of the DENV-2, which might be responsible for binding small molecules between domain I/III interface, as an important residue conserved in all the dengue serotypes.

  11. A domain unique to plant RanGAP is responsible for its targeting to the plant nuclear rim

    PubMed Central

    Rose, Annkatrin; Meier, Iris

    2001-01-01

    Ran is a small signaling GTPase that is involved in nucleocytoplasmic transport. Two additional functions of animal Ran in the formation of spindle asters and the reassembly of the nuclear envelope in mitotic cells have been recently reported. In contrast to Ras or Rho, Ran is not associated with membranes. Instead, the spatial sequestering of its accessory proteins, the Ran GTPase-activating protein RanGAP and the nucleotide exchange factor RCC1, appears to define the local concentration of RanGTP vs. RanGDP involved in signaling. Mammalian RanGAP is bound to the nuclear pore by a mechanism involving the attachment of small ubiquitin-related modifier protein (SUMO) to its C terminus and the subsequent binding of the SUMOylated domain to the nucleoporin Nup358. Here we show that plant RanGAP utilizes a different mechanism for nuclear envelope association, involving a novel targeting domain that appears to be unique to plants. The N-terminal WPP domain is highly conserved among plant RanGAPs and the small, plant-specific nuclear envelope-associated protein MAF1, but not present in yeast or animal RanGAP. Confocal laser scanning microscopy of green fluorescent protein (GFP) fusion proteins showed that it is necessary for RanGAP targeting and sufficient to target the heterologous protein GFP to the plant nuclear rim. The highly conserved tryptophan and proline residues of the WPP motif are necessary for its function. The 110-aa WPP domain is the first nuclear-envelope targeting domain identified in plants. Its fundamental difference to its mammalian counterpart implies that different mechanisms have evolved in plants and animals to anchor RanGAP at the nuclear surface. PMID:11752475

  12. Real-Time Investigation of Referential Domains in Unscripted Conversation: A Targeted Language Game Approach

    ERIC Educational Resources Information Center

    Brown-Schmidt, Sarah; Tanenhaus, Michael K.

    2008-01-01

    Two experiments examined the restriction of referential domains during unscripted conversation by analyzing the modification and online interpretation of referring expressions. Experiment 1 demonstrated that from the earliest moments of processing, addressees interpreted referring expressions with respect to referential domains constrained by the…

  13. Targeting extracellular domains D4 and D7 of vascular endothelial growth factor receptor 2 reveals allosteric receptor regulatory sites.

    PubMed

    Hyde, Caroline A C; Giese, Alexandra; Stuttfeld, Edward; Abram Saliba, Johan; Villemagne, Denis; Schleier, Thomas; Binz, H Kaspar; Ballmer-Hofer, Kurt

    2012-10-01

    Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory "designed ankyrin repeat proteins" (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies.

  14. Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites

    PubMed Central

    Hyde, Caroline A. C.; Giese, Alexandra; Stuttfeld, Edward; Abram Saliba, Johan; Villemagne, Denis; Schleier, Thomas; Binz, H. Kaspar

    2012-01-01

    Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory “designed ankyrin repeat proteins” (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies. PMID:22801374

  15. Real-time investigation of referential domains in unscripted conversation: a targeted language game approach

    PubMed Central

    Brown-Schmidt, Sarah; Tanenhaus, Michael K.

    2009-01-01

    Two experiments examined the restriction of referential domains during unscripted conversation by analyzing the modification and on-line interpretation of referring expressions. Experiment 1 demonstrated that from the earliest moments of processing, addressees interpreted referring expressions with respect to referential domains constrained by the conversation. Analysis of eye movements during the conversation showed elimination of standard competition effects seen with scripted language. Results from Experiment 2 pinpointed two pragmatic factors responsible for restriction of the referential domains used by speakers to design referential expressions and demonstrated that the same factors predict whether addressees consider local competitors to be potential referents during on-line interpretation of the same expressions. These experiments demonstrate for the first time that on-line interpretation of referring expressions in conversation is facilitated by referential domains constrained by pragmatic factors which predict when addressees are likely to encounter temporary ambiguity in language processing. PMID:19890480

  16. The C1 and C2 domains target human type 6 adenylyl cyclase to lipid rafts and caveolae.

    PubMed

    Thangavel, Muthusamy; Liu, Xiaoqiu; Sun, Shu Qiang; Kaminsky, Joseph; Ostrom, Rennolds S

    2009-02-01

    Previous data has shown that adenylyl cyclase type 6 (AC6) is expressed principally in lipid rafts or caveolae of cardiac myocytes and other cell types while certain other isoforms of AC are excluded from these microdomains. The mechanism by which AC6 is localized to lipid rafts or caveolae is unknown. In this study, we show AC6 is localized in lipid rafts of COS-7 cells (expressing caveolin-1) and in HEK-293 cells or cardiac fibroblasts isolated from caveolin-1 knock-out mice (both of which lack prototypical caveolins). To determine the region of AC6 that confers raft localization, we independently expressed each of the major intracellular domains, the N-terminus, C1 and C2 domains, and examined their localization with various approaches. The N-terminus did not associate with lipid rafts or caveolae of either COS-7 or HEK-293 cells nor did it immunoprecipitate with caveolin-1 when expressed in COS-7 cells. By contrast, the C1 and C2 domains each associated with lipid rafts to varying degrees and were present in caveolin-1 immunoprecipitates. There were no differences in the pattern of localization of either the C1 or C2 domains between COS-7 and HEK-293 cells. Further dissection of the C1 domain into four individual proteins indicated that the N-terminal half of this domain is responsible for its raft localization. To probe for a role of a putative palmitoylation motif in the C-terminal portion of the C2 domain, we expressed various truncated forms of AC6 lacking most or all of the C-terminal 41 amino acids. These truncated AC6 proteins were not altered in terms of their localization in lipid rafts or their catalytic activity, implying that this C-terminal region is not required for lipid raft targeting of AC6. We conclude that while the C1 domain may be most important, both the C1 and C2 domains of AC6 play a role in targeting AC6 to lipid rafts.

  17. Engineered domain based assays to identify individual antibodies in oligoclonal combinations targeting the same protein

    PubMed Central

    Meng, Q.; Garcia-Rodriguez, C.; Manzanarez, G.; Silberg, M.A.; Conrad, F.; Bettencourt, J.; Pan, X.; Breece, T.; To, R.; Li, M.; Lee, D.; Thorner, L.; Tomic, M.T.; Marks, J.D.

    2014-01-01

    Quantitation of individual mAbs within a combined antibody drug product is required for preclinical and clinical drug development. We have developed two antitoxins (XOMA 3B and XOMA 3E) each consisting of three monoclonal antibodies (mAbs) that neutralize type B and type E botulinum neurotoxin (BoNT/B and BoNT/E) to treat serotype B and E botulism. To develop mAb-specific binding assays for each antitoxin, we mapped the epitopes of the six mAbs. Each mAb bound an epitope on either the BoNT light chain (LC) or translocation domain (HN). Epitope mapping data was used to design LC-HN domains with orthogonal mutations to make them specific for only one mAb in either XOMA 3B or 3E. Mutant LC-HN domains were cloned, expressed, and purified from E. coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. Further engineering of domains allowed construction of ELISAs that could characterize the integrity, binding affinity, and identity of each of the six mAbs in XOMA 3B, and 3E without interference from the three BoNT/A mAbs in XOMA 3AB. Such antigen engineering is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein. PMID:22922799

  18. Time-domain response of a metal detector to a target buried in soil with frequency-dependent magnetic susceptibility

    NASA Astrophysics Data System (ADS)

    Das, Y.

    2006-05-01

    The work reported in this paper is a part of on-going studies to clarify how and to what extent soil electromagnetic properties affect the performance of induction metal detectors widely used in humanitarian demining. This paper studies the specific case of the time-domain response of a small metallic sphere buried in a non-conducting soil half-space with frequency-dependent complex magnetic susceptibility. The sphere is chosen as a simple prototype for the small metal parts in low-metal landmines, while soil with dispersive magnetic susceptibility is a good model for some soils that are known to adversely affect the performance of metal detectors. The included analysis and computations extend previous work which has been done mostly in the frequency domain. Approximate theoretical expressions for weakly magnetic soils are found to fit the experimental data very well, which allowed the estimation of soil model parameters, albeit in an ad hoc manner. Soil signal is found to exceed target signal (due to an aluminum sphere of radius 0.0127 m) in many cases, even for the weakly magnetic Cambodian laterite used in the experiments. How deep a buried target is detected depends on many other factors in addition to the relative strength of soil and target signals. A general statement cannot thus be made regarding detectability of a target in soil based on the presented results. However, computational results complemented with experimental data extend the understanding of the effect that soil has on metal detectors.

  19. Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers.

    PubMed

    Lin, Yi; Mori, Eiichiro; Kato, Masato; Xiang, Siheng; Wu, Leeju; Kwon, Ilmin; McKnight, Steven L

    2016-10-20

    Two complementary approaches were used in search of the intracellular targets of the toxic PR poly-dipeptide encoded by the repeat sequences expanded in the C9orf72 form of amyotrophic lateral sclerosis. The top categories of PRn-bound proteins include constituents of non-membrane invested cellular organelles and intermediate filaments. PRn targets are enriched for the inclusion of low complexity (LC) sequences. Evidence is presented indicating that LC sequences represent the direct target of PRn binding and that interaction between the PRn poly-dipeptide and LC domains is polymer-dependent. These studies indicate that PRn-mediated toxicity may result from broad impediments to the dynamics of cell structure and information flow from gene to message to protein.

  20. Toxic PR poly-dipeptides encoded by the C9orf72 repeat expansion target LC domain polymers

    PubMed Central

    Lin, Yi; Mori, Eiichiro; Kato, Masato; Xiang, Siheng; Wu, Leeju; Kwon, Ilmin; McKnight, Steven L.

    2016-01-01

    Summary Two complementary approaches were used in search of the intracellular targets of the toxic PR poly-dipeptide encoded by the repeat sequences expanded in the C9orf72 form of amyotrophic lateral sclerosis. The top categories of PRn-bound proteins include constituents of non-membrane invested cellular organelles and intermediate filaments. PRn targets are enriched for the inclusion of low complexity (LC) sequences. Evidence is presented indicating that LC sequences represent the direct target of PRn binding, and that interaction between the PRn poly-dipeptide and LC domains is polymer-dependent. These studies indicate that PRn-mediated toxicity may result from broad impediments to the dynamics of cell structure and information flow from gene to message to protein. PMID:27768897

  1. Domain based assays of individual antibody concentrations in an oligoclonal combination targeting a single protein

    PubMed Central

    Meng, Q.; Li, M.; Silberg, M.A.; Conrad, F.; Bettencourt, J.; To, R.; Huang, C.; Ma, J.; Meyer, K.; Shimizu, R.; Cao, L.; Tomic, M.T.; Marks, J.D.

    2014-01-01

    Quantitation of individual mAbs within a combined antibody drug product is required for preclinical and clinical drug development including pharmacokinetics (PK), toxicology, stability and biochemical characterization studies of such drugs. We have developed an antitoxin (XOMA 3AB) consisting of three recombinant monoclonal antibodies (mAbs) that potently neutralizes the known subtypes of type A botulinum neurotoxin (BoNT/A). The three mAbs bind non-overlapping BoNT/A epitopes with high affinity. XOMA3AB is being developed as a treatment for botulism resulting from BoNT/A. To develop antibody-specific assays, we cloned, expressed, and purified BoNT/A domains from E. coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. MAb specific domains were used to develop an ELISA for characterization of the integrity and binding activity of the three mAbs in the drug product. An electrochemiluminescence bridging assay was also developed that is robust to interference from components in serum and we demonstrate that it can be used for PK assays. This type of antigen engineering to generate mAb-specific domains is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein and is superior to anti-idiotype approaches. PMID:22037290

  2. Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata

    SciTech Connect

    Sampathkumar, Parthasarathy; Kim, Seung Joong; Manglicmot, Danalyn; Bain, Kevin T.; Gilmore, Jeremiah; Gheyi, Tarun; Phillips, Jeremy; Pieper, Ursula; Fernandez-Martinez, Javier; Franke, Josef D.; Matsui, Tsutomu; Tsuruta, Hiro; Atwell, Shane; Thompson, Devon A.; Emtage, J. Spencer; Wasserman, Stephen R.; Rout, Michael P.; Sali, Andrej; Sauder, J. Michael; Almo, Steven C.; Burley, Stephen K.

    2012-10-23

    The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of {approx}456 polypeptide chains contributed by {approx}30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal 'FG' repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 {angstrom} resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed.

  3. GT-2: in vivo transcriptional activation activity and definition of novel twin DNA binding domains with reciprocal target sequence selectivity.

    PubMed

    Ni, M; Dehesh, K; Tepperman, J M; Quail, P H

    1996-06-01

    GT-2 is a novel DNA binding protein that interacts with a triplet functionally defined, positively acting GT-box motifs (GT1-bx, GT2-bx, and GT3-bx) in the rice phytochrome A gene (PHYA) promoter. Data from a transient transfection assay used here show that recombinant GT-2 enhanced transcription from both homologous and heterologous GT-box-containing promoters, thereby indicating that this protein can function as a transcriptional activator in vivo. Previously, we have shown that GT-2 contains separate DNA binding determinants in its N- and C-terminal halves, with binding site preferences for the GT3-bx and GT2-bx promoter motifs, respectively. Here, we demonstrate that the minimal DNA binding domains reside within dual 90-amino acid polypeptide segments encompassing duplicated sequences, termed trihelix regions, in each half of the molecule, plus 15 additional immediately adjacent amino acids downstream. These minimal binding domains retained considerable target sequence selectivity for the different GT-box motifs, but this selectivity was enhanced by a separate polypeptide segment farther downstream on the C-terminal side of each trihelix region. Therefore, the data indicate that the twin DNA binding domains of GT-2 each consist of a general GT-box recognition core with intrinsic differential binding activity toward closely related target motifs and a modified sequence conferring higher resolution reciprocal selectivity between these motifs.

  4. Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata.

    PubMed

    Sampathkumar, Parthasarathy; Kim, Seung Joong; Manglicmot, Danalyn; Bain, Kevin T; Gilmore, Jeremiah; Gheyi, Tarun; Phillips, Jeremy; Pieper, Ursula; Fernandez-Martinez, Javier; Franke, Josef D; Matsui, Tsutomu; Tsuruta, Hiro; Atwell, Shane; Thompson, Devon A; Emtage, J Spencer; Wasserman, Stephen R; Rout, Michael P; Sali, Andrej; Sauder, J Michael; Almo, Steven C; Burley, Stephen K

    2012-08-01

    The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of ~456 polypeptide chains contributed by ~30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal "FG" repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 Å resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed.

  5. Fast analysis of wide-band scattering from electrically large targets with time-domain parabolic equation method

    NASA Astrophysics Data System (ADS)

    He, Zi; Chen, Ru-Shan

    2016-03-01

    An efficient three-dimensional time domain parabolic equation (TDPE) method is proposed to fast analyze the narrow-angle wideband EM scattering properties of electrically large targets. The finite difference (FD) of Crank-Nicolson (CN) scheme is used as the traditional tool to solve the time-domain parabolic equation. However, a huge computational resource is required when the meshes become dense. Therefore, the alternating direction implicit (ADI) scheme is introduced to discretize the time-domain parabolic equation. In this way, the reduced transient scattered fields can be calculated line by line in each transverse plane for any time step with unconditional stability. As a result, less computational resources are required for the proposed ADI-based TDPE method when compared with both the traditional CN-based TDPE method and the finite-different time-domain (FDTD) method. By employing the rotating TDPE method, the complete bistatic RCS can be obtained with encouraging accuracy for any observed angle. Numerical examples are given to demonstrate the accuracy and efficiency of the proposed method.

  6. The rotaviral NSP3 protein stimulates translation of polyadenylated target mRNAs independently of its RNA-binding domain

    SciTech Connect

    Keryer-Bibens, Cecile; Legagneux, Vincent; Namanda-Vanderbeken, Allen; Cosson, Bertrand; Paillard, Luc; Poncet, Didier; Osborne, H. Beverley

    2009-12-11

    The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3' extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3' end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.

  7. Time-Domain Modeling of Electromagnetic Pulses Returned from Targets in Dispersive and Dissipative Media

    NASA Astrophysics Data System (ADS)

    Gaunaurd, G. C.; Strifors, H. C.; Sullivan, A.

    2008-06-01

    Using a Method-of-Moments (MoM) code, we earlier simulated ultra-wideband (UWB) returned echoes from two targets, one penetrable and one impenetrable, buried in a soil with known electric properties. The simple shape of mines, coupled with their predictable deployment in the ground, has also provided a fundamental understanding of the underlying backscatter phenomenology. In particular, the backscattered waveform from a mine can be decomposed into a collection of closely spaced copies of an elemental wave object with different amplitudes and time delays. This wave object is here defined as the derivative of the waveform incident on the target. The spacing of the copies or replicas of the wave object could be determined by the round trip time delay between scattering centers. This methodology was previously applied to impenetrable targets and is now applied also to a penetrable target. The previously computed returns from each target for a given incident pulse are modeled by a few copies of the elemental wave object with the time delay and amplitude of each copy taken as unknown parameters. These parameters are then determined by minimizing in the least square sense the difference between the MoM computed signal and the model signal using the differential evolution method (DEM). The methodology is extended by way of our previously developed target translated method (TTM) to approximate the computation of the backscattered model signal when the target is buried at a different depth in the soil with a different moisture content.

  8. Mechanism of intermediate filament recognition by plakin repeat domains revealed by envoplakin targeting of vimentin

    NASA Astrophysics Data System (ADS)

    Fogl, Claudia; Mohammed, Fiyaz; Al-Jassar, Caezar; Jeeves, Mark; Knowles, Timothy J.; Rodriguez-Zamora, Penelope; White, Scott A.; Odintsova, Elena; Overduin, Michael; Chidgey, Martyn

    2016-03-01

    Plakin proteins form critical connections between cell junctions and the cytoskeleton; their disruption within epithelial and cardiac muscle cells cause skin-blistering diseases and cardiomyopathies. Envoplakin has a single plakin repeat domain (PRD) which recognizes intermediate filaments through an unresolved mechanism. Herein we report the crystal structure of envoplakin's complete PRD fold, revealing binding determinants within its electropositive binding groove. Four of its five internal repeats recognize negatively charged patches within vimentin via five basic determinants that are identified by nuclear magnetic resonance spectroscopy. Mutations of the Lys1901 or Arg1914 binding determinants delocalize heterodimeric envoplakin from intracellular vimentin and keratin filaments in cultured cells. Recognition of vimentin is abolished when its residues Asp112 or Asp119 are mutated. The latter slot intermediate filament rods into basic PRD domain grooves through electrosteric complementarity in a widely applicable mechanism. Together this reveals how plakin family members form dynamic linkages with cytoskeletal frameworks.

  9. PERIPARTUM DEPRESSION AND ANXIETY AS AN INTEGRATIVE CROSS DOMAIN TARGET FOR PSYCHIATRIC PREVENTATIVE MEASURES

    PubMed Central

    Babb, Jessica A.; Deligiannidis, Kristina M.; Murgatroyd, Christopher A.

    2014-01-01

    Exposure to high levels of early life stress has been identified as a potent risk factor for neurodevelopmental delays in infants, behavioral problems and autism in children, but also for several psychiatric illnesses in adulthood, such as depression, anxiety, autism, and posttraumatic stress disorder. Despite having robust adverse effects on both mother and infant, the pathophysiology of peripartum depression and anxiety are poorly understood. The objective of this review is to highlight the advantages of using an integrated approach addressing several behavioral domains in both animal and clinical studies of peripartum depression and anxiety. It is postulated that a greater focus on integrated cross domain studies will lead to advances in treatments and preventative measures for several disorders associated with peripartum depression and anxiety. PMID:24709228

  10. Mechanism of intermediate filament recognition by plakin repeat domains revealed by envoplakin targeting of vimentin

    PubMed Central

    Fogl, Claudia; Mohammed, Fiyaz; Al-Jassar, Caezar; Jeeves, Mark; Knowles, Timothy J.; Rodriguez-Zamora, Penelope; White, Scott A.; Odintsova, Elena; Overduin, Michael; Chidgey, Martyn

    2016-01-01

    Plakin proteins form critical connections between cell junctions and the cytoskeleton; their disruption within epithelial and cardiac muscle cells cause skin-blistering diseases and cardiomyopathies. Envoplakin has a single plakin repeat domain (PRD) which recognizes intermediate filaments through an unresolved mechanism. Herein we report the crystal structure of envoplakin's complete PRD fold, revealing binding determinants within its electropositive binding groove. Four of its five internal repeats recognize negatively charged patches within vimentin via five basic determinants that are identified by nuclear magnetic resonance spectroscopy. Mutations of the Lys1901 or Arg1914 binding determinants delocalize heterodimeric envoplakin from intracellular vimentin and keratin filaments in cultured cells. Recognition of vimentin is abolished when its residues Asp112 or Asp119 are mutated. The latter slot intermediate filament rods into basic PRD domain grooves through electrosteric complementarity in a widely applicable mechanism. Together this reveals how plakin family members form dynamic linkages with cytoskeletal frameworks. PMID:26935805

  11. Target selectivity of vertebrate notch proteins. Collaboration between discrete domains and CSL-binding site architecture determines activation probability.

    PubMed

    Ong, Chin-Tong; Cheng, Hui-Teng; Chang, Li-Wei; Ohtsuka, Toshiyuki; Kageyama, Ryoichiro; Stormo, Gary D; Kopan, Raphael

    2006-02-24

    All four mammalian Notch proteins interact with a single DNA-binding protein (RBP-jkappa), yet they are not equivalent in activating target genes. Parallel assays of three Notch-responsive promoters in several cell lines revealed that relative activation strength is dependent on protein module and promoter context more than the cellular context. Each Notch protein reads binding site orientation and distribution on the promoter differently; Notch1 performs extremely well on paired sites, and Notch3 prefers single sites in conjunction with a proximal zinc finger transcription factor. Although head-head sites can elicit a Notch response on their own, use of CBS (CSL binding site) in tail-tail orientation is context-dependent. Bias for specific DNA elements is achieved by interplay between the N-terminal RAM (RBP-jkappa-associated molecule/ankyrin region), which interprets CBS proximity and orientation, and the C-terminal transactivation domain that interacts specifically with the transcription machinery or nearby factors. To confirm the prediction that modular design underscores the evolution of functional divergence between Notch proteins, we generated a synthetic Notch protein (Notch1 ankyrin with Notch3 transactivation domain) that displayed superior signaling strength on the hes5 promoter. Consistent with the prediction that "preferred" targets (Hes1) should respond faster and at lower Notch concentration than other targets, we showed that Hes5-GFP was extinguished fast and recovered slowly, whereas Hes1-GFP was inhibited late and recovered quickly after a pulse of DAPT in metanephroi cultures.

  12. Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle.

    PubMed

    Zhang, Kathy; Geddie, Melissa L; Kohli, Neeraj; Kornaga, Tad; Kirpotin, Dmitri B; Jiao, Yang; Rennard, Rachel; Drummond, Daryl C; Nielsen, Ulrik B; Xu, Lihui; Lugovskoy, Alexey A

    2015-01-01

    Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.

  13. Improving Maritime Domain Awareness Using Neural Networks for Target of Interest Classification

    DTIC Science & Technology

    2015-03-01

    Lieutenant, United States Navy B.S., United States Naval Academy, 2008 Submitted in partial fulfillment of the requirements for the degree of...Awareness (MDA). MDA encompasses anything associated with the global maritime domain that could impact the United States [1]. The efforts made to...increase MDA are critical to sustaining global economic stability and growth and are vital to the interests of the United States . Due to its complexity

  14. Diagnostic nanoparticle targeting of the EGF-receptor in complex biological conditions using single-domain antibodies

    NASA Astrophysics Data System (ADS)

    Zarschler, K.; Prapainop, K.; Mahon, E.; Rocks, L.; Bramini, M.; Kelly, P. M.; Stephan, H.; Dawson, K. A.

    2014-05-01

    For effective localization of functionalized nanoparticles at diseased tissues such as solid tumours or metastases through biorecognition, appropriate targeting vectors directed against selected tumour biomarkers are a key prerequisite. The diversity of such vector molecules ranges from proteins, including antibodies and fragments thereof, through aptamers and glycans to short peptides and small molecules. Here, we analyse the specific nanoparticle targeting capabilities of two previously suggested peptides (D4 and GE11) and a small camelid single-domain antibody (sdAb), representing potential recognition agents for the epidermal growth factor receptor (EGFR). We investigate specificity by way of receptor RNA silencing techniques and look at increasing complexity in vitro by introducing increasing concentrations of human or bovine serum. Peptides D4 and GE11 proved problematic to employ and conjugation resulted in non-receptor specific uptake into cells. Our results show that sdAb-functionalized particles can effectively target the EGFR, even in more complex bovine and human serum conditions where targeting specificity is largely conserved for increasing serum concentration. In human serum however, an inhibition of overall nanoparticle uptake is observed with increasing protein concentration. For highly affine targeting ligands such as sdAbs, targeting a receptor such as EGFR with low serum competitor abundance, receptor recognition function can still be partially realised in complex conditions. Here, we stress the value of evaluating the targeting efficiency of nanoparticle constructs in realistic biological milieu, prior to more extensive in vivo studies.For effective localization of functionalized nanoparticles at diseased tissues such as solid tumours or metastases through biorecognition, appropriate targeting vectors directed against selected tumour biomarkers are a key prerequisite. The diversity of such vector molecules ranges from proteins, including

  15. A DNA vaccine targeting the receptor-binding domain of Clostridium difficile toxin A.

    PubMed

    Gardiner, David F; Rosenberg, Talia; Zaharatos, Jerry; Franco, David; Ho, David D

    2009-06-02

    Clostridium difficile is a pathogen with increasing severity for which host antibody responses provide protection from disease. DNA vaccination has several advantages compared to traditional vaccine methods, however no study has examined this platform against C. difficile toxins. A synthetic gene was created encoding the receptor-binding domain (RBD) of C. difficile toxin A, optimized for expression in human cells. Gene expression was examined in vitro. Mice were inoculated and then challenged with parenteral toxin A. Vaccination provided high titer antibodies and protected mice from death. This represents the first report of DNA vaccine inducing neutralizing antibodies to C. difficile toxin A.

  16. The role of symptom induction in the treatment of panic and anxiety. Identifiable domains, conditional properties, and treatment targets.

    PubMed

    Rudd, M D; Joiner, T

    1998-01-01

    Although the importance of affectively charged material in the treatment of panic and anxiety has been emphasized and implicitly viewed as essential for effective therapeutic change, a general framework for organizing, understanding, implementing, and evaluating symptom induction techniques has yet to be offered. This article offers a framework for organizing symptoms induction techniques, categorizing treatment targets, and, accordingly, assessing therapeutic change in the treatment of panic and anxiety. Symptom induction techniques are examined in three exposure domains: physiological, cognitive, and situational/circumstantial; treatment targets fall into five categories: (a) poor symptom tolerance and resultant hypersensitivity and hypervigilance, (b) avoidance of internal and external triggers, (c) the emergence of specific catastrophic thoughts and related misinterpretations, (d) diminished adaptive coping skills, and (e) a reduction in general self-efficacy. Additionally, a distinction is proposed between conditional and unconditional properties of symptom induction, with an emphasis on the potential deleterious role conditional properties play during the course of treatment.

  17. Experimental Study of High-Range-Resolution Medical Acoustic Imaging for Multiple Target Detection by Frequency Domain Interferometry

    NASA Astrophysics Data System (ADS)

    Kimura, Tomoki; Taki, Hirofumi; Sakamoto, Takuya; Sato, Toru

    2009-07-01

    We employed frequency domain interferometry (FDI) for use as a medical acoustic imager to detect multiple targets with high range resolution. The phase of each frequency component of an echo varies with the frequency, and target intervals can be estimated from the phase variance. This processing technique is generally used in radar imaging. When the interference within a range gate is coherent, the cross correlation between the desired signal and the coherent interference signal is nonzero. The Capon method works under the guiding principle that output power minimization cancels the desired signal with a coherent interference signal. Therefore, we utilize frequency averaging to suppress the correlation of the coherent interference. The results of computational simulations using a pseudoecho signal show that the Capon method with adaptive frequency averaging (AFA) provides a higher range resolution than a conventional method. These techniques were experimentally investigated and we confirmed the effectiveness of the proposed method of processing by FDI.

  18. Therapeutic targeting of two-pore-domain potassium (K(2P)) channels in the cardiovascular system.

    PubMed

    Wiedmann, Felix; Schmidt, Constanze; Lugenbiel, Patrick; Staudacher, Ingo; Rahm, Ann-Kathrin; Seyler, Claudia; Schweizer, Patrick A; Katus, Hugo A; Thomas, Dierk

    2016-05-01

    The improvement of treatment strategies in cardiovascular medicine is an ongoing process that requires constant optimization. The ability of a therapeutic intervention to prevent cardiovascular pathology largely depends on its capacity to suppress the underlying mechanisms. Attenuation or reversal of disease-specific pathways has emerged as a promising paradigm, providing a mechanistic rationale for patient-tailored therapy. Two-pore-domain K(+) (K(2P)) channels conduct outward K(+) currents that stabilize the resting membrane potential and facilitate action potential repolarization. K(2P) expression in the cardiovascular system and polymodal K2P current regulation suggest functional significance and potential therapeutic roles of the channels. Recent work has focused primarily on K(2P)1.1 [tandem of pore domains in a weak inwardly rectifying K(+) channel (TWIK)-1], K(2P)2.1 [TWIK-related K(+) channel (TREK)-1], and K(2P)3.1 [TWIK-related acid-sensitive K(+) channel (TASK)-1] channels and their role in heart and vessels. K(2P) currents have been implicated in atrial and ventricular arrhythmogenesis and in setting the vascular tone. Furthermore, the association of genetic alterations in K(2P)3.1 channels with atrial fibrillation, cardiac conduction disorders and pulmonary arterial hypertension demonstrates the relevance of the channels in cardiovascular disease. The function, regulation and clinical significance of cardiovascular K(2P) channels are summarized in the present review, and therapeutic options are emphasized.

  19. Distinct domains of the spinal muscular atrophy protein SMN are required for targeting to Cajal bodies in mammalian cells.

    PubMed

    Renvoisé, Benoît; Khoobarry, Kevinee; Gendron, Marie-Claude; Cibert, Christian; Viollet, Louis; Lefebvre, Suzie

    2006-02-15

    Mutations of the survival motor neuron gene SMN1 cause the inherited disease spinal muscular atrophy (SMA). The ubiquitous SMN protein facilitates the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs). The protein is detected in the cytoplasm, nucleoplasm and enriched with snRNPs in nuclear Cajal bodies. It is structurally divided into at least an amino-terminal region rich in basic amino acid residues, a central Tudor domain, a self-association tyrosine-glycine-box and an exon7-encoded C-terminus. To examine the domains required for the intranuclear localization of SMN, we have used fluorescently tagged protein mutants transiently overexpressed in mammalian cells. The basic amino acid residues direct nucleolar localization of SMN mutants. The Tudor domain promotes localization of proteins in the nucleus and it cooperates with the basic amino acid residues and the tyrosine-glycine-box for protein localization in Cajal bodies. Moreover, the most frequent disease-linked mutant SMNDeltaex7 reduces accumulation of snRNPs in Cajal bodies, suggesting that the C-terminus of SMN participates in targeting to Cajal bodies. A reduced number of Cajal bodies in patient fibroblasts associates with the absence of snRNPs in Cajal bodies, revealing that intranuclear snRNA organization is modified in disease. These results indicate that direct and indirect mechanisms regulate localization of SMN in Cajal bodies.

  20. An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain

    PubMed Central

    Ranjitkar, Prerana; Press, Maximilian O.; Yi, Xianhua; Baker, Richard; MacCoss, Michael J.; Biggins, Sue

    2010-01-01

    Summary Proper centromere function is critical to maintain genomic stability and to prevent aneuploidy, a hallmark of tumors and birth defects. A conserved feature of all eukaryotic centromeres is an essential histone H3 variant called CENP-A that requires a centromere targeting domain (CATD) for its localization. Although proteolysis prevents CENP-A from mislocalizing to euchromatin, regulatory factors have not been identified. Here, we identify an E3 ubiquitin ligase called Psh1 that leads to the degradation of Cse4, the budding yeast CENP-A homolog. Cse4 overexpression is toxic to psh1Δ cells and results in euchromatic localization. Strikingly, the Cse4 centromere targeting domain is a key regulator of its stability and helps Psh1 discriminate Cse4 from histone H3. Taken together, we propose that the CATD has a previously unknown role in maintaining the exclusive localization of Cse4 by preventing its mislocalization to euchromatin via Psh1-mediated degradation. PMID:21070971

  1. Acquisition of a single EZH2 D1 domain mutation confers acquired resistance to EZH2-targeted inhibitors

    PubMed Central

    Baker, Theresa; Nerle, Sujata; Pritchard, Justin; Zhao, Boyang; Rivera, Victor M.

    2015-01-01

    Although targeted therapies have revolutionized cancer treatment, overcoming acquired resistance remains a major clinical challenge. EZH2 inhibitors (EZH2i), EPZ-6438 and GSK126, are currently in the early stages of clinical evaluation and the first encouraging signs of efficacy have recently emerged in the clinic. To anticipate mechanisms of resistance to EZH2i, we used a forward genetic platform combining a mutagenesis screen with next generation sequencing technology and identified a hotspot of secondary mutations in the EZH2 D1 domain (Y111 and I109). Y111D mutation within the WT or A677G EZH2 allele conferred robust resistance to both EPZ-6438 and GSK126, but it only drove a partial resistance within the Y641F allele. EZH2 mutants required histone methyltransferase (HMT) catalytic activity and the polycomb repressive complex 2 (PRC2) components, SUZ12 and EED, to drive drug resistance. Furthermore, D1 domain mutations not only blocked the ability of EZH2i to bind to WT and A677G mutant, but also abrogated drug binding to the Y641F mutant. These data provide the first cellular validation of the mechanistic model underpinning the oncogenic function of WT and mutant EZH2. Importantly, our findings suggest that acquired-resistance to EZH2i may arise in WT and mutant EZH2 patients through a single mutation that remains targetable by second generation EZH2i. PMID:26360609

  2. Targeting the hepatitis B virus precore antigen with a novel IgNAR single variable domain intrabody.

    PubMed

    Walsh, Renae; Nuttall, Stewart; Revill, Peter; Colledge, Danni; Cabuang, Liza; Soppe, Sally; Dolezal, Olan; Griffiths, Kate; Bartholomeusz, Angeline; Locarnini, Stephen

    2011-03-01

    The Hepatitis B virus precore protein is processed in the endoplasmic reticulum (ER) into secreted hepatitis B e antigen (HBeAg), which acts as an immune tolerogen to establish chronic infection. Downregulation of secreted HBeAg should improve clinical outcome, as patients who effectively respond to current treatments (IFN-α) have significantly lower serum HBeAg levels. Here, we describe a novel reagent, a single variable domain (V(NAR)) of the shark immunoglobulin new antigen receptor (IgNAR) antibodies. V(NAR)s possess advantages in stability, size (~14 kDa) and cryptic epitope recognition compared to conventional antibodies. The V(NAR) domain displayed biologically useful affinity for recombinant and native HBeAg, and recognised a unique conformational epitope. To assess therapeutic potential in targeting intracellular precore protein to reduce secreted HBeAg, the V(NAR) was engineered for ER-targeted in vitro delivery to function as an intracellular antibody (intrabody). In vitro data from HBV/precore hepatocyte cell lines demonstrated effective intrabody regulation of precore/HBeAg.

  3. Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis.

    PubMed

    Hiratsu, Keiichiro; Matsui, Kyoko; Koyama, Tomotsugu; Ohme-Takagi, Masaru

    2003-06-01

    The redundancy of genes for plant transcription factors often interferes with efforts to identify the biologic functions of such factors. We show here that four different transcription factors fused to the EAR motif, a repression domain of only 12 amino acids, act as dominant repressors in transgenic Arabidopsis and suppress the expression of specific target genes, even in the presence of the redundant transcription factors, with resultant dominant loss-of-function phenotypes. Chimeric EIN3, CUC1, PAP1, and AtMYB23 repressors that included the EAR motif dominantly suppressed the expression of their target genes and caused insensitivity to ethylene, cup-shaped cotyledons, reduction in the accumulation of anthocyanin, and absence of trichomes, respectively. This chimeric repressor silencing technology (CRES-T), exploiting the EAR-motif repression domain, is simple and effective and can overcome genetic redundancy. Thus, it should be useful not only for the rapid analysis of the functions of redundant plant transcription factors but also for the manipulation of plant traits via the suppression of gene expression that is regulated by specific transcription factors.

  4. Computational modeling of novel inhibitors targeting the Akt pleckstrin homology domain.

    PubMed

    Du-Cuny, Lei; Song, Zuohe; Moses, Sylvestor; Powis, Garth; Mash, Eugene A; Meuillet, Emmanuelle J; Zhang, Shuxing

    2009-10-01

    Computational modeling continues to play an important role in novel therapeutics discovery and development. In this study, we have investigated the use of in silico approaches to develop inhibitors of the pleckstrin homology (PH) domain of AKT (protein kinase B). Various docking/scoring schemes have been evaluated, and the best combination was selected to study the system. Using this strategy, two hits were identified and their binding behaviors were investigated. Robust and predictive QSAR models were built using the k nearest neighbor (kNN) method to study their cellular permeability. Based on our in silico results, long flexible aliphatic tails were proposed to improve the Caco-2 penetration without affecting the binding mode. The modifications enhanced the AKT inhibitory activity of the compounds in cell-based assays, and increased their activity as in vivo antitumor testing.

  5. Probing Dense Plasmas Created from Intense Irradiation of Solid Target in the XUV Domain

    SciTech Connect

    Dobosz, S.; Doumy, G.; Stabile, H.; Monot, P.; Bougeard, M.; Reau, F.; Martin, Ph.

    2006-04-07

    In this paper, electronic density and temperature have been inferred from XUV transmission through hot solid-density plasma created by high temporal contrast femtosecond irradiation of thin plastic foil target in the 1018W/cm2 intensity range. High order harmonics generated in pulsed gas jet are used as a probe beam. The initial plasma parameters are determined with an accuracy better than 15% on the 100fs time scale, by comparison of the transmission of two consecutive harmonics.

  6. Targeted yield concept and a framework of fertilizer recommendation in irrigated rice domains of subtropical India*

    PubMed Central

    Bera, R.; Seal, A.; Bhattacharyya, P.; Das, T.H.; Sarkar, D.; Kangjoo, K.

    2006-01-01

    Soil test crop response (STCR) correlation studies were carried out in Vindhyan alluvial plain during 2001 to 2004 taking IR-36 as test crop to quantify rice production in the context of the variability of soil properties and use of balanced fertilizers based on targeted yield concept. The soils were developed on gently sloping alluvial plain with different physiographic settings and notable variation in drainage condition. Soil properties show moderate variation in texture (loamy to clay), organic carbon content (4.4 to 9.8 g/kg), cation exchange capacity (10.2 to 22.4 cmol (p+)/kg) and pH (5.3 to 6.4). Soil fertility status for N is low to medium (224 to 348 kg/ha), P is medium to high (87 to 320 kg/ha) and K ranges from medium to high (158 to 678 kg/ha). Database regarding nutrient requirement in kg/t of grain produce (NR), the percent contribution from the soil available nutrients [CS (%)] and the percent contribution from the applied fertilizer nutrients [CF (%)] were computed for calibrating and formulating fertilizer recommendations. Validity of the yield target for 7 and 8 t/ha was tested in farmers’ fields and yields targets varied at less than 10%. The percent achievement of targets aimed at different level was more than 90%, indicating soil test based fertilizer recommendation approach was economically viable within the agro-ecological zone with relatively uniform cropping practices and socio-economic conditions. PMID:17111464

  7. Light Curves as Predictors of Good Radial Velocity Planet Search Targets in New Stellar Domains

    NASA Astrophysics Data System (ADS)

    Bastien, Fabienne A.; Wright, Jason; Sigurdsson, Steinn; Dumusque, Xavier; Luhn, Jacob K.; Howard, Andrew

    2017-01-01

    As Kepler and K2 have collectively found thousands of exoplanet candidates, their discoveries have strained ground-based radial velocity (RV) follow-up resources, which are unable to simultaneously keep up with the pace of transit discoveries by measuring masses for all of the candidates and maintain vigorous RV searches for planets that do not transit their parent star. The burden to the RV community is expected to worsen with the upcoming TESS mission, even as new RV instruments are slated to come online in the coming years. Observations that can enable the RV community to prioritize targets on the basis of their stellar RV variability in advance and, ideally, independently of the RV instruments themselves, can therefore permit us to reserve our RV resources for the stars most likely to yield the highest payoff. We show that the light curves from space-based transit surveys may not only be used as predictors of good RV search targets for the stars predominantly targeted by the exoplanet community but also for stars usually avoided by both RV and transit surveys due to their high intrinsic levels of stellar variability. We also briefly present recommendations to the RV planet search community on how to improve prospects for finding Earth analogs from the recent workshop at the Aspen Center for Physics, “Approaching the Stellar Astrophysical Limits of Exoplanet Detection: Getting to 10cm/s.”

  8. Isolation and characterization of signermycin B, an antibiotic that targets the dimerization domain of histidine kinase WalK.

    PubMed

    Watanabe, Takafumi; Igarashi, Masayuki; Okajima, Toshihide; Ishii, Eiji; Kino, Hirokazu; Hatano, Masaki; Sawa, Ryuichi; Umekita, Maya; Kimura, Tomoyuki; Okamoto, Sho; Eguchi, Yoko; Akamatsu, Yuzuru; Utsumi, Ryutaro

    2012-07-01

    The WalK (histidine kinase)/WalR (response regulator) two-component signal transduction system is a master regulatory system for cell wall metabolism and growth. This system is conserved in low G+C Gram-positive bacteria, including Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, and Streptococcus mutans. In this study, we found the first antibiotic that functions as a WalK inhibitor (signermycin B) by screening 10,000 Streptomyces extracts. The chemical structure (C(23)H(35)NO(4); molecular weight, 389.5) comprises a tetramic acid moiety and a decalin ring. Signermycin B exhibited antimicrobial activity, with MIC values ranging from 3.13 μg/ml (8 μM) to 6.25 μg/ml (16 μM) against Gram-positive bacteria that possess the WalK/WalR two-component signal transduction system, including the drug-resistant bacteria methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. The half-maximal inhibitory concentrations of signermycin B against WalK in these organisms ranged from 37 to 61 μM. To determine the mechanism of action of signermycin B, surface plasmon resonance response analysis with the two WalK domains of Bacillus subtilis and competition assay with ATP were performed. The results showed that signermycin B binds to the dimerization domain but not the ATP-binding domain of WalK. In the presence of the cross-linker glutaraldehyde, signermycin B did not cause protein aggregation but interfered with the cross-linking of WalK dimers. These results suggest that signermycin B targets the conserved dimerization domain of WalK to inhibit autophosphorylation. In Bacillus subtilis and Staphylococcus aureus, signermycin B preferentially controlled the WalR regulon, thereby inhibiting cell division. These phenotypes are consistent with those of cells starved for the WalK/WalR system.

  9. Isolation and Characterization of Signermycin B, an Antibiotic That Targets the Dimerization Domain of Histidine Kinase WalK

    PubMed Central

    Watanabe, Takafumi; Igarashi, Masayuki; Okajima, Toshihide; Ishii, Eiji; Kino, Hirokazu; Hatano, Masaki; Sawa, Ryuichi; Umekita, Maya; Kimura, Tomoyuki; Okamoto, Sho; Eguchi, Yoko; Akamatsu, Yuzuru

    2012-01-01

    The WalK (histidine kinase)/WalR (response regulator) two-component signal transduction system is a master regulatory system for cell wall metabolism and growth. This system is conserved in low G+C Gram-positive bacteria, including Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, and Streptococcus mutans. In this study, we found the first antibiotic that functions as a WalK inhibitor (signermycin B) by screening 10,000 Streptomyces extracts. The chemical structure (C23H35NO4; molecular weight, 389.5) comprises a tetramic acid moiety and a decalin ring. Signermycin B exhibited antimicrobial activity, with MIC values ranging from 3.13 μg/ml (8 μM) to 6.25 μg/ml (16 μM) against Gram-positive bacteria that possess the WalK/WalR two-component signal transduction system, including the drug-resistant bacteria methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. The half-maximal inhibitory concentrations of signermycin B against WalK in these organisms ranged from 37 to 61 μM. To determine the mechanism of action of signermycin B, surface plasmon resonance response analysis with the two WalK domains of Bacillus subtilis and competition assay with ATP were performed. The results showed that signermycin B binds to the dimerization domain but not the ATP-binding domain of WalK. In the presence of the cross-linker glutaraldehyde, signermycin B did not cause protein aggregation but interfered with the cross-linking of WalK dimers. These results suggest that signermycin B targets the conserved dimerization domain of WalK to inhibit autophosphorylation. In Bacillus subtilis and Staphylococcus aureus, signermycin B preferentially controlled the WalR regulon, thereby inhibiting cell division. These phenotypes are consistent with those of cells starved for the WalK/WalR system. PMID:22526318

  10. A bi-functional antibody-receptor domain fusion protein simultaneously targeting IGF-IR and VEGF for degradation

    PubMed Central

    Shen, Yang; Zeng, Lin; Novosyadlyy, Ruslan; Forest, Amelie; Zhu, Aiping; Korytko, Andrew; Zhang, Haifan; Eastman, Scott W; Topper, Michael; Hindi, Sagit; Covino, Nicole; Persaud, Kris; Kang, Yun; Burtrum, Douglas; Surguladze, David; Prewett, Marie; Chintharlapalli, Sudhakar; Wroblewski, Victor J; Shen, Juqun; Balderes, Paul; Zhu, Zhenping; Snavely, Marshall; Ludwig, Dale L

    2015-01-01

    Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor – type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique “capture-for-degradation” mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions. PMID:26073904

  11. A bi-functional antibody-receptor domain fusion protein simultaneously targeting IGF-IR and VEGF for degradation.

    PubMed

    Shen, Yang; Zeng, Lin; Novosyadlyy, Ruslan; Forest, Amelie; Zhu, Aiping; Korytko, Andrew; Zhang, Haifan; Eastman, Scott W; Topper, Michael; Hindi, Sagit; Covino, Nicole; Persaud, Kris; Kang, Yun; Burtrum, Douglas; Surguladze, David; Prewett, Marie; Chintharlapalli, Sudhakar; Wroblewski, Victor J; Shen, Juqun; Balderes, Paul; Zhu, Zhenping; Snavely, Marshall; Ludwig, Dale L

    2015-01-01

    Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor - type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique "capture-for-degradation" mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.

  12. Human CENP-A contains a histone H3 related histone fold domain that is required for targeting to the centromere

    PubMed Central

    1994-01-01

    Centromeres are the differentiated chromosomal domains that specify the mitotic behavior of chromosomes. To examine the molecular basis for the specification of centromeric chromatin, we have cloned a human cDNA that encodes the 17-kD histone-like centromere antigen, CENP-A. Two domains are evident in the 140 aa CENP-A polypeptide: a unique NH2- terminal domain and a 93-amino acid COOH-terminal domain that shares 62% identity with nucleosomal core protein, histone H3. An epitope tagged derivative of CENP-A was faithfully targeted to centromeres when expressed in a variety of animal cells and this targeting activity was shown to reside in the histone-like COOH-terminal domain of CENP-A. These data clearly indicate that the assembly of centromeres is driven, at least in part, by the incorporation of a novel core histone into centromeric chromatin. PMID:7962047

  13. AlzPlatform: An Alzheimer’s Disease Domain-Specific Chemogenomics Knowledgebase for Polypharmacology and Target Identification Research

    PubMed Central

    2015-01-01

    Alzheimer’s disease (AD) is one of the most complicated progressive neurodegeneration diseases that involve many genes, proteins, and their complex interactions. No effective medicines or treatments are available yet to stop or reverse the progression of the disease due to its polygenic nature. To facilitate discovery of new AD drugs and better understand the AD neurosignaling pathways involved, we have constructed an Alzheimer’s disease domain-specific chemogenomics knowledgebase, AlzPlatform (www.cbligand.org/AD/) with cloud computing and sourcing functions. AlzPlatform is implemented with powerful computational algorithms, including our established TargetHunter, HTDocking, and BBB Predictor for target identification and polypharmacology analysis for AD research. The platform has assembled various AD-related chemogenomics data records, including 928 genes and 320 proteins related to AD, 194 AD drugs approved or in clinical trials, and 405 188 chemicals associated with 1 023 137 records of reported bioactivities from 38 284 corresponding bioassays and 10 050 references. Furthermore, we have demonstrated the application of the AlzPlatform in three case studies for identification of multitargets and polypharmacology analysis of FDA-approved drugs and also for screening and prediction of new AD active small chemical molecules and potential novel AD drug targets by our established TargetHunter and/or HTDocking programs. The predictions were confirmed by reported bioactivity data and our in vitro experimental validation. Overall, AlzPlatform will enrich our knowledge for AD target identification, drug discovery, and polypharmacology analyses and, also, facilitate the chemogenomics data sharing and information exchange/communications in aid of new anti-AD drug discovery and development. PMID:24597646

  14. Targeting a novel domain in podoplanin for inhibiting platelet-mediated tumor metastasis

    PubMed Central

    Sekiguchi, Takaya; Takemoto, Ai; Takagi, Satoshi; Takatori, Kazuki; Sato, Shigeo; Takami, Miho; Fujita, Naoya

    2016-01-01

    Podoplanin/Aggrus is a sialoglycoprotein expressed in various cancers. We previously identified podoplanin as a key factor in tumor-induced platelet aggregation. Podoplanin-mediated platelet aggregation enhances tumor growth and metastasis by secreting growth factors and by forming tumor emboli in the microvasculature. Thus, precise analysis of the mechanisms of podoplanin-mediated platelet aggregation is critical for developing anti-tumor therapies. Here we report the discovery of a novel platelet aggregation-inducing domain, PLAG4 (81-EDLPT-85). PLAG4 has high homology to the previously reported PLAG3 and contributes to the binding of its platelet receptor CLEC-2. Mutant analyses indicated that PLAG4 exhibits a predominant platelet-aggregating function relative to PLAG3 and that conserved Glu81/Asp82/Thr85 residues in PLAG4 are indispensable for CLEC-2 binding. By establishing anti-PLAG4-neutralizing monoclonal antibodies, we confirmed its role in CLEC-2 binding, platelet aggregation, and tumor emboli formation. Our results suggest the requirement of simultaneous inhibition of PLAG3/4 for complete suppression of podoplanin-mediated tumor growth and metastasis. PMID:26684030

  15. High-Throughput, Protein-Targeted Biomolecular Detection Using Frequency-Domain Faraday Rotation Spectroscopy.

    PubMed

    Murdock, Richard J; Putnam, Shawn A; Das, Soumen; Gupta, Ankur; Chase, Elyse D Z; Seal, Sudipta

    2017-01-16

    A clinically relevant magneto-optical technique (fd-FRS, frequency-domain Faraday rotation spectroscopy) for characterizing proteins using antibody-functionalized magnetic nanoparticles (MNPs) is demonstrated. This technique distinguishes between the Faraday rotation of the solvent, iron oxide core, and functionalization layers of polyethylene glycol polymers (spacer) and model antibody-antigen complexes (anti-BSA/BSA, bovine serum albumin). A detection sensitivity of ≈10 pg mL(-1) and broad detection range of 10 pg mL(-1) ≲ cBSA ≲ 100 µg mL(-1) are observed. Combining this technique with predictive analyte binding models quantifies (within an order of magnitude) the number of active binding sites on functionalized MNPs. Comparative enzyme-linked immunosorbent assay (ELISA) studies are conducted, reproducing the manufacturer advertised BSA ELISA detection limits from 1 ng mL(-1) ≲ cBSA ≲ 500 ng mL(-1) . In addition to the increased sensitivity, broader detection range, and similar specificity, fd-FRS can be conducted in less than ≈30 min, compared to ≈4 h with ELISA. Thus, fd-FRS is shown to be a sensitive optical technique with potential to become an efficient diagnostic in the chemical and biomolecular sciences.

  16. Targeted inhibition of disheveled PDZ domain via NSC668036 depresses fibrotic process

    SciTech Connect

    Wang, Cong; Dai, Jinghong; Sun, Zhaorui; Shi, Chaowen; Cao, Honghui; and others

    2015-02-01

    In this study, we determined the effects of transforming growth factor-beta (TGF-β) and Wnt/β-catenin signaling on myofibroblast differentiation of NIH/3T3 fibroblasts in vitro and evaluated the therapeutic efficacy of NSC668036 in bleomycin-induced pulmonary fibrosis murine model. In vitro study, NSC668036, a small organic inhibitor of the PDZ domain in Dvl, suppressed β-catenin-driven gene transcription and abolished TGF-β1-induced migration, expression of collagen I and α-smooth muscle actin (α-SMA) in fibroblasts. In vivo study, we found that NSC668036 significantly suppressed accumulation of collagen I, α-SMA, and TGF-β1 but increased the expression of CK19, Occludin and E-cadherin that can inhibit pulmonary fibrogenesis. Because fibrotic lung exhibit aberrant activation of Wnt/β-catenin signaling, these data collectively suggest that inhibition of Wnt/β-catenin signaling at the Dvl level may be an effective approach to the treatment of fibrotic lung diseases. - Highlights: • NSC668036 inhibited the proliferation and migration of NIH/3T3 fibroblasts. • NSC668036 suppressed the Wnt/β-catenin signaling pathway. • TGF-β-induced stimulation of profibrotic responses were inhibited by NSC668036. • NSC668036 can inhibit the development of bleomycin-induced pulmonary fibrosis.

  17. Targeted induction of meiotic double-strand breaks reveals chromosomal domain-dependent regulation of Spo11 and interactions among potential sites of meiotic recombination.

    PubMed

    Fukuda, Tomoyuki; Kugou, Kazuto; Sasanuma, Hiroyuki; Shibata, Takehiko; Ohta, Kunihiro

    2008-02-01

    Meiotic recombination is initiated by programmed DNA double-strand break (DSB) formation mediated by Spo11. DSBs occur with frequency in chromosomal regions called hot domains but are seldom seen in cold domains. To obtain insights into the determinants of the distribution of meiotic DSBs, we examined the effects of inducing targeted DSBs during yeast meiosis using a UAS-directed form of Spo11 (Gal4BD-Spo11) and a meiosis-specific endonuclease, VDE (PI-SceI). Gal4BD-Spo11 cleaved its target sequence (UAS) integrated in hot domains but rarely in cold domains. However, Gal4BD-Spo11 did bind to UAS and VDE efficiently cleaved its recognition sequence in either context, suggesting that a cold domain is not a region of inaccessible or uncleavable chromosome structure. Importantly, self-association of Spo11 occurred at UAS in a hot domain but not in a cold domain, raising the possibility that Spo11 remains in an inactive intermediate state in cold domains. Integration of UAS adjacent to known DSB hotspots allowed us to detect competitive interactions among hotspots for activation. Moreover, the presence of VDE-introduced DSB repressed proximal hotspot activity, implicating DSBs themselves in interactions among hotspots. Thus, potential sites for Spo11-mediated DSB are subject to domain-specific and local competitive regulations during and after DSB formation.

  18. Discoidin Domain Receptor 2 as a Potential Therapeutic Target for Development of Disease-Modifying Osteoarthritis Drugs.

    PubMed

    Manning, Lauren B; Li, Yefu; Chickmagalur, Nithya S; Li, Xiaolong; Xu, Lin

    2016-11-01

    Osteoarthritis (OA) is the most common form of arthritis disorders, but the identification of therapeutic targets to effectively prevent OA has been increasingly difficult. The goal of this investigation is to provide experimental evidence that discoidin domain receptor 2 (DDR2) may be an ideal target for the development of disease-modifying OA drugs. Ddr2 was conditionally deleted from articular cartilage of adult mouse knee joints. Aggrecan-CreERT2;floxed Ddr2 mice, which were generated by crossing Aggrecan-CreERT2 mice with floxed Ddr2 mice, then received tamoxifen injections at the age of 8 weeks. The mice were then subjected to destabilization of the medial meniscus (DMM) surgery. At 8 and 16 weeks after DMM, mice were euthanized for the collection of knee joints. In a separate experiment, Aggrecan-CreERT2;floxed Ddr2 mice were subjected to DMM at the age of 10 weeks. The mice then received tamoxifen injections at 8 weeks after DMM. The mice were euthanized for the collection of knee joints at 16 weeks after DMM. The progressive process of articular cartilage degeneration was significantly delayed in the knee joints of Ddr2-deficient mice in comparison to their control littermates. Articular cartilage damage in the knee joints of the mice was associated with increased expression profiles of both Ddr2 and matrix metalloproteinase 13. These findings suggest that DDR2 may be an ideal target for the development of disease-modifying OA drugs.

  19. A bivalent tarantula toxin activates the capsaicin receptor, TRPV1, by targeting the outer pore domain.

    PubMed

    Bohlen, Christopher J; Priel, Avi; Zhou, Sharleen; King, David; Siemens, Jan; Julius, David

    2010-05-28

    Toxins have evolved to target regions of membrane ion channels that underlie ligand binding, gating, or ion permeation, and have thus served as invaluable tools for probing channel structure and function. Here, we describe a peptide toxin from the Earth Tiger tarantula that selectively and irreversibly activates the capsaicin- and heat-sensitive channel, TRPV1. This high-avidity interaction derives from a unique tandem repeat structure of the toxin that endows it with an antibody-like bivalency. The "double-knot" toxin traps TRPV1 in the open state by interacting with residues in the presumptive pore-forming region of the channel, highlighting the importance of conformational changes in the outer pore region of TRP channels during activation.

  20. Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration.

    PubMed

    Ibrahim, Hisham R; Imazato, Kenta; Ono, Hajime

    2011-09-28

    Human milk lysozyme is thought to be a key defense factor in protecting the gastrointestinal tract of newborns against bacterial infection. Recently, evidence was found that pepsin, under conditions relevant to the newborn stomach, cleaves chicken lysozyme (cLZ) at specific loops to generate five antimicrobial peptide motifs. This study explores the antimicrobial role of the corresponding peptides of human lysozyme (hLZ), the actual protein in breast milk. Five peptide motifs of hLZ, one helix-loop-helix (HLH), its two helices (H1 and H2), and two helix-sheet motifs, H2-β-strands 1-2 (H2-S12) or H2-β-strands 1-3 (H2-S13), were synthesized and examined for antimicrobial action. The five peptides of hLZ exhibit microbicidal activity to various degrees against several bacterial strains. The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria and the fungus Candida albicans . Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. This finding is the first to describe that hLZ possesses multiple antimicrobial peptide motifs within its N-terminal domain, providing insight into new classes of antibiotic peptides with potential use in the treatment of infectious diseases.

  1. Targeting of Discoidin Domain Receptor 2 (DDR2) Prevents Myofibroblast Activation and Neovessel Formation During Pulmonary Fibrosis.

    PubMed

    Zhao, Hu; Bian, Huan; Bu, Xin; Zhang, Shuya; Zhang, Pan; Yu, Jiangtian; Lai, Xiaofeng; Li, Di; Zhu, Chuchao; Yao, Libo; Su, Jin

    2016-10-01

    Idiopathic pulmonary fibrosis (IPF) is a lethal human disease with short survival time and few treatment options. Herein, we demonstrated that discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase that predominantly transduces signals from fibrillar collagens, plays a critical role in the induction of fibrosis and angiogenesis in the lung. In vitro cell studies showed that DDR2 can synergize the actions of both transforming growth factor (TGF)-β and fibrillar collagen to stimulate lung fibroblasts to undergo myofibroblastic changes and vascular endothelial growth factor (VEGF) expression. In addition, we confirmed that late treatment of the injured mice with specific siRNA against DDR2 or its kinase inhibitor exhibited therapeutic efficacy against lung fibrosis. Thus, this study not only elucidated novel mechanisms by which DDR2 controls the development of pulmonary fibrosis, but also provided candidate target for the intervention of this stubborn disease.

  2. Effect of single-chain antibody targeting of the ligand-binding domain in the anaplastic lymphoma kinase receptor

    PubMed Central

    Stylianou, DC; Auf der Maur, A; Kodack, DP; Henke, RT; Hohn, S; Toretsky, JA; Riegel, AT; Wellstein, A

    2013-01-01

    The tyrosine kinase receptor anaplastic lymphoma kinase (ALK) and its ligand, the growth factor pleiotrophin (PTN), are highly expressed during the development of the nervous system and have been implicated in the malignant progression of different tumor types. Here, we describe human single-chain variable fragment (scFv) antibodies that target the ligand-binding domain (LBD) in ALK and show the effect in vitro and in vivo. The ALK LBD was used as a bait in a yeast two-hybdrid system to select human scFv from a library with randomized complementarity-determining region 3 domains. Surface plasmon resonance showed high-affinity binding of the selected scFv. The anti-ALK scFv competed for binding of PTN to ALK in intact cells and inhibited PTN-dependent signal transduction through endogenous ALK. Invasion of an intact endothelial cell monolayer by U87MG human glioblastoma cells was inhibited by the anti-ALK scFv. In addition, the growth of established tumor xenografts in mice was reversed after the induction of the conditional expression of the anti-ALK scFv. In archival malignant brain tumors expression levels of ALK and PTN were found elevated and appear correlated with poor patient survival. This suggests a rate-limiting function of the PTN/ALK interaction that may be exploited therapeutically. PMID:19633684

  3. Clustering of mammalian Hox genes with other H3K27me3 targets within an active nuclear domain.

    PubMed

    Vieux-Rochas, Maxence; Fabre, Pierre J; Leleu, Marion; Duboule, Denis; Noordermeer, Daan

    2015-04-14

    Embryogenesis requires the precise activation and repression of many transcriptional regulators. The Polycomb group proteins and the associated H3K27me3 histone mark are essential to maintain the inactive state of many of these genes. Mammalian Hox genes are targets of Polycomb proteins and form local 3D clusters centered on the H3K27me3 mark. More distal contacts have also been described, yet their selectivity, dynamics, and relation to other layers of chromatin organization remained elusive. We report that repressed Hox genes form mutual intra- and interchromosomal interactions with other genes located in strong domains labeled by H3K27me3. These interactions occur in a central and active nuclear environment that consists of the HiC compartment A, away from peripheral lamina-associated domains. Interactions are independent of nearby H3K27me3-marked loci and determined by chromosomal distance and cell-type-specific scaling factors, thus inducing a moderate reorganization during embryogenesis. These results provide a simplified view of nuclear organization whereby Polycomb proteins may have evolved to repress genes located in gene-dense regions whose position is restricted to central, active, nuclear environments.

  4. Ig-like domain 6 of VCAM-1 is a potential therapeutic target in TNFα-induced angiogenesis

    PubMed Central

    Kim, Taek-Keun; Park, Chang Sik; Na, Hee-Jun; Lee, Kangseung; Yoon, Aerin; Chung, Junho; Lee, Sukmook

    2017-01-01

    Tumor necrosis factor alpha (TNFα)-induced angiogenesis plays important roles in the progression of various diseases, including cancer, wet age-related macular degeneration, and rheumatoid arthritis. However, the relevance and role of vascular cell adhesion molecule-1 (VCAM-1) in angiogenesis have not yet been clearly elucidated. In this study, VCAM-1 knockdown shows VCAM-1 involvement in TNFα-induced angiogenesis. Through competitive blocking experiments with VCAM-1 Ig-like domain 6 (VCAM-1-D6) protein, we identified VCAM-1-D6 as a key domain regulating TNFα-induced vascular tube formation. We demonstrated that a monoclonal antibody specific to VCAM-1-D6 suppressed TNFα-induced endothelial cell migration and tube formation and TNFα-induced vessel sprouting in rat aortas. We also found that the antibody insignificantly affected endothelial cell viability, morphology and activation. Finally, the antibody specifically blocked VCAM-1-mediated cell–cell contacts by directly inhibiting VCAM-1-D6-mediated interaction between VCAM-1 molecules. These findings suggest that VCAM-1-D6 may be a potential novel therapeutic target in TNFα-induced angiogenesis and that antibody-based modulation of VCAM-1-D6 may be an effective strategy to suppress TNFα-induced angiogenesis. PMID:28209985

  5. Ig-like domain 6 of VCAM-1 is a potential therapeutic target in TNFα-induced angiogenesis.

    PubMed

    Kim, Taek-Keun; Park, Chang Sik; Na, Hee-Jun; Lee, Kangseung; Yoon, Aerin; Chung, Junho; Lee, Sukmook

    2017-02-17

    Tumor necrosis factor alpha (TNFα)-induced angiogenesis plays important roles in the progression of various diseases, including cancer, wet age-related macular degeneration, and rheumatoid arthritis. However, the relevance and role of vascular cell adhesion molecule-1 (VCAM-1) in angiogenesis have not yet been clearly elucidated. In this study, VCAM-1 knockdown shows VCAM-1 involvement in TNFα-induced angiogenesis. Through competitive blocking experiments with VCAM-1 Ig-like domain 6 (VCAM-1-D6) protein, we identified VCAM-1-D6 as a key domain regulating TNFα-induced vascular tube formation. We demonstrated that a monoclonal antibody specific to VCAM-1-D6 suppressed TNFα-induced endothelial cell migration and tube formation and TNFα-induced vessel sprouting in rat aortas. We also found that the antibody insignificantly affected endothelial cell viability, morphology and activation. Finally, the antibody specifically blocked VCAM-1-mediated cell-cell contacts by directly inhibiting VCAM-1-D6-mediated interaction between VCAM-1 molecules. These findings suggest that VCAM-1-D6 may be a potential novel therapeutic target in TNFα-induced angiogenesis and that antibody-based modulation of VCAM-1-D6 may be an effective strategy to suppress TNFα-induced angiogenesis.

  6. Constructs of human neuropathy target esterase catalytic domain containing mutations related to motor neuron disease have altered enzymatic properties.

    PubMed

    Hein, Nichole D; Stuckey, Jeanne A; Rainier, Shirley R; Fink, John K; Richardson, Rudy J

    2010-07-01

    Neuropathy target esterase (NTE) is a phospholipase/lysophospholipase associated with organophosphorus (OP) compound-induced delayed neurotoxicity (OPIDN). Distal degeneration of motor axons occurs in both OPIDN and the hereditary spastic paraplegias (HSPs). Recently, mutations within the esterase domain of NTE were identified in patients with a novel type of HSP (SPG39) designated NTE-related motor neuron disease (NTE-MND). Two of these mutations, arginine 890 to histidine (R890H) and methionine 1012 to valine (M1012V), were created in human recombinant NTE catalytic domain (NEST) to measure possible changes in catalytic properties. These mutated enzymes had decreased specific activities for hydrolysis of the artificial substrate, phenyl valerate. In addition, the M1012V mutant exhibited a reduced bimolecular rate constant of inhibition (k(i)) for all three inhibitors tested: mipafox, diisopropylphosphorofluoridate, and chlorpyrifos oxon. Finally, while both mutated enzymes inhibited by OP compounds exhibited altered time-dependent loss of their ability to be reactivated by nucleophiles (aging), more pronounced effects were seen with the M1012V mutant. Taken together, the results from specific activity, inhibition, and aging experiments suggest that the mutations found in association with NTE-MND have functional correlates in altered enzymological properties of NTE.

  7. Direct inhibition of NF-κB activation by peptide targeting the NOA ubiquitin binding domain of NEMO.

    PubMed

    Chiaravalli, Jeanne; Fontan, Elisabeth; Fsihi, Hafida; Coic, Yves-Marie; Baleux, Françoise; Véron, Michel; Agou, Fabrice

    2011-11-01

    Aberrant and constitutive NF-κB activation are frequently reported in numerous tumor types, making its inhibition an attractive target for the treatment of certain cancers. NEMO (NF-κB essential modulator) is the crucial component of the canonical NF-κB pathway that mediates IκB kinase (IKK) complex activation. IKK activation resides in the ability of the C-terminal domain of NEMO to properly dimerize and interact with linear and K63-linked polyubiquitin chains. Here, we have identified a new NEMO peptide inhibitor, termed UBI (ubiquitin binding inhibitor) that derives from the NOA/NUB/UBAN ubiquitin binding site located in the CC2-LZ domain of NEMO. UBI specifically inhibits the NF-κB pathway at the IKK level in different cell types stimulated by a variety of NF-κB signals. Circular dichroïsm and fluorescence studies showed that UBI exhibits an increased α-helix character and direct, good-affinity binding to the NOA-LZ region of NEMO. We also showed that UBI targets NEMO in cells but its mode of inhibition is completely different from the previously reported LZ peptide (herein denoted NOA-LZ). UBI does not promote dissociation of NEMO subunits in cells but impairs the interaction between the NOA UBD of NEMO and polyubiquitin chains. Importantly, we showed that UBI efficiently competes with the in vitro binding of K63-linked chains, but not with linear chains. The identification of this new NEMO inhibitor emphasizes the important contribution of K63-linked chains for IKK activation in NF-κB signaling and would provide a new tool for studying the complex role of NF-κB in inflammation and cancer.

  8. Inhibition of HIV-1 Maturation via Small Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein.

    PubMed

    Wang, Weifeng; Zhou, Jing; Halambage, Upul D; Jurado, Kellie A; Jamin, Augusta V; Wang, Yujie; Engelman, Alan N; Aiken, Christopher

    2017-02-15

    The HIV-1 capsid protein is an attractive therapeutic target owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsid to HIV-1 infectivity. To date, small molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, BI compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor Bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant crosslinks in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle.IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here we show that one such compound, Compound 1, interferes with assembly of the conical viral capsid during virion maturation, and results in perturbations at a specific protein-protein interface in the capsid lattice. We also identify and characterize a

  9. C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA

    PubMed Central

    Santiago-Frangos, Andrew; Kavita, Kumari; Schu, Daniel J.; Gottesman, Susan

    2016-01-01

    The bacterial Sm protein and RNA chaperone Hfq stabilizes small noncoding RNAs (sRNAs) and facilitates their annealing to mRNA targets involved in stress tolerance and virulence. Although an arginine patch on the Sm core is needed for Hfq’s RNA chaperone activity, the function of Hfq’s intrinsically disordered C-terminal domain (CTD) has remained unclear. Here, we use stopped flow spectroscopy to show that the CTD of Escherichia coli Hfq is not needed to accelerate RNA base pairing but is required for the release of dsRNA. The Hfq CTD also mediates competition between sRNAs, offering a kinetic advantage to sRNAs that contact both the proximal and distal faces of the Hfq hexamer. The change in sRNA hierarchy caused by deletion of the Hfq CTD in E. coli alters the sRNA accumulation and the kinetics of sRNA regulation in vivo. We propose that the Hfq CTD displaces sRNAs and annealed sRNA⋅mRNA complexes from the Sm core, enabling Hfq to chaperone sRNA–mRNA interactions and rapidly cycle between competing targets in the cell. PMID:27681631

  10. Discovery of small-molecule inhibitors selectively targeting the DNA-binding domain of the human androgen receptor.

    PubMed

    Li, Huifang; Ban, Fuqiang; Dalal, Kush; Leblanc, Eric; Frewin, Kate; Ma, Dennis; Adomat, Hans; Rennie, Paul S; Cherkasov, Artem

    2014-08-14

    The human androgen receptor (AR) is considered as a master regulator in the development and progression of prostate cancer (PCa). As resistance to clinically used anti-AR drugs remains a major challenge for the treatment of advanced PCa, there is a pressing need for new anti-AR therapeutic avenues. In this study, we identified a binding site on the DNA binding domain (DBD) of the receptor and utilized virtual screening to discover a set of micromolar hits for the target. Through further exploration of the most potent hit (1), a structural analogue (6) was identified demonstrating 10-fold improved anti-AR potency. Further optimization resulted in a more potent synthetic analogue (25) with anti-AR potency comparable to a newly FDA-approved drug Enzalutamide. Site-directed mutagenesis demonstrated that the developed inhibitors do interact with the intended target site. Importantly, the AR DBD inhibitors could effectively inhibit the growth of Enzalutamide-resistant cells as well as block the transcriptional activity of constitutively active AR splice variants, such as V7.

  11. Imaging site-specific peptide-targeting in tumor tissues using spectral-domain optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Ma, Lixin; Zhang, Miao; Yu, Ping

    2011-03-01

    We report imaging studies on site-specific peptide-targeting in tumor tissues using newly developed optical peptide probes and spectral-domain optical coherence tomography (SD-OCT). The system used two broadband superluminescent light emission diodes with different central wavelengths. An electro-optic modulation in the reference beam was used to get full-range deep imaging inside tumor tissues. The optical probes were based on Bombesin (BBN) that is a fourteen amino acid peptide. BBN has high binding affinity to gastrin-releasing peptide (GRP) receptors overexpressed on several human cancer cell lines. Fluorescence BBN probes were developed by conjugating the last eight residues of BBN, -Q-W-A-V-G-H-L-M-(NH2), with Alexa Flour 680 or Alexa Fluor 750 dye molecules via amino acid linker -G-G-G. The SD-OCT imaging can identify normal tissue and tumor tissue through the difference in scattering coefficient, and trace the BBN conjugate probes through the absorption of the dye molecules using the twowavelength algorithm. We performed the specific uptake and receptor-blocking experiments of the optical BBN probes in severely compromised immunodeficient mouse model bearing human PC-3 prostate tumor xenografts. Tumor and muscle tissues were collected and used for SD-OCT imaging. The SD-OCT images showed fluorescence traces of the BBN probes in the peptide-targeted tumor tissues. Our results demonstrated that SD-OCT is a potential tool for preclinical and clinical early cancer detection.

  12. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

    PubMed Central

    Gust, Bertolt; Challis, Greg L.; Fowler, Kay; Kieser, Tobias; Chater, Keith F.

    2003-01-01

    Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and λ-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriTRK2 for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriTRK2 to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate. PMID:12563033

  13. Structural and functional analyses of minimal phosphopeptides targeting the polo-box domain of polo-like kinase 1

    SciTech Connect

    Yun, Sang-Moon; Moulaei, Tinoush; Lim, Dan; Bang, Jeong K.; Park, Jung-Eun; Shenoy, Shilpa R.; Liu, Fa; Kang, Young H.; Liao, Chenzhong; Soung, Nak-Kyun; Lee, Sunhee; Yoon, Do-Young; Lim, Yoongho; Lee, Dong-Hee; Otaka, Akira; Appella, Ettore; McMahon, James B.; Nicklaus, Marc C.; Burke, Jr., Terrence R.; Yaffe, Michael B.; Wlodawer, Alexander; Lee, Kyung S.

    2009-09-14

    Polo-like kinase-1 (Plk1) has a pivotal role in cell proliferation and is considered a potential target for anticancer therapy. The noncatalytic polo-box domain (PBD) of Plk1 forms a phosphoepitope binding module for protein-protein interaction. Here, we report the identification of minimal phosphopeptides that specifically interact with the PBD of human PLK1, but not those of the closely related PLK2 and PLK3. Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction. Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death. The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

  14. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin.

    PubMed

    Gust, Bertolt; Challis, Greg L; Fowler, Kay; Kieser, Tobias; Chater, Keith F

    2003-02-18

    Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and lambda-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriT(RK2) for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT(RK2) to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.

  15. The GRP1 PH domain, like the AKT1 PH domain, possesses a sentry glutamate residue essential for specific targeting to plasma membrane PI(3,4,5)P(3).

    PubMed

    Pilling, Carissa; Landgraf, Kyle E; Falke, Joseph J

    2011-11-15

    During the appearance of the signaling lipid PI(3,4,5)P(3), an important subset of pleckstrin homology (PH) domains target signaling proteins to the plasma membrane. To ensure proper pathway regulation, such PI(3,4,5)P(3)-specific PH domains must exclude the more prevalant, constitutive plasma membrane lipid PI(4,5)P(2) and bind the rare PI(3,4,5)P(3) target lipid with sufficiently high affinity. Our previous study of the E17K mutant of the protein kinase B (AKT1) PH domain, together with evidence from Carpten et al. [Carpten, J. D., et al. (2007) Nature 448, 439-444], revealed that the native AKT1 E17 residue serves as a sentry glutamate that excludes PI(4,5)P(2), thereby playing an essential role in specific PI(3,4,5)P(3) targeting [Landgraf, K. E., et al. (2008) Biochemistry 47, 12260-12269]. The sentry glutamate hypothesis proposes that an analogous sentry glutamate residue is a widespread feature of PI(3,4,5)P(3)-specific PH domains, and that charge reversal mutation at the sentry glutamate position will yield both increased PI(4,5)P(2) affinity and constitutive plasma membrane targeting. To test this hypothesis, we investigated the E345 residue, a putative sentry glutamate, of the general receptor for phosphoinositides 1 (GRP1) PH domain. The results show that incorporation of the E345K charge reversal mutation into the GRP1 PH domain enhances PI(4,5)P(2) affinity 8-fold and yields constitutive plasma membrane targeting in cells, reminiscent of the effects of the E17K mutation in the AKT1 PH domain. Hydrolysis of plasma membrane PI(4,5)P(2) releases the E345K GRP1 PH domain into the cytoplasm, and the efficiency of this release increases when Arf6 binding is disrupted. Overall, the findings provide strong support for the sentry glutamate hypothesis and suggest that the GRP1 E345K mutation will be linked to changes in cell physiology and human pathologies, as demonstrated for AKT1 E17K [Carpten, J. D., et al. (2007) Nature 448, 439-444; Lindhurst, M. J., et al

  16. Arabidopsis myosin XI sub-domains homologous to the yeast myo2p organelle inheritance sub-domain target subcellular structures in plant cells

    PubMed Central

    Sattarzadeh, Amirali; Schmelzer, Elmon; Hanson, Maureen R.

    2013-01-01

    Myosin XI motor proteins transport plant organelles on the actin cytoskeleton. The Arabidopsis gene family that encodes myosin XI has 13 members, 12 of which have sub-domains within the tail region that are homologous to well-characterized cargo-binding domains in the yeast myosin V myo2p. Little is presently known about the cargo-binding domains of plant myosin XIs. Prior experiments in which most or all of the tail regions of myosin XIs have been fused to yellow fluorescent protein (YFP) and transiently expressed have often not resulted in fluorescent labeling of plant organelles. We identified 42 amino-acid regions within 12 Arabidopsis myosin XIs that are homologous to the yeast myo2p tail region known to be essential for vacuole and mitochondrial inheritance. A YFP fusion of the yeast region expressed in plants did not label tonoplasts or mitochondria. We investigated whether the homologous Arabidopsis regions, termed by us the “PAL” sub-domain, could associate with subcellular structures following transient expression of fusions with YFP in Nicotiana benthamiana. Seven YFP::PAL sub-domain fusions decorated Golgi and six were localized to mitochondria. In general, the myosin XI PAL sub-domains labeled organelles whose motility had previously been observed to be affected by mutagenesis or dominant negative assays with the respective myosins. Simultaneous transient expression of the PAL sub-domains of myosin XI-H, XI-I, and XI-K resulted in inhibition of movement of mitochondria and Golgi. PMID:24187546

  17. Arsenite Targets the Zinc Finger Domains of Tet Proteins and Inhibits Tet-Mediated Oxidation of 5-Methylcytosine.

    PubMed

    Liu, Shuo; Jiang, Ji; Li, Lin; Amato, Nicholas J; Wang, Zi; Wang, Yinsheng

    2015-10-06

    Arsenic toxicity is a serious public health problem worldwide that brings more than 100 million people into the risk of arsenic exposure from groundwater and food contamination. Although there is accumulating evidence linking arsenic exposure with aberrant cytosine methylation in the global genome or at specific genomic loci, very few have investigated the impact of arsenic on the oxidation of 5-methylcytosine (5-mC) mediated by the Ten-eleven translocation (Tet) family of proteins. Owing to the high binding affinity of As(III) toward cysteine residues, we reasoned that the highly conserved C3H-type zinc fingers situated in Tet proteins may constitute potential targets for arsenic binding. Herein, we found that arsenite could bind directly to the zinc fingers of Tet proteins in vitro and in cells, and this interaction substantially impaired the catalytic efficiency of Tet proteins in oxidizing 5-mC to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC). Treatments with arsenite also led to a dose-dependent decrease in the level of 5-hmC, but not 5-mC, in DNA isolated from HEK293T cells overexpressing the catalytic domain of any of the three Tet proteins and from mouse embryonic stem cells. Together, our study unveiled, for the first time, that arsenite could alter epigenetic signaling by targeting the zinc fingers of Tet proteins and perturbing the Tet-mediated oxidation of 5-mC in vitro and in cells. Our results offer important mechanistic understanding of arsenic epigenotoxicity and carcinogenesis in mammalian systems and may lead to novel approaches for the chemoprevention of arsenic toxicity.

  18. Effects of targeted phosphorylation site mutations in the DNA-PKcs phosphorylation domain on low and high LET radiation sensitivity.

    PubMed

    Cartwright, Ian M; Bell, Justin J; Maeda, Junko; Genet, Matthew D; Romero, Ashley; Fujii, Yoshihiro; Fujimori, Akira; Kitamuta, Hisashi; Kamada, Tadashi; Chen, David J; Kato, Takamitsu A

    2015-04-01

    The present study investigated the effect of targeted mutations in the DNA-dependent protein kinase catalytic subunit and phosphorylation domains on the survival of cells in response to different qualities of ionizing radiation. Mutated Chinese hamster ovary V3 cells were exposed to 500 MeV/nucleon initial energy and 200 keV/μm monoenergetic Fe ions; 290 MeV/nucleon initial energy and average 50 keV/μm spread-out Bragg peak C ions; 70 MeV/nucleon initial energy and 1 keV/μm monoenergetic protons; and 0.663 MeV initial energy and 0.3 keV/μm Cs(137) γ radiation. The results demonstrated that sensitivity to high linear energy transfer radiation is increased when both S2056 and T2609 clusters each contain a point mutation or multiple mutations are present in either cluster, whereas the phosphoinositide 3 kinase cluster only requires a single mutation to induce the sensitized phenotype of V3 cells. Additionally, the present study demonstrated that sensitivity to DNA cross-linking damage by cisplatin only requires a single mutation in one of the three clusters and that additional point mutations do not increase cell sensitivity.

  19. Transcription factor TFIID is a direct functional target of the adenovirus E1A transcription-repression domain.

    PubMed Central

    Song, C Z; Loewenstein, P M; Toth, K; Green, M

    1995-01-01

    The 243-amino acid adenovirus E1A oncoprotein both positively and negatively modulates the expression of cellular genes involved in the regulation of cell growth. The E1A transcription repression function appears to be linked with its ability to induce cellular DNA synthesis, cell proliferation, and cell transformation, as well as to inhibit cell differentiation. The mechanism by which E1A represses the transcription of various promoters has proven enigmatic. Here we provide several lines of evidence that the "TATA-box" binding protein (TBP) component of transcription factor TFIID is a cellular target of the E1A repression function encoded within the E1A N-terminal 80 amino acids. (i) The E1A N-terminal 80 amino acids [E1A-(1-80)protein] efficiently represses basal transcription from TATA-containing core promoters in vitro. (ii) TBP reverses completely E1A repression in vitro. (iii) TBP restores transcriptional activity to E1A-(1-80) protein affinity-depleted nuclear extracts. (iv) The N-terminal repression domain of E1A interacts directly and specifically with TBP in vitro. These results may help explain how E1A represses a set of genes that lack common upstream promoter elements. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7479778

  20. Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes.

    PubMed

    Win, Joe; Morgan, William; Bos, Jorunn; Krasileva, Ksenia V; Cano, Liliana M; Chaparro-Garcia, Angela; Ammar, Randa; Staskawicz, Brian J; Kamoun, Sophien

    2007-08-01

    Oomycete plant pathogens deliver effector proteins inside host cells to modulate plant defense circuitry and to enable parasitic colonization. These effectors are defined by a conserved motif, termed RXLR (for Arg, any amino acid, Leu, Arg), that is located downstream of the signal peptide and that has been implicated in host translocation. Because the phenotypes of RXLR effectors extend to plant cells, their genes are expected to be the direct target of the evolutionary forces that drive the antagonistic interplay between pathogen and host. We used the draft genome sequences of three oomycete plant pathogens, Phytophthora sojae, Phytophthora ramorum, and Hyaloperonospora parasitica, to generate genome-wide catalogs of RXLR effector genes and determine the extent to which these genes are under positive selection. These analyses revealed that the RXLR sequence is overrepresented and positionally constrained in the secretome of Phytophthora relative to other eukaryotes. The three examined plant pathogenic oomycetes carry complex and diverse sets of RXLR effector genes that have undergone relatively rapid birth and death evolution. We obtained robust evidence of positive selection in more than two-thirds of the examined paralog families of RXLR effectors. Positive selection has acted for the most part on the C-terminal region, consistent with the view that RXLR effectors are modular, with the N terminus involved in secretion and host translocation and the C-terminal domain dedicated to modulating host defenses inside plant cells.

  1. Incorporation of Peptides Targeting EGFR and FGFR1 into the Adenoviral Fiber Knob Domain and Their Evaluation as Targeted Cancer Therapies

    PubMed Central

    Uusi-Kerttula, Hanni; Legut, Mateusz; Davies, James; Jones, Rachel; Hudson, Emma; Hanna, Louise; Stanton, Richard J.; Chester, John D.

    2015-01-01

    Abstract Oncolytic virotherapies based on adenovirus 5 (Ad5) hold promise as adjunctive cancer therapies; however, their efficacy when delivered systemically is hampered by poor target cell specificity and preexisting anti-Ad5 immunity. Ovarian cancer represents a promising target for virotherapy, since the virus can be delivered locally into the peritoneal cavity. Both epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1) are overexpressed in the majority of human tumors, including ovarian cancer. To generate adenoviral vectors with improved tumor specificity, we generated a panel of Ad5 vectors with altered tropism for EGFR and FGFR, rather than the natural Ad5 receptor, hCAR. We have included mutations within AB loop of the viral fiber knob (KO1 mutation) to preclude interaction with hCAR, combined with insertions in the HI loop to incorporate peptides that bind either EGFR (peptide YHWYGYTPQNVI, GE11) or FGFR1 (peptides MQLPLAT, M*, and LSPPRYP, LS). Viruses were produced to high titers, and the integrity of the fiber protein was validated by Western blotting. The KO1 mutation efficiently ablated hCAR interactions, and significantly increased transduction was observed in hCARlow/EGFRhigh cell lines using Ad5.GE11, while transduction levels using Ad5.M* or Ad5.LS were not increased. In the presence of physiological concentrations of human blood clotting factor X (hFX), significantly increased levels of transduction via the hFX-mediated pathway were observed in cell lines, but not in primary tumor cells derived from epithelial ovarian cancer (EOC) ascites samples. Ad5-mediated transduction of EOC cells was completely abolished by the presence of 2.5% serum from patients, while, surprisingly, incorporation of the GE11 peptide resulted in significant evasion of neutralization in the same samples. We thus speculate that incorporation of the YHWYGYTPQNVI dodecapeptide within the fiber knob domain may provide a novel means of

  2. Incorporation of Peptides Targeting EGFR and FGFR1 into the Adenoviral Fiber Knob Domain and Their Evaluation as Targeted Cancer Therapies.

    PubMed

    Uusi-Kerttula, Hanni; Legut, Mateusz; Davies, James; Jones, Rachel; Hudson, Emma; Hanna, Louise; Stanton, Richard J; Chester, John D; Parker, Alan L

    2015-05-01

    Oncolytic virotherapies based on adenovirus 5 (Ad5) hold promise as adjunctive cancer therapies; however, their efficacy when delivered systemically is hampered by poor target cell specificity and preexisting anti-Ad5 immunity. Ovarian cancer represents a promising target for virotherapy, since the virus can be delivered locally into the peritoneal cavity. Both epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1) are overexpressed in the majority of human tumors, including ovarian cancer. To generate adenoviral vectors with improved tumor specificity, we generated a panel of Ad5 vectors with altered tropism for EGFR and FGFR, rather than the natural Ad5 receptor, hCAR. We have included mutations within AB loop of the viral fiber knob (KO1 mutation) to preclude interaction with hCAR, combined with insertions in the HI loop to incorporate peptides that bind either EGFR (peptide YHWYGYTPQNVI, GE11) or FGFR1 (peptides MQLPLAT, M*, and LSPPRYP, LS). Viruses were produced to high titers, and the integrity of the fiber protein was validated by Western blotting. The KO1 mutation efficiently ablated hCAR interactions, and significantly increased transduction was observed in hCAR(low)/EGFR(high) cell lines using Ad5.GE11, while transduction levels using Ad5.M* or Ad5.LS were not increased. In the presence of physiological concentrations of human blood clotting factor X (hFX), significantly increased levels of transduction via the hFX-mediated pathway were observed in cell lines, but not in primary tumor cells derived from epithelial ovarian cancer (EOC) ascites samples. Ad5-mediated transduction of EOC cells was completely abolished by the presence of 2.5% serum from patients, while, surprisingly, incorporation of the GE11 peptide resulted in significant evasion of neutralization in the same samples. We thus speculate that incorporation of the YHWYGYTPQNVI dodecapeptide within the fiber knob domain may provide a novel means of circumventing

  3. In vitro and in vivo activity of novel small-molecule inhibitors targeting the pleckstrin homology domain of protein kinase B/AKT.

    PubMed

    Moses, Sylvestor A; Ali, M Ahad; Zuohe, Song; Du-Cuny, Lei; Zhou, Li Li; Lemos, Robert; Ihle, Nathan; Skillman, A Geoffrey; Zhang, Shuxing; Mash, Eugene A; Powis, Garth; Meuillet, Emmanuelle J

    2009-06-15

    The phosphatidylinositol 3-kinase/AKT signaling pathway plays a critical role in activating survival and antiapoptotic pathways within cancer cells. Several studies have shown that this pathway is constitutively activated in many different cancer types. The goal of this study was to discover novel compounds that bind to the pleckstrin homology (PH) domain of AKT, thereby inhibiting AKT activation. Using proprietary docking software, 22 potential PH domain inhibitors were identified. Surface plasmon resonance spectroscopy was used to measure the binding of the compounds to the expressed PH domain of AKT followed by an in vitro activity screen in Panc-1 and MiaPaCa-2 pancreatic cancer cell lines. We identified a novel chemical scaffold in several of the compounds that binds selectively to the PH domain of AKT, inducing a decrease in AKT activation and causing apoptosis at low micromolar concentrations. Structural modifications of the scaffold led to compounds with enhanced inhibitory activity in cells. One compound, 4-dodecyl-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide, inhibited AKT and its downstream targets in cells as well as in pancreatic cancer cell xenografts in immunocompromised mice; it also exhibited good antitumor activity. In summary, a pharmacophore for PH domain inhibitors targeting AKT function was developed. Computer-aided modeling, synthesis, and testing produced novel AKT PH domain inhibitors that exhibit promising preclinical properties.

  4. Cooperation of two-domain Ca2+ channel fragments in triad targeting and restoration of excitation– contraction coupling in skeletal muscle

    PubMed Central

    Flucher, Bernhard E.; Weiss, Regina G.; Grabner, Manfred

    2002-01-01

    The specific incorporation of the skeletal muscle voltage-dependent Ca2+ channel in the triad is a prerequisite of normal excitation–contraction (EC) coupling. Sequences involved in membrane expression and in targeting of Ca2+ channels into skeletal muscle triads have been described in different regions of the α1S subunit. Here we studied the targeting properties of two-domain α1S fragments, green fluorescent protein (GFP)-I⋅II (1–670) and III⋅IV (691–1873) expressed alone or in combination in dysgenic (α1S-null) myotubes. Immunofluorescence analysis showed that GFP-I⋅II or III⋅IV expressed separately were not targeted into triads. In contrast, on coexpression the two α1S fragments were colocalized with one another and with the ryanodine receptor in the triads. Coexpression of GFP-I⋅II and III⋅IV also fully restored Ca2+ currents and depolarization-induced Ca2+ transients, despite the severed connection between the two channel halves and the absence of amino acids 671–690 from either α1S fragment. Thus, triad targeting, like the rescue of function, requires the cooperation and coassembly of the two complementary channel fragments. Transferring the C terminus of α1S to the N-terminal two-domain fragment (GFP-I⋅II⋅tail), or transferring the I–II connecting loop containing the β interaction domain to the C-terminal fragment (III⋅IV⋅βin) did not improve the targeting properties of the individually expressed two-domain channel fragments. Thus, the cooperation of GFP-I⋅II and III⋅IV in targeting cannot be explained solely by a sequential action of the β subunit by means of the I–II loop in releasing the channel from the sarcoplasmic reticulum and of the C terminus in triad targeting. PMID:12119388

  5. The PGRS Domain from PE_PGRS33 of Mycobacterium tuberculosis is Target of Humoral Immune Response in Mice and Humans.

    PubMed

    Cohen, Ingrid; Parada, Cristina; Acosta-Gío, Enrique; Espitia, Clara

    2014-01-01

    The PE_PGRS33 protein is a member of the PE family, which encompasses the PE and the PE_PGRS subfamilies. Among PE_PGRS's, this protein is one of the most studied antigens and its immunomodulatory properties are influence by both PE and PGRS domains. However, the contribution of these domains to the host immune recognition of the PE_PGRS33 protein and their potential role in latent tuberculosis infection in humans is still unknown. In this study, the immunogenic properties of the complete PE_PGRS33 protein and each domain separately were evaluated in BALB/c mice and latent tuberculosis infected (LTBI) humans. In mice, PE_PGRS33 and its domains induced similar antibody production and secretion of IFN-γ. PE_PGRS33 and the PE domain stimulated higher CD4(+) and CD8(+) T-cell proliferation compared to the PGRS domain. This demonstrated that the principal difference in the immune recognition of the domains is the higher activation of T-cell subpopulations involved in the control of tuberculosis. In humans, the secretion of IFN-γ in response to PE_PGRS33 was detected in both LTBI and in non-infected vaccinated individuals. The same was observed for antibody response, which targets epitopes located in the PGRS domain but not in the PE domain. These observations suggest that T and B cell responses to PE_PGRS33 are induced by BCG vaccination and can be maintained for many years in non-infected individuals. This also indicates that the IFN-γ response detected might not be associated with latent tuberculosis infection. These results contribute to the elucidation of the role of the PE_PGRS33 protein and its PE and PGRS domains in the immune response against Mycobacterium tuberculosis.

  6. The PGRS Domain from PE_PGRS33 of Mycobacterium tuberculosis is Target of Humoral Immune Response in Mice and Humans

    PubMed Central

    Cohen, Ingrid; Parada, Cristina; Acosta-Gío, Enrique; Espitia, Clara

    2014-01-01

    The PE_PGRS33 protein is a member of the PE family, which encompasses the PE and the PE_PGRS subfamilies. Among PE_PGRS’s, this protein is one of the most studied antigens and its immunomodulatory properties are influence by both PE and PGRS domains. However, the contribution of these domains to the host immune recognition of the PE_PGRS33 protein and their potential role in latent tuberculosis infection in humans is still unknown. In this study, the immunogenic properties of the complete PE_PGRS33 protein and each domain separately were evaluated in BALB/c mice and latent tuberculosis infected (LTBI) humans. In mice, PE_PGRS33 and its domains induced similar antibody production and secretion of IFN-γ. PE_PGRS33 and the PE domain stimulated higher CD4+ and CD8+ T-cell proliferation compared to the PGRS domain. This demonstrated that the principal difference in the immune recognition of the domains is the higher activation of T-cell subpopulations involved in the control of tuberculosis. In humans, the secretion of IFN-γ in response to PE_PGRS33 was detected in both LTBI and in non-infected vaccinated individuals. The same was observed for antibody response, which targets epitopes located in the PGRS domain but not in the PE domain. These observations suggest that T and B cell responses to PE_PGRS33 are induced by BCG vaccination and can be maintained for many years in non-infected individuals. This also indicates that the IFN-γ response detected might not be associated with latent tuberculosis infection. These results contribute to the elucidation of the role of the PE_PGRS33 protein and its PE and PGRS domains in the immune response against Mycobacterium tuberculosis. PMID:24904584

  7. Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe

    PubMed Central

    Li, John J.; Cao, Chune; Fixsen, Sarah M.; Young, Janet M.; Bando, Hisanori; Elde, Nels C.; Katsuma, Susumu; Dever, Thomas E.; Sicheri, Frank

    2015-01-01

    Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by eIF2α family kinases is a conserved mechanism to limit protein synthesis under specific stress conditions. The baculovirus-encoded protein PK2 inhibits eIF2α family kinases in vivo, thereby increasing viral fitness. However, the precise mechanism by which PK2 inhibits eIF2α kinase function remains an enigma. Here, we probed the mechanism by which PK2 inhibits the model eIF2α kinase human RNA-dependent protein kinase (PKR) as well as native insect eIF2α kinases. Although PK2 structurally mimics the C-lobe of a protein kinase domain and possesses the required docking infrastructure to bind eIF2α, we show that PK2 directly binds the kinase domain of PKR (PKRKD) but not eIF2α. The PKRKD–PK2 interaction requires a 22-residue N-terminal extension preceding the globular PK2 body that we term the “eIF2α kinase C-lobe mimic” (EKCM) domain. The functional insufficiency of the N-terminal extension of PK2 implicates a role for the adjacent EKCM domain in binding and inhibiting PKR. Using a genetic screen in yeast, we isolated PK2-activating mutations that cluster to a surface of the EKCM domain that in bona fide protein kinases forms the catalytic cleft through sandwiching interactions with a kinase N-lobe. Interaction assays revealed that PK2 associates with the N- but not the C-lobe of PKRKD. We propose an inhibitory model whereby PK2 engages the N-lobe of an eIF2α kinase domain to create a nonfunctional pseudokinase domain complex, possibly through a lobe-swapping mechanism. Finally, we show that PK2 enhances baculovirus fitness in insect hosts by targeting the endogenous insect heme-regulated inhibitor (HRI)–like eIF2α kinase. PMID:26216977

  8. The interaction between the pleckstrin homology domain of ceramide kinase and phosphatidylinositol 4,5-bisphosphate regulates the plasma membrane targeting and ceramide 1-phosphate levels

    SciTech Connect

    Kim, Tack-Joong; Mitsutake, Susumu; Igarashi, Yasuyuki . E-mail: yigarash@pharm.hokudai.ac.jp

    2006-04-07

    Ceramide kinase (CERK) converts ceramide to ceramide-1-phosphate (C1P), which has recently emerged as a new bioactive molecule capable of regulating diverse cellular functions. The N-terminus of the CERK protein encompasses a sequence motif known as a pleckstrin homology (PH) domain. Although the PH domain was previously demonstrated to be an important domain for the subcellular localization of CERK, the precise properties of this domain remained unclear. In this study, we reveal that the PH domain of CERK exhibits high affinity for phosphatidylinositol 4,5-bisphosphate (PI(4,5)P{sub 2}), among other lipids. Furthermore, in COS7 cells, GFP-fused CERK translocated rapidly from the cytoplasm to the plasma membrane in response to hyper-osmotic stress, which is known to increase the intracellular PI(4,5)P{sub 2} levels, whereas a PH domain deletion mutant did not. Additionally, in [{sup 32}P]orthophosphate-labeled COS7 cells, the translocation of CERK to the plasma membrane induced a 2.8-fold increase in C1P levels. The study presented here provides insight into the crucial role of the CERK-PH domain in plasma membrane targeting, through its binding to PI(4,5)P{sub 2}, and subsequent induction of C1P production in the vicinity of the membrane.

  9. The matricellular protein CCN1 controls retinal angiogenesis by targeting VEGF, Src homology 2 domain phosphatase-1 and Notch signaling.

    PubMed

    Chintala, Hemabindu; Krupska, Izabela; Yan, Lulu; Lau, Lester; Grant, Maria; Chaqour, Brahim

    2015-07-01

    Physiological angiogenesis depends on the highly coordinated actions of multiple angiogenic regulators. CCN1 is a secreted cysteine-rich and integrin-binding matricellular protein required for proper cardiovascular development. However, our understanding of the cellular origins and activities of this molecule is incomplete. Here, we show that CCN1 is predominantly expressed in angiogenic endothelial cells (ECs) at the leading front of actively growing vessels in the mouse retina. Endothelial deletion of CCN1 in mice using a Cre-Lox system is associated with EC hyperplasia, loss of pericyte coverage and formation of dense retinal vascular networks lacking the normal hierarchical arrangement of arterioles, capillaries and venules. CCN1 is a product of an immediate-early gene that is transcriptionally induced in ECs in response to stimulation by vascular endothelial growth factor (VEGF). We found that CCN1 activity is integrated with VEGF receptor 2 (VEGF-R2) activation and downstream signaling pathways required for tubular network formation. CCN1-integrin binding increased the expression of and association between Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) and VEGF-R2, which leads to rapid dephosphorylation of VEGF-R2 tyrosine, thus preventing EC hyperproliferation. Predictably, CCN1 further brings receptors/signaling molecules into proximity that are otherwise spatially separated. Furthermore, CCN1 induces integrin-dependent Notch activation in cultured ECs, and its targeted gene inactivation in vivo alters Notch-dependent vascular specification and remodeling, suggesting that functional levels of Notch signaling requires CCN1 activity. These data highlight novel functions of CCN1 as a naturally optimized molecule, fine-controlling key processes in physiological angiogenesis and safeguarding against aberrant angiogenic responses.

  10. The auto-inhibitory state of Rho guanine nucleotide exchange factor ARHGEF5/TIM can be relieved by targeting its SH3 domain with rationally designed peptide aptamers.

    PubMed

    He, Ping; Tan, De-Li; Liu, Hong-Xiang; Lv, Feng-Lin; Wu, Wei

    2015-04-01

    The short isoform of Rho guanine nucleotide exchange factor ARHGEF5 is known as TIM, which plays diverse roles in, for example, tumorigenesis, neuronal development and Src-induced podosome formation through the activation of its substrates, the Rho family of GTPases. The activation is auto-inhibited by a putative helix N-terminal to the DH domain of TIM, which is stabilized by the intramolecular interaction of C-terminal SH3 domain with a poly-proline sequence between the putative helix and the DH domain. In this study, we systematically investigated the structural basis, energetic landscape and biological implication underlying TIM auto-inhibition by using atomistic molecular dynamics simulations and binding free energy analysis. The computational study revealed that the binding of SH3 domain to poly-proline sequence is the prerequisite for the stabilization of TIM auto-inhibition. Thus, it is suggested that targeting SH3 domain with competitors of the poly-proline sequence would be a promising strategy to relieve the auto-inhibitory state of TIM. In this consideration, we rationally designed a number of peptide aptamers for competitively inhibiting the SH3 domain based on modeled TIM structure and computationally generated data. Peptide binding test and guanine nucleotide exchange analysis solidified that these designed peptides can both bind to the SH3 domain potently and activate TIM-catalyzed RhoA exchange reaction effectively. Interestingly, a positive correlation between the peptide affinity and induced exchange activity was observed. In addition, separate mutation of three conserved residues Pro49, Pro52 and Lys54 - they are required for peptide recognition by SH3 domain -- in a designed peptide to Ala would completely abolish the capability of this peptide activating TIM. All these come together to suggest an intrinsic relationship between peptide binding to SH3 domain and the activation of TIM.

  11. Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

    SciTech Connect

    Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.; Wang, Yu-Hsiu; Slochower, David; Janmey, Paul A.; Lemmon, Mark A.

    2011-09-28

    Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.

  12. Enhancement of hERG channel activity by scFv antibody fragments targeted to the PAS domain

    PubMed Central

    Harley, Carol A.; Starek, Greg; Jones, David K.; Fernandes, Andreia S.; Robertson, Gail A.; Morais-Cabral, João H.

    2016-01-01

    The human human ether-à-go-go–related gene (hERG) potassium channel plays a critical role in the repolarization of the cardiac action potential. Changes in hERG channel function underlie long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. A striking feature of this channel and KCNH channels in general is the presence of an N-terminal Per-Arnt-Sim (PAS) domain. In other proteins, PAS domains bind ligands and modulate effector domains. However, the PAS domains of KCNH channels are orphan receptors. We have uncovered a family of positive modulators of hERG that specifically bind to the PAS domain. We generated two single-chain variable fragments (scFvs) that recognize different epitopes on the PAS domain. Both antibodies increase the rate of deactivation but have different effects on channel activation and inactivation. Importantly, we show that both antibodies, on binding to the PAS domain, increase the total amount of current that permeates the channel during a ventricular action potential and significantly reduce the action potential duration recorded in human cardiomyocytes. Overall, these molecules constitute a previously unidentified class of positive modulators and establish that allosteric modulation of hERG channel function through ligand binding to the PAS domain can be attained. PMID:27516548

  13. A synthetic peptide targeting the BH4 domain of Bcl-2 induces apoptosis in multiple myeloma and follicular lymphoma cells alone or in combination with agents targeting the BH3-binding pocket of Bcl-2.

    PubMed

    Lavik, Andrew R; Zhong, Fei; Chang, Ming-Jin; Greenberg, Edward; Choudhary, Yuvraj; Smith, Mitchell R; McColl, Karen S; Pink, John; Reu, Frederic J; Matsuyama, Shigemi; Distelhorst, Clark W

    2015-09-29

    Bcl-2 inhibits apoptosis by two distinct mechanisms but only one is targeted to treat Bcl-2-positive malignancies. In this mechanism, the BH1-3 domains of Bcl-2 form a hydrophobic pocket, binding and inhibiting pro-apoptotic proteins, including Bim. In the other mechanism, the BH4 domain mediates interaction of Bcl-2 with inositol 1,4, 5-trisphosphate receptors (IP3Rs), inhibiting pro-apoptotic Ca2+ signals. The current anti-Bcl-2 agents, ABT-263 (Navitoclax) and ABT-199 (Venetoclax), induce apoptosis by displacing pro-apoptotic proteins from the hydrophobic pocket, but do not inhibit Bcl-2-IP3R interaction. Therefore, to target this interaction we developed BIRD-2 (Bcl-2 IP3 Receptor Disruptor-2), a decoy peptide that binds to the BH4 domain, blocking Bcl-2-IP3R interaction and thus inducing Ca2+-mediated apoptosis in chronic lymphocytic leukemia, multiple myeloma, and follicular lymphoma cells, including cells resistant to ABT-263, ABT-199, or the Bruton's tyrosine kinase inhibitor Ibrutinib. Moreover, combining BIRD-2 with ABT-263 or ABT-199 enhances apoptosis induction compared to single agent treatment. Overall, these findings provide strong rationale for developing novel therapeutic agents that mimic the action of BIRD-2 in targeting the BH4 domain of Bcl-2 and disrupting Bcl-2-IP3R interaction.

  14. Pleckstrin Homology Domain of Akt Kinase: A Proof of Principle for Highly Specific and Effective Non-Enzymatic Anti-Cancer Target

    PubMed Central

    Joh, Eun-Ha; Hollenbaugh, Joseph A.; Kim, Baek; Kim, Dong-Hyun

    2012-01-01

    While pharmacological inhibition of Akt kinase has been regarded as a promising anti-cancer strategy, most of the Akt inhibitors that have been developed are enzymatic inhibitors that target the kinase active site of Akt. Another key cellular regulatory event for Akt activation is the translocation of Akt kinase to the cell membrane from the cytoplasm, which is accomplished through the pleckstrin homology (PH) domain of Akt. However, compounds specifically interacting with the PH domain of Akt to inhibit Akt activation are currently limited. Here we identified a compound, lancemaside A (LAN-A), which specifically binds to the PH domain of Akt kinase. First, our mass spectra analysis of cellular Akt kinase isolated from cells treated with LAN-A revealed that LAN-A specifically binds to the PH domain of cellular Akt kinase. Second, we observed that LAN-A inhibits the translocation of Akt kinase to the membrane and thus Akt activation, as examined by the phosphorylation of various downstream targets of Akt such as GSK3β, mTOR and BAD. Third, in a co-cultured cell model containing human lung epithelial cancer cells (A549) and normal human primary lung fibroblasts, LAN-A specifically restricts the growth of the A549 cells. LAN-A also displayed anti-proliferative effects on various human cancer cell lines. Finally, in the A549-luciferase mouse transplant model, LAN-A effectively inhibited A549 cell growth with little evident cytotoxicity. Indeed, the therapeutic index of LAN-A in this mouse model was >250, supporting that LAN-A is a potential lead compound for PH domain targeting as a safe anti-cancer Akt inhibitor. PMID:23189201

  15. A novel bispecific peptide HIV-1 fusion inhibitor targeting the N-terminal heptad repeat and fusion peptide domains in gp41.

    PubMed

    Jiang, Xifeng; Jia, Qiyan; Lu, Lu; Yu, Fei; Zheng, Jishen; Shi, Weiguo; Cai, Lifeng; Jiang, Shibo; Liu, Keliang

    2016-12-01

    HIV-1 fusion with the target cell is initiated by the insertion of the gp41 fusion peptide (FP) into the target cell membrane and the interaction between the gp41 N- and C-terminal heptad repeats (NHR and CHR), followed by the formation of the six-helix bundle (6-HB) fusion core. Therefore, both FP and NHR are important targets for HIV-1 fusion inhibitors. Here, we designed and synthesized a dual-target peptidic HIV-1 fusion inhibitor, 4HR-LBD-VIRIP, in which 4HR-LBD is able to bind to the gp41 NHR domain, while VIRIP is able to interact with gp41 FP. We found that 4HR-LBD-VIRIP is about tenfold more potent than 4HR-LBD and VIRIP in inhibiting HIV-1IIIB infection and HIV-1 envelope glycoprotein (Env)-mediated cell-cell fusion, suggesting that this dual-target HIV-1 fusion inhibitor possesses a strong synergistic antiviral effect. A biophysical analysis indicates that 4HR-LBD-VIRIP can interact with N70 peptide that contains the gp41 NHR and FP domains and binds with lipid membrane. This study provides a new approach for designing novel viral fusion inhibitors against HIV and other enveloped viruses with class I membrane fusion proteins.

  16. An antigenic domain within a catalytically active Leishmania infantum nucleoside triphosphate diphosphohydrolase (NTPDase 1) is a target of inhibitory antibodies.

    PubMed

    Maia, Ana Carolina Ribeiro Gomes; Porcino, Gabriane Nascimento; Detoni, Michelle de Lima; Emídio, Nayara Braga; Marconato, Danielle Gomes; Faria-Pinto, Priscila; Fessel, Melissa Regina; Reis, Alexandre Barbosa; Juliano, Luiz; Juliano, Maria Aparecida; Marques, Marcos José; Vasconcelos, Eveline Gomes

    2013-02-01

    We identified a shared B domain within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived from the potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived from the Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83-122). By immunoprecipitation assays and Western blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87-99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.

  17. Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor.

    PubMed

    Sahu, Sarata C; Swanson, Kurt A; Kang, Richard S; Huang, Kai; Brubaker, Kurt; Ratcliff, Kathleen; Radhakrishnan, Ishwar

    2008-02-01

    The recruitment of chromatin-modifying coregulator complexes by transcription factors to specific sites of the genome constitutes an important step in many eukaryotic transcriptional regulatory pathways. The histone deacetylase-associated Sin3 corepressor complex is recruited by a large and diverse array of transcription factors through direct interactions with the N-terminal PAH domains of Sin3. Here, we describe the solution structures of the mSin3A PAH1 domain in the apo form and when bound to SAP25, a component of the corepressor complex. Unlike the apo-mSin3A PAH2 domain, the apo-PAH1 domain is conformationally pure and is largely, but not completely, folded. Portions of the interacting segments of both mSin3A PAH1 and SAP25 undergo folding upon complex formation. SAP25 binds through an amphipathic helix to a predominantly hydrophobic cleft on the surface of PAH1. Remarkably, the orientation of the helix is reversed compared to that adopted by NRSF, a transcription factor unrelated to SAP25, upon binding to the mSin3B PAH1 domain. The reversal in helical orientations is correlated with a reversal in the underlying PAH1-interaction motifs, echoing a theme previously described for the mSin3A PAH2 domain. The definition of these so-called type I and type II PAH1-interaction motifs has allowed us to predict the precise location of these motifs within previously experimentally characterized PAH1 binders. Finally, we explore the specificity determinants of protein-protein interactions involving the PAH1 and PAH2 domains. These studies reveal that even conservative replacements of PAH2 residues with equivalent PAH1 residues are sufficient to alter the affinity and specificity of these protein-protein interactions dramatically.

  18. FERM Domain Phosphoinositide Binding Targets Merlin to the Membrane and Is Essential for Its Growth-Suppressive Function ▿

    PubMed Central

    Mani, Timmy; Hennigan, Robert F.; Foster, Lauren A.; Conrady, Deborah G.; Herr, Andrew B.; Ip, Wallace

    2011-01-01

    The neurofibromatosis type 2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of plasma membrane-actin cytoskeleton linkers. For ezrin, phosphatidylinositol 4,5-bisphosphate (PIP2) binding to the amino-terminal FERM domain is required for its conformational activation, proper subcellular localization, and function, but less is known about the role of phosphoinositide binding for merlin. Current evidence indicates that association with the membrane is important for merlin to function as a growth regulator; however, the mechanisms by which merlin localizes to the membrane are less clear. Here, we report that merlin binds phosphoinositides, including PIP2, via a conserved binding motif in its FERM domain. Abolition of FERM domain-mediated phosphoinositide binding of merlin displaces merlin from the membrane and releases it into the cytosol without altering the folding of merlin. Importantly, a merlin protein whose FERM domain cannot bind phosphoinositide is defective in growth suppression. Retargeting the mutant merlin into the membrane using a dual-acylated amino-terminal decapeptide from Fyn is sufficient to restore the growth-suppressive properties to the mutant merlin. Thus, FERM domain-mediated phosphoinositide binding and membrane association are critical for the growth-regulatory function of merlin. PMID:21402777

  19. Structure-based identification of CaMKIIα-interacting MUPP1 PDZ domains and rational design of peptide ligands to target such interaction in human fertilization.

    PubMed

    Zhang, Yi-Le; Han, Zhao-Feng; Sun, Ying-Pu

    2016-06-01

    The recognition and association between Ca(2+)/calmodulin-activated protein kinase II-α (CaMKIIα) and multi-PDZ domain protein 1 (MUPP1) plays an important role in sperm acrosome reaction and human fertilization, which is mediated by the binding of CaMKIIα's C-terminal tail to one or more PDZ domains of the scaffolding protein MUPP1. In this study, we attempt to identify the CaMKIIα-interacting MUPP1 PDZ domains and to design peptide ligands that can potently target and then competitively disrupt such interaction. Here, a synthetic biology approach was proposed to systematically characterize the structural basis, energetic property, dynamic behavior and biological implication underlying the intermolecular interactions between the C-terminal peptide of CaMKIIα and all the 13 PDZ domains of MUPP1. These domains can be grouped into four clusters in terms of their sequence, structure and physiochemical profile; different clusters appear to recognize different classes of PDZ-binding motifs. The cluster 3 includes two members, i.e. MUPP1 PDZ 5 and 11 domains, which were suggested to bind class II motif Φ-X-Φ(-COOH) of the C-terminal peptide SGAPSV(-COOH) of CaMKIIα. Subsequently, the two domains were experimentally measured as the moderate- and high-affinity binders of the peptide by using fluorescence titration (dissociation constants K d = 25.2 ± 4.6 and 0.47 ± 0.08 µM for peptide binding to PDZ 5 and 11, respectively), which was in line with theoretical prediction (binding free energies ΔG total = -7.6 and -9.2 kcal/mol for peptide binding to PDZ 5 and 11, respectively). A systematic mutation of SGAPSV(-COOH) residues suggested few favorable amino acids at different residue positions of the peptide, which were then combined to generate a number of potent peptide mutants for PDZ 11 domain. Consequently, two peptides (SIAPNV(-COOH) and SIVMNV(-COOH)) were identified to have considerably improved affinity with K d increase by ~tenfold relative to

  20. Finding a Needle in a Haystack: The Role of Electrostatics in Target Lipid Recognition by PH Domains

    PubMed Central

    Lumb, Craig N.; Sansom, Mark S. P.

    2012-01-01

    Interactions between protein domains and lipid molecules play key roles in controlling cell membrane signalling and trafficking. The pleckstrin homology (PH) domain is one of the most widespread, binding specifically to phosphatidylinositol phosphates (PIPs) in cell membranes. PH domains must locate specific PIPs in the presence of a background of approximately 20% anionic lipids within the cytoplasmic leaflet of the plasma membrane. We investigate the mechanism of such recognition via a multiscale procedure combining Brownian dynamics (BD) and molecular dynamics (MD) simulations of the GRP1 PH domain interacting with phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3). The interaction of GRP1-PH with PI(3,4,5)P3 in a zwitterionic bilayer is compared with the interaction in bilayers containing different levels of anionic ‘decoy’ lipids. BD simulations reveal both translational and orientational electrostatic steering of the PH domain towards the PI(3,4,5)P3-containing anionic bilayer surface. There is a payoff between non-PIP anionic lipids attracting the PH domain to the bilayer surface in a favourable orientation and their role as ‘decoys’, disrupting the interaction of GRP1-PH with the PI(3,4,5)P3 molecule. Significantly, approximately 20% anionic lipid in the cytoplasmic leaflet of the bilayer is nearly optimal to both enhance orientational steering and to localise GRP1-PH proximal to the surface of the membrane without sacrificing its ability to locate PI(3,4,5)P3 within the bilayer plane. Subsequent MD simulations reveal binding to PI(3,4,5)P3, forming protein-phosphate contacts comparable to those in X-ray structures. These studies demonstrate a computational framework which addresses lipid recognition within a cell membrane environment, offering a link between structural and cell biological characterisation. PMID:22844242

  1. Essential Role of the EF-hand Domain in Targeting Sperm Phospholipase Cζ to Membrane Phosphatidylinositol 4,5-Bisphosphate (PIP2)*

    PubMed Central

    Nomikos, Michail; Sanders, Jessica R.; Parthimos, Dimitris; Buntwal, Luke; Calver, Brian L.; Stamatiadis, Panagiotis; Smith, Adrian; Clue, Matthew; Sideratou, Zili; Swann, Karl; Lai, F. Anthony

    2015-01-01

    Sperm-specific phospholipase C-ζ (PLCζ) is widely considered to be the physiological stimulus that triggers intracellular Ca2+ oscillations and egg activation during mammalian fertilization. Although PLCζ is structurally similar to PLCδ1, it lacks a pleckstrin homology domain, and it remains unclear how PLCζ targets its phosphatidylinositol 4,5-bisphosphate (PIP2) membrane substrate. Recently, the PLCδ1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLCδ1 are notably conserved in PLCζ. We investigated the potential role of these conserved cationic residues in PLCζ by generating a series of mutants that sequentially neutralized three positively charged residues (Lys-49, Lys-53, and Arg-57) within the mouse PLCζ EF-hand domain. Microinjection of the PLCζ EF-hand mutants into mouse eggs enabled their Ca2+ oscillation inducing activities to be compared with wild-type PLCζ. Furthermore, the mutant proteins were purified, and the in vitro PIP2 hydrolysis and binding properties were monitored. Our analysis suggests that PLCζ binds significantly to PIP2, but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the first EF-hand domain of PLCζ significantly alters in vivo Ca2+ oscillation inducing activity and in vitro interaction with PIP2 without affecting its Ca2+ sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca2+ frequency and the binding ability of different PLCζ mutants to PIP2. Moreover, a PLCζ mutant with mutations in the cationic residues within the first EF-hand domain and the XY linker region dramatically reduces the binding of PLCζ to PIP2, leading to complete abolishment of its Ca2+ oscillation inducing activity. PMID:26429913

  2. Long-range repression by multiple polycomb group (PcG) proteins targeted by fusion to a defined DNA-binding domain in Drosophila.

    PubMed Central

    Roseman, R R; Morgan, K; Mallin, D R; Roberson, R; Parnell, T J; Bornemann, D J; Simon, J A; Geyer, P K

    2001-01-01

    A tethering assay was developed to study the effects of Polycomb group (PcG) proteins on gene expression in vivo. This system employed the Su(Hw) DNA-binding domain (ZnF) to direct PcG proteins to transposons that carried the white and yellow reporter genes. These reporters constituted naive sensors of PcG effects, as bona fide PcG response elements (PREs) were absent from the constructs. To assess the effects of different genomic environments, reporter transposons integrated at nearly 40 chromosomal sites were analyzed. Three PcG fusion proteins, ZnF-PC, ZnF-SCM, and ZnF-ESC, were studied, since biochemical analyses place these PcG proteins in distinct complexes. Tethered ZnF-PcG proteins repressed white and yellow expression at the majority of sites tested, with each fusion protein displaying a characteristic degree of silencing. Repression by ZnF-PC was stronger than ZnF-SCM, which was stronger than ZnF-ESC, as judged by the percentage of insertion lines affected and the magnitude of the conferred repression. ZnF-PcG repression was more effective at centric and telomeric reporter insertion sites, as compared to euchromatic sites. ZnF-PcG proteins tethered as far as 3.0 kb away from the target promoter produced silencing, indicating that these effects were long range. Repression by ZnF-SCM required a protein interaction domain, the SPM domain, which suggests that this domain is not primarily used to direct SCM to chromosomal loci. This targeting system is useful for studying protein domains and mechanisms involved in PcG repression in vivo. PMID:11333237

  3. Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin.

    PubMed Central

    Schäfer, W; Stroh, A; Berghöfer, S; Seiler, J; Vey, M; Kruse, M L; Kern, H F; Klenk, H D; Garten, W

    1995-01-01

    Furin, a subtilisin-like eukaryotic endoprotease, is responsible for proteolytic cleavage of cellular and viral proteins transported via the constitutive secretory pathway. Cleavage occurs at the C-terminus of basic amino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golgi network (TGN), but also in clathrin-coated vesicles dispatched from the TGN, on the plasma membrane as an integral membrane protein and in the medium as an anchorless enzyme. When furin was vectorially expressed in normal rat kidney (NRK) cells it accumulated in the TGN similarly to the endogenous glycoprotein TGN38, often used as a TGN marker protein. The signals determining TGN targeting of furin were investigated by mutational analysis of the cytoplasmic tail of furin and by using the hemagglutinin (HA) of fowl plague virus, a protein with cell surface destination, as a reporter molecule, in which membrane anchor and cytoplasmic tail were replaced by the respective domains of furin. The membrane-spanning domain of furin grafted to HA does not localize the chimeric molecule to the TGN, whereas the cytoplasmic domain does. Results obtained on furin mutants with substitutions and deletions of amino acids in the cytoplasmic tail indicate that wild-type furin is concentrated in the TGN by a mechanism involving two independent targeting signals, which consist of the acidic peptide CPSDSEEDEG783 and the tetrapeptide YKGL765. The acidic signal in the cytoplasmic domain of a HA-furin chimera is necessary and sufficient to localize the reporter molecule to the TGN, whereas YKGL is a determinant for targeting to the endosomes. The data support the concept that the acidic signal, which is the dominant one, retains furin in the TGN, whereas the YKGL motif acts as a retrieval signal for furin that has escaped to the cell surface. Images PMID:7781597

  4. Identification of Novel Molecular Targets for Pleckstrin Homology (PH) Domains Found in Oncogenes Implicated in Breast Cancer

    DTIC Science & Technology

    2008-03-01

    2006; Rajala et al, 2005; Yan et al, 2005; Skowronek et al, 2004; Saxena et al, 2002; Dowler et al, 2000 ; Fleming et al, 2000 ). As expected from...PtdIns4P in vivo (Várnai and Balla, 1998; Dowler et al, 2000 ). In contrast to reported PtdIns4P specificity, I have determined that the PH domains of...PH structures exhibit a standard PH domain fold (Ferguson et al 2000 ; Lietzke et al 2000 ), with seven β-strands forming two orthogonal anti-parallel

  5. In Vitro and In Vivo Assessment of Targeting Lipid-based Nanoparticles to the Epidermal Growth Factor-Receptor (EGFR) Using a Novel Heptameric ZEGFR Domain

    PubMed Central

    Benhabbour, S. Rahima; Luft, J. Christopher; Kim, Dongwook; Jain, Anekant; Wadhwa, Saurabh; Parrott, Matthew C.; Liu, Rihe; DeSimone, Joseph; Mumper, Russell J.

    2011-01-01

    Lipid-based oil-filled nanoparticles (NPs) with a high concentration of surface-chelated nickel (Ni-NPs) were successfully prepared using a Brij78-NTA-Ni conjugate synthesized with Brij 78 (Polyoxyethylene (20) stearyl ether) and nitrilotriacetic acid (NTA). The facile incorporation of the Brij 78-NTA-Ni conjugate into the NP formulation allowed up to 90% Ni incorporation, which was a significant improvement over the previously used standard agent DOGS-NTA-Ni which led to ~6% Ni incorporation. The Ni-NPs were targeted to the highly epidermal growth factor receptor (EGFR)-overexpressing epidermoid carcinoma cells A431. This was accomplished using a novel high affinity histidine×6-tagged EGFR-binding Z domain (heptameric ZEGFR domain). In vitro cell uptake studies showed enhanced internalization (up to 90%) of the targeted Ni-NPs in A431 cells with only ≤10% internalization of the of untargeted Ni-NPs. ICP-MS analysis used to quantify the amount of Ni in the cells were in close agreement with flow cytometry studies, which showed a dose dependent increase in the amount of Ni with the targeted Ni-NPs. Cell uptake competition studies showed that internalization of the targeted Ni-NPs within the cells was competed off with free heptameric ZEGFR domain at concentrations of 8.75 ng/mL or higher. In vivo studies were carried out in nude mice bearing A431 tumors to determine the biodistribution and intracellular delivery. Near Infrared (NIR) optical imaging studies using Alexa750-labeled heptameric ZEGFR domain showed localization of 19% of the total detected fluorescence intensity in the tumor tissue, 28% in the liver and 42% in the kidneys 16 h post i.v. injection. ICP-MS analysis showed almost a two-fold increase in the amount of intracellular Ni with the targeted Ni-NPs. These new Ni-NPs could be a very useful tool for targeting and drug delivery to a wide range of EGFR positive cancers. PMID:22037106

  6. Time-domain Response of a Metal Detector to a Target Buried in Soil with Frequency-dependent Magnetic Susceptibility

    DTIC Science & Technology

    2016-07-06

    The work reported in this paper is a part of on-going studies to clarify how and to what extent soil electromagnetic properties affect the...performance of induction metal detectors widely used in humanitarian demining. This paper studies the specific case of the time-domain response of a small

  7. Crystal Structure of Human Profilaggrin S100 Domain and Identification of Target Proteins Annexin II, Stratifin, and HSP27.

    PubMed

    Bunick, Christopher G; Presland, Richard B; Lawrence, Owen T; Pearton, David J; Milstone, Leonard M; Steitz, Thomas A

    2015-07-01

    The fused-type S100 protein profilaggrin and its proteolytic products including filaggrin are important in the formation of a normal epidermal barrier; however, the specific function of the S100 calcium-binding domain in profilaggrin biology is poorly understood. To explore its molecular function, we determined a 2.2 Å-resolution crystal structure of the N-terminal fused-type S100 domain of human profilaggrin with bound calcium ions. The profilaggrin S100 domain formed a stable dimer, which contained two hydrophobic pockets that provide a molecular interface for protein interactions. Biochemical and molecular approaches demonstrated that three proteins, annexin II/p36, stratifin/14-3-3 sigma, and heat shock protein 27, bind to the N-terminal domain of human profilaggrin; one protein (stratifin) co-localized with profilaggrin in the differentiating granular cell layer of human skin. Together, these findings suggest a model where the profilaggrin N-terminus uses calcium-dependent and calcium-independent protein-protein interactions to regulate its involvement in keratinocyte terminal differentiation and incorporation into the cornified cell envelope.

  8. A parallel panning scheme used for selection of a GluA4-specific Fab targeting the ligand-binding domain.

    PubMed

    Clausen, Rasmus P; Mohr, Andreas Ø; Riise, Erik; Jensen, Anders A; Gill, Avinash; Madden, Dean R; Kastrup, Jette S; Skottrup, Peter D

    2016-11-01

    A method for development of murine Fab fragments towards extracellular domains of a surface receptor is presented. The GluA4 ionotropic glutamate receptor is used as a model system. Recombinant GluA4 ectodomain comprising both the N-terminal domain (NTD) and the ligand-binding domain (LBD) in one molecule was used for immunization. A Fab-phage library was constructed and a parallel panning approach enabled selection of murine Fab fragments towards either intact ectodomain or the isolated LBD of the GluA4 receptor. One LBD-Fab (FabL9) showed exclusive selectivity for the GluA4 LBD, over a panel of LBDs from GluA2, GluK1, GluK2 and GluD2. Soluble FabL9 was produced in amounts suitable for characterization. Competitive ELISA and rat-brain immunoprecipitation experiments confirmed that the FabL9 epitope is conserved in the LBD and in the intact native receptor. By an alignment of GluA2 and GluA4, the likely binding epitope for FabL9 was predicted. This study demonstrates a simple approach for development of antibody fragments towards specific sub-domains of a large ligand-gated ion channel, and this method could be utilized for all multi-domain surface receptors where antibody domain-selectivity may be desirable. Furthermore, we present for the first time a GluA4 subtype-specific murine Fab fragment targeting the LBD of the receptor.

  9. Passive immunization targeting the N-terminal projection domain of tau decreases tau pathology and improves cognition in a transgenic mouse model of Alzheimer disease and tauopathies.

    PubMed

    Dai, Chun-ling; Chen, Xia; Kazim, Syed Faraz; Liu, Fei; Gong, Cheng-Xin; Grundke-Iqbal, Inge; Iqbal, Khalid

    2015-04-01

    Intraneuronal accumulation of abnormally hyperphosphorylated tau in the brain is a histopathological hallmark of Alzheimer's disease and a family of related neurodegenerative disorders collectively called tauopathies. At present there is no effective treatment available for these progressive neurodegenerative diseases which are clinically characterized by dementia in mid to old-age. Here we report the treatment of 14-17-months-old 3xTg-AD mice with tau antibodies 43D (tau 6-18) and 77E9 (tau 184-195) to the N-terminal projection domain of tau or mouse IgG as a control by intraperitoneal injection once a week for 4 weeks, and the effects of the passive immunization on reduction of hyperphosphorylated tau, Aβ accumulation and cognitive performance in these animals. We found that treatment with tau antibodies 43D and 77E9 reduced total tau level, decreased tau hyperphosphorylated at Ser199, Ser202/Thr205 (AT8), Thr205, Ser262/356 (12E8), and Ser396/404 (PHF-1) sites, and a trend to reduce Aβ pathology. Most importantly, targeting N-terminal tau especially by 43D (tau 6-18) improved reference memory in the Morris water maze task in 3xTg-AD mice. We did not observe any abnormality in general physical characteristics of the treated animals with either of the two antibodies during the course of this study. Taken together, our studies demonstrate for the first time (1) that passive immunization targeting normal tau can effectively clear the hyperphosphorylated protein and possibly reduce Aβ pathology from the brain and (2) that targeting N-terminal projection domain of tau containing amino acid 6-18 is especially beneficial. Thus, targeting selective epitopes of N-terminal domain of tau may present a novel effective therapeutic opportunity for Alzheimer disease and other tauopathies.

  10. MZF-1/Elk-1 interaction domain as therapeutic target for protein kinase Cα-based triple-negative breast cancer cells

    PubMed Central

    Lee, Chia-Jen; Hsu, Li-Sung; Yue, Chia-Herng; Lin, Ho; Chiu, Yung-Wei; Lin, Yu-Yu; Huang, Chih-Yang; Hung, Mien-Chie; Liu, Jer-Yuh

    2016-01-01

    Recent reports demonstrate that the expression of protein kinase C alpha (PKCα) in triple-negative breast cancer (TNBC) correlates with decreased survival outcomes. However, off-target effects of targeting PKCα and limited understanding of the signaling mechanisms upstream of PKCα have hampered previous efforts to manipulate this ubiquitous gene. This study shows that the expression of both myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) correlates with PKCα expression in TNBC. We found that the acidic domain of MZF-1 and the heparin-binding domain of Elk-1 facilitate the heterodimeric interaction between the two genes before the complex formation binds to the PKCα promoter. Blocking the formation of the heterodimer by transfection of MZF-160–72 or Elk-1145–157 peptide fragments at the MZF-1 / Elk-1 interface decreases DNA-binding activity of the MZF-1 / Elk-1 complex at the PKCα promoter. Subsequently, PKCα expression, migration, tumorigenicity, and the epithelial–mesenchymal transition potential of TNBC cells decrease. These subsequent effects are reversed by transfection with full-length PKCα, confirming that the MZF-1/Elk-1 heterodimer is a mediator of PKCα in TNBC cells. These data suggest that the next therapeutic strategy in treating PKCα-related cancer will be developed from blocking MZF-1/Elk-1 interaction through their binding domain. PMID:27542222

  11. Role of the N-terminal transmembrane domain in the endo-lysosomal targeting and function of the human ABCB6 protein

    PubMed Central

    Kiss, Katalin; Kucsma, Nora; Brozik, Anna; Tusnady, Gabor E.; Bergam, Ptissam; vanNiel, Guillaume; Szakacs, Gergely

    2015-01-01

    ATP-binding cassette, subfamily B (ABCB) 6 is a homodimeric ATP-binding cassette (ABC) transporter present in the plasma membrane and in the intracellular organelles. The intracellular localization of ABCB6 has been a matter of debate, as it has been suggested to reside in the mitochondria and the endo-lysosomal system. Using a variety of imaging modalities, including confocal microscopy and EM, we confirm the endo-lysosomal localization of ABCB6 and show that the protein is internalized from the plasma membrane through endocytosis, to be distributed to multivesicular bodies and lysosomes. In addition to the canonical nucleotide-binding domain (NBD) and transmembrane domain (TMD), ABCB6 contains a unique N-terminal TMD (TMD0), which does not show sequence homology to known proteins. We investigated the functional role of these domains through the molecular dissection of ABCB6. We find that the folding, dimerization, membrane insertion and ATP binding/hydrolysis of the core–ABCB6 complex devoid of TMD0 are preserved. However, in contrast with the full-length transporter, the core–ABCB6 construct is retained at the plasma membrane and does not appear in Rab5-positive endosomes. TMD0 is directly targeted to the lysosomes, without passage to the plasma membrane. Collectively, our results reveal that TMD0 represents an independently folding unit, which is dispensable for catalysis, but has a crucial role in the lysosomal targeting of ABCB6. PMID:25627919

  12. MZF-1/Elk-1 interaction domain as therapeutic target for protein kinase Cα-based triple-negative breast cancer cells.

    PubMed

    Lee, Chia-Jen; Hsu, Li-Sung; Yue, Chia-Herng; Lin, Ho; Chiu, Yung-Wei; Lin, Yu-Yu; Huang, Chih-Yang; Hung, Mien-Chie; Liu, Jer-Yuh

    2016-09-13

    Recent reports demonstrate that the expression of protein kinase C alpha (PKCα) in triple-negative breast cancer (TNBC) correlates with decreased survival outcomes. However, off-target effects of targeting PKCα and limited understanding of the signaling mechanisms upstream of PKCα have hampered previous efforts to manipulate this ubiquitous gene. This study shows that the expression of both myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) correlates with PKCα expression in TNBC. We found that the acidic domain of MZF-1 and the heparin-binding domain of Elk-1 facilitate the heterodimeric interaction between the two genes before the complex formation binds to the PKCα promoter. Blocking the formation of the heterodimer by transfection of MZF-160-72 or Elk-1145-157 peptide fragments at the MZF-1 / Elk-1 interface decreases DNA-binding activity of the MZF-1 / Elk-1 complex at the PKCα promoter. Subsequently, PKCα expression, migration, tumorigenicity, and the epithelial-mesenchymal transition potential of TNBC cells decrease. These subsequent effects are reversed by transfection with full-length PKCα, confirming that the MZF-1/Elk-1 heterodimer is a mediator of PKCα in TNBC cells. These data suggest that the next therapeutic strategy in treating PKCα-related cancer will be developed from blocking MZF-1/Elk-1 interaction through their binding domain.

  13. VERDANDI Is a Direct Target of the MADS Domain Ovule Identity Complex and Affects Embryo Sac Differentiation in Arabidopsis[W

    PubMed Central

    Matias-Hernandez, Luis; Battaglia, Raffaella; Galbiati, Francesca; Rubes, Marco; Eichenberger, Christof; Grossniklaus, Ueli; Kater, Martin M.; Colombo, Lucia

    2010-01-01

    In Arabidopsis thaliana, the three MADS box genes SEEDSTICK (STK), SHATTERPROOF1 (SHP1), and SHP2 redundantly regulate ovule development. Protein interaction studies have shown that a multimeric complex composed of the ovule identity proteins together with the SEPALLATA MADS domain proteins is necessary to determine ovule identity. Despite the extensive knowledge that has become available about these MADS domain transcription factors, little is known regarding the genes that they regulate. Here, we show that STK, SHP1, and SHP2 redundantly regulate VERDANDI (VDD), a putative transcription factor that belongs to the plant-specific B3 superfamily. The vdd mutant shows defects during the fertilization process resulting in semisterility. Analysis of the vdd mutant female gametophytes indicates that antipodal and synergid cell identity and/or differentiation are affected. Our results provide insights into the pathways regulated by the ovule identity factors and the role of the downstream target gene VDD in female gametophyte development. PMID:20581305

  14. Protein phosphatase PP1/GLC7 interaction domain in yeast eIF2γ bypasses targeting subunit requirement for eIF2α dephosphorylation.

    PubMed

    Rojas, Margarito; Gingras, Anne-Claude; Dever, Thomas E

    2014-04-08

    Whereas the protein kinases GCN2, HRI, PKR, and PERK specifically phosphorylate eukaryotic translation initiation factor 2 (eIF2α) on Ser51 to regulate global and gene-specific mRNA translation, eIF2α is dephosphorylated by the broadly acting serine/threonine protein phosphatase 1 (PP1). In mammalian cells, the regulatory subunits GADD34 and CReP target PP1 to dephosphorylate eIF2α; however, as there are no homologs of these targeting subunits in yeast, it is unclear how GLC7, the functional homolog of PP1 in yeast, is recruited to dephosphorylate eIF2α. Here, we show that a novel N-terminal extension on yeast eIF2γ contains a PP1-binding motif (KKVAF) that enables eIF2γ to pull down GLC7 and target it to dephosphorylate eIF2α. Truncation or point mutations designed to eliminate the KKVAF motif in eIF2γ impair eIF2α dephosphorylation in vivo and in vitro and enhance expression of GCN4. Replacement of the N terminus of eIF2γ with the GLC7-binding domain from GAC1 or fusion of heterologous dimerization domains to eIF2γ and GLC7, respectively, maintained eIF2α phosphorylation at basal levels. Taken together, these results indicate that, in contrast to the paradigm of distinct PP1-targeting or regulatory subunits, the unique N terminus of yeast eIF2γ functions in cis to target GLC7 to dephosphorylate eIF2α.

  15. Modular elements of the TPR domain in the Mps1 N terminus differentially target Mps1 to the centrosome and kinetochore

    PubMed Central

    Marquardt, Joseph R.; Perkins, Jennifer L.; Beuoy, Kyle J.; Fisk, Harold A.

    2016-01-01

    Faithful segregation of chromosomes to two daughter cells is regulated by the formation of a bipolar mitotic spindle and the spindle assembly checkpoint, ensuring proper spindle function. Here we show that the proper localization of the kinase Mps1 (monopolar spindle 1) is critical to both these processes. Separate elements in the Mps1 N-terminal extension (NTE) and tetratricopeptide repeat (TPR) domains govern localization to either the kinetochore or the centrosome. The third TPR (TPR3) and the TPR-capping helix (C-helix) are each sufficient to target Mps1 to the centrosome. TPR3 binds to voltage-dependent anion channel 3, but although this is sufficient for centrosome targeting of Mps1, it is not necessary because of the presence of the C-helix. A version of Mps1 lacking both elements cannot localize to or function at the centrosome, but maintains kinetochore localization and spindle assembly checkpoint function, indicating that TPR3 and the C-helix define a bipartite localization determinant that is both necessary and sufficient to target Mps1 to the centrosome but dispensable for kinetochore targeting. In contrast, elements required for kinetochore targeting (the NTE and first two TPRs) are dispensable for centrosomal localization and function. These data are consistent with a separation of Mps1 function based on localization determinants within the N terminus. PMID:27339139

  16. Molecular cloning and cold shock induced overexpression of the DNA encoding phor sensor domain from Mycobacterium tuberculosis as a target molecule for novel anti-tubercular drugs

    NASA Astrophysics Data System (ADS)

    Langi, Gladys Emmanuella Putri; Moeis, Maelita R.; Ihsanawati, Giri-Rachman, Ernawati Arifin

    2014-03-01

    Mycobacterium tuberculosis (Mtb), the sole cause of Tuberculosis (TB), is still a major global problem. The discovery of new anti-tubercular drugs is needed to face the increasing TB cases, especially to prevent the increase of cases with resistant Mtb. A potential novel drug target is the Mtb PhoR sensor domain protein which is the histidine kinase extracellular domain for receiving environmental signals. This protein is the initial part of the two-component system PhoR-PhoP regulating 114 genes related to the virulence of Mtb. In this study, the gene encoding PhoR sensor domain (SensPhoR) was subcloned from pGEM-T SensPhoR from the previous study (Suwanto, 2012) to pColdII. The construct pColdII SensPhoR was confirmed through restriction analysis and sequencing. Using the construct, SensPhoR was overexpressed at 15°C using Escherichia coli BL21 (DE3). Low temperature was chosen because according to the solubility prediction program of recombinant proteins from The University of Oklahama, the PhoR sensor domain has a chance of 79.8% to be expressed as insoluble proteins in Escherichia coli's (E. coli) cytoplasm. This prediction is also supported by other similar programs: PROSO and PROSO II. The SDS PAGE result indicated that the PhoR sensor domain recombinant protein was overexpressed. For future studies, this protein will be purified and used for structure analysis which can be used to find potential drugs through rational drug design.

  17. Receptor-binding domains of spike proteins of emerging or re-emerging viruses as targets for development of antiviral vaccines

    PubMed Central

    Jiang, Shibo; Lu, Lu; Liu, Qi; Xu, Wei; Du, Lanying

    2012-01-01

    A number of emerging and re-emerging viruses have caused epidemics or pandemics of infectious diseases leading to major devastations throughout human history. Therefore, developing effective and safe vaccines against these viruses is clearly important for the protection of at-risk populations. Our previous studies have shown that the receptor-binding domain (RBD) in the spike protein of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) is a key target for the development of SARS vaccines. In this review, we highlight some key advances in the development of antiviral vaccines targeting the RBDs of spike proteins of emerging and re-emerging viruses, using SARS-CoV, influenza virus, Hendra virus (HeV) and Nipah virus (NiV) as examples. PMID:26038424

  18. CIGB-300, a synthetic peptide-based drug that targets the CK2 phosphoaceptor domain. Translational and clinical research.

    PubMed

    Perea, Silvio E; Baladron, Idania; Garcia, Yanelda; Perera, Yasser; Lopez, Adlin; Soriano, Jorge L; Batista, Noyde; Palau, Aley; Hernández, Ignacio; Farina, Hernán; Garcia, Idrian; Gonzalez, Lidia; Gil, Jeovanis; Rodriguez, Arielis; Solares, Margarita; Santana, Agueda; Cruz, Marisol; Lopez, Matilde; Valenzuela, Carmen; Reyes, Osvaldo; López-Saura, Pedro A; González, Carlos A; Diaz, Alina; Castellanos, Lila; Sanchez, Aniel; Betancourt, Lazaro; Besada, Vladimir; González, Luis J; Garay, Hilda; Gómez, Roberto; Gómez, Daniel E; Alonso, Daniel F; Perrin, Phillipe; Renualt, Jean-Yves; Sigman, Hugo; Herrera, Luis; Acevedo, Boris

    2011-10-01

    CK2 represents an oncology target scientifically validated. However, clinical research with inhibitors of the CK2-mediated phosphorylation event is still insufficient to recognize it as a clinically validated target. CIGB-300, an investigational peptide-based drug that targets the phosphoaceptor site, binds to a CK2 substrate array in vitro but mainly to B23/nucleophosmin in vivo. The CIGB-300 proapoptotic effect is preceded by its nucleolar localization, inhibition of the CK2-mediated phosphorylation on B23/nucleophosmin and nucleolar disassembly. Importantly, CIGB-300 shifted a protein array linked to apoptosis, ribosome biogenesis, cell proliferation, glycolisis, and cell motility in proteomic studies which helped to understand its mechanism of action. In the clinical ground, CIGB-300 has proved to be safe and well tolerated in a First-in-Human trial in women with cervical malignancies who also experienced signs of clinical benefit. In a second Phase 1 clinical trial in women with cervical cancer stage IB2/II, the MTD and DLT have been also identified in the clinical setting. Interestingly, in cervical tumors the B23/nucleophosmin protein levels were significantly reduced after CIGB-300 treatment at the nucleus compartment. In addition, expanded use of CIGB-300 in case studies has evidenced antitumor activity when administered as compassional option. Collectively, our data outline important clues on translational and clinical research from this novel peptide-based drug reinforcing its perspectives to treat cancer and paving the way to validate CK2 as a promising target in oncology.

  19. Porcine bocavirus NP1 negatively regulates interferon signaling pathway by targeting the DNA-binding domain of IRF9

    SciTech Connect

    Zhang, Ruoxi; Fang, Liurong; Wang, Dang; Cai, Kaimei; Zhang, Huan; Xie, Lilan; Li, Yi; Chen, Huanchun; Xiao, Shaobo

    2015-11-15

    To subvert host antiviral immune responses, many viruses have evolved countermeasures to inhibit IFN signaling pathway. Porcine bocavirus (PBoV), a newly identified porcine parvovirus, has received attention because it shows clinically high co-infection prevalence with other pathogens in post-weaning multisystemic wasting syndrome (PWMS) and diarrheic piglets. In this study, we screened the structural and non-structural proteins encoded by PBoV and found that the non-structural protein NP1 significantly suppressed IFN-stimulated response element (ISRE) activity and subsequent IFN-stimulated gene (ISG) expression. However, NP1 affected neither the activation and translocation of STAT1/STAT2, nor the formation of the heterotrimeric transcription factor complex ISGF3 (STAT1/STAT2/IRF9). Detailed analysis demonstrated that PBoV NP1 blocked the ISGF3 DNA-binding activity by combining with the DNA-binding domain (DBD) of IRF9. In summary, these results indicate that PBoV NP1 interferes with type I IFN signaling pathway by blocking DNA binding of ISGF3 to attenuate innate immune responses. - Highlights: • Porcine bocavirus (PBoV) NP1 interferes with the IFN α/β signaling pathway. • PBoV NP1 does not prevent STAT1/STAT2 phosphorylation and nuclear translocation. • PBoV NP1 inhibits the DNA-binding activity of ISGF3. • PBoV NP1 interacts with the DNA-binding domain of IRF9.

  20. Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

    PubMed Central

    Heintz, Udo; Schlichting, Ilme

    2016-01-01

    The design of synthetic optogenetic tools that allow precise spatiotemporal control of biological processes previously inaccessible to optogenetic control has developed rapidly over the last years. Rational design of such tools requires detailed knowledge of allosteric light signaling in natural photoreceptors. To understand allosteric communication between sensor and effector domains, characterization of all relevant signaling states is required. Here, we describe the mechanism of light-dependent DNA binding of the light-oxygen-voltage (LOV) transcription factor Aureochrome 1a from Phaeodactylum tricornutum (PtAu1a) and present crystal structures of a dark state LOV monomer and a fully light-adapted LOV dimer. In combination with hydrogen/deuterium-exchange, solution scattering data and DNA-binding experiments, our studies reveal a light-sensitive interaction between the LOV and basic region leucine zipper DNA-binding domain that together with LOV dimerization results in modulation of the DNA affinity of PtAu1a. We discuss the implications of these results for the design of synthetic LOV-based photosensors with application in optogenetics. DOI: http://dx.doi.org/10.7554/eLife.11860.001 PMID:26754770

  1. A novel domain cassette identifies Plasmodium falciparum PfEMP1 proteins binding ICAM-1 and is a target of cross-reactive, adhesion-inhibitory antibodies.

    PubMed

    Bengtsson, Anja; Joergensen, Louise; Rask, Thomas S; Olsen, Rebecca W; Andersen, Marianne A; Turner, Louise; Theander, Thor G; Hviid, Lars; Higgins, Matthew K; Craig, Alister; Brown, Alan; Jensen, Anja T R

    2013-01-01

    Cerebral Plasmodium falciparum malaria is characterized by adhesion of infected erythrocytes (IEs) to the cerebral microvasculature. This has been linked to parasites expressing the structurally related group A subset of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of IE adhesion ligands and to IEs with affinity for ICAM-1. However, recent evidence has cast doubt on both these associations, tempering hopes of the feasibility of developing a vaccine based on ICAM-1-binding PfEMP1. In this study, we report the identification of a domain cassette (DC) present in group A var genes from six genetically distinct P. falciparum parasites. The three domains in the cassette, which we call DC4, had a high level of sequence identity and cluster together phylogenetically. Erythrocytes infected by these parasites and selected in vitro for expression of DC4 adhered specifically to ICAM-1. The ICAM-1-binding capacity of DC4 was mapped to the C-terminal third of its Duffy-binding-like β3 domain. DC4 was the target of broadly cross-reactive and adhesion-inhibitory IgG Abs, and levels of DC4-specific and adhesion-inhibitory IgG increased with age among P. falciparum-exposed children. Our study challenges earlier conclusions that group A PfEMP1 proteins are not central to ICAM-1-specific IE adhesion and support the feasibility of developing a vaccine preventing cerebral malaria by inhibiting cerebral IE sequestration.

  2. Resveratrol induces apoptosis by directly targeting Ras-GTPase activating protein SH3 domain binding protein 1 (G3BP1)

    PubMed Central

    Oi, Naomi; Yuan, Jian; Malakhova, Margarita; Luo, Kuntian; Li, Yunhui; Ryu, Joohyun; Zhang, Lei; Bode, Ann M.; Xu, Zengguang; Li, Yan; Lou, Zhenkun; Dong, Zigang

    2014-01-01

    Resveratrol possesses a strong anticancer activity exhibited as the induction of apoptosis through p53 activation. However, the molecular mechanism and direct target(s) of resveratrol-induced p53 activation remain elusive. Here, the Ras-GTPase activating protein SH3 domain binding protein 1 (G3BP1) was identified as a potential target of resveratrol, and in vitro binding assay results using resveratrol (RSVL)-conjugated Sepharose 4B beads confirmed their direct binding. Depletion of G3BP1 significantly diminishes resveratrol-induced p53 expression and apoptosis. We also found that G3BP1 negatively regulates p53 expression by interacting with ubiquitin-specific protease 10 (USP10), a deubiquitinating enzyme of p53. Disruption of the interaction of p53 with USP10 by G3BP1 interference leads to suppression of p53 deubiquitination. Resveratrol, on the other hand, directly binds to G3BP1 and prevents the G3BP1/USP10 interaction, resulting in enhanced USP10-mediated deubiquitination of p53 and consequently increased p53 expression. These findings disclose a novel mechanism of resveratrol-induced p53 activation and resveratrol-induced apoptosis by direct targeting of G3BP1. PMID:24998844

  3. Preferential Entry of Botulinum Neurotoxin A Hc Domain through Intestinal Crypt Cells and Targeting to Cholinergic Neurons of the Mouse Intestine

    PubMed Central

    Couesnon, Aurélie; Molgó, Jordi; Connan, Chloé; Popoff, Michel R.

    2012-01-01

    Botulism, characterized by flaccid paralysis, commonly results from botulinum neurotoxin (BoNT) absorption across the epithelial barrier from the digestive tract and then dissemination through the blood circulation to target autonomic and motor nerve terminals. The trafficking pathway of BoNT/A passage through the intestinal barrier is not yet fully understood. We report that intralumenal administration of purified BoNT/A into mouse ileum segment impaired spontaneous muscle contractions and abolished the smooth muscle contractions evoked by electric field stimulation. Entry of BoNT/A into the mouse upper small intestine was monitored with fluorescent HcA (half C-terminal domain of heavy chain) which interacts with cell surface receptor(s). We show that HcA preferentially recognizes a subset of neuroendocrine intestinal crypt cells, which probably represent the entry site of the toxin through the intestinal barrier, then targets specific neurons in the submucosa and later (90–120 min) in the musculosa. HcA mainly binds to certain cholinergic neurons of both submucosal and myenteric plexuses, but also recognizes, although to a lower extent, other neuronal cells including glutamatergic and serotoninergic neurons in the submucosa. Intestinal cholinergic neuron targeting by HcA could account for the inhibition of intestinal peristaltism and secretion observed in botulism, but the consequences of the targeting to non-cholinergic neurons remains to be determined. PMID:22438808

  4. An amphiphilic region in the cytoplasmic domain of KdpD is recognized by the signal recognition particle and targeted to the Escherichia coli membrane

    PubMed Central

    Maier, Katja S; Hubich, Stefanie; Liebhart, Helga; Krauss, Susanne; Kuhn, Andreas; Facey, Sandra J

    2008-01-01

    The sensor protein KdpD of Escherichia coli is composed of a large N-terminal hydrophilic region (aa 1–400), four transmembrane regions (aa 401–498) and a large hydrophilic region (aa 499–894) at the C-terminus. KdpD requires the signal recognition particle (SRP) for its targeting to the membrane. Deletions within KdpD show that the first 50 residues are required for SRP-driven membrane insertion. A fusion protein of the green fluorescent protein (GFP) with KdpD is found localized at the membrane only when SRP is present. The membrane targeting of GFP was not observed when the first 50 KdpD residues were deleted. A truncated mutant of KdpD containing only the first 25 amino acids fused to GFP lost its ability to specifically interact with SRP, whereas a specific interaction between SRP and the first 48 amino acids of KdpD fused to GFP was confirmed by pull-down experiments. Conclusively, a small amphiphilic region of 27 residues within the amino-terminal domain of KdpD (aa 22–48) is recognized by SRP and targets the protein to the membrane. This shows that membrane proteins with a large N-terminal region in the cytoplasm can be membrane-targeted early on to allow co-translational membrane insertion of their distant transmembrane regions. PMID:18433452

  5. The α2 helix in the DNA ligase IV BRCT-1 domain is required for targeted degradation of ligase IV during adenovirus infection.

    PubMed

    Gilson, Timra; Greer, Amy E; Vindigni, Alessandro; Ketner, Gary; Hanakahi, Leslyn A

    2012-07-05

    In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood. Through genetic analysis, we identified the α2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 α2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 α2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV.

  6. Endocytotic routes of cobra cardiotoxins depend on spatial distribution of positively charged and hydrophobic domains to target distinct types of sulfated glycoconjugates on cell surface.

    PubMed

    Lee, Shao-Chen; Lin, Chien-Chu; Wang, Chia-Hui; Wu, Po-Long; Huang, Hsuan-Wei; Chang, Chung-I; Wu, Wen-guey

    2014-07-18

    Cobra cardiotoxins (CTX) are a family of three-fingered basic polypeptides known to interact with diverse targets such as heparan sulfates, sulfatides, and integrins on cell surfaces. After CTX bind to the membrane surface, they are internalized to intracellular space and exert their cytotoxicity via an unknown mechanism. By the combined in vitro kinetic binding, three-dimensional x-ray structure determination, and cell biology studies on the naturally abundant CTX homologues from the Taiwanese cobra, we showed that slight variations on the spatial distribution of positively charged or hydrophobic domains among CTX A2, A3, and A4 could lead to significant changes in their endocytotic pathways and action mechanisms via distinct sulfated glycoconjugate-mediated processes. The intracellular locations of these structurally similar CTX after internalization are shown to vary between the mitochondria and lysosomes via either dynamin2-dependent or -independent processes with distinct membrane cholesterol sensitivity. Evidence is presented to suggest that the shifting between the sulfated glycoconjugates as distinct targets of CTX A2, A3, and A4 might play roles in the co-evolutionary arms race between venomous snake toxins to cope with different membrane repair mechanisms at the cellular levels. The sensitivity of endocytotic routes to the spatial distribution of positively charged or hydrophobic domains may provide an explanation for the diverse endocytosis pathways of other cell-penetrating basic polypeptides.

  7. A stably expressed llama single-domain intrabody targeting Rev displays broad-spectrum anti-HIV activity.

    PubMed

    Boons, Eline; Li, Guangdi; Vanstreels, Els; Vercruysse, Thomas; Pannecouque, Christophe; Vandamme, Anne-Mieke; Daelemans, Dirk

    2014-12-01

    The HIV Rev protein mediates the transport of partially and unspliced HIV mRNA from the nucleus to the cytoplasm. Rev multimerizes on a secondary stem-loop structure present in the viral intron-containing mRNA species and recruits the cellular karyopherin CRM1 to export viral mRNAs from the nucleus to the cytoplasm. Previously we have identified a single-domain intrabody (Nb(190)), derived from a llama heavy-chain antibody, which efficiently inhibits Rev multimerization and suppresses the production of infectious virus. We recently mapped the epitope of this nanobody and demonstrated that Rev residues K20 and Y23 are crucial for interaction while residues V16, H53 and L60 are important to a lesser extent. Here, we generated cell lines stably expressing Nb(190) and assessed the capacity of these cell lines to suppress the replication of different HIV-1 subtypes. These cells stably expressing the single-domain antibody are protected from virus-induced cytopathogenic effect even in the context of high multiplicity of infection. In addition, the replication of different subtypes of group M and one strain of group O is significantly suppressed in these cell lines. Next, we analysed the natural variations of Rev amino acids in sequence samples from HIV-1 infected patients worldwide and assessed the effect of Nb(190) on the most prevalent polymorphisms occurring at the key epitope positions (K20 and Y23) in Rev. We found that Nb(190) was able to suppress the function of these Rev variants except for the K20N mutant, which was present in only 0.7% of HIV-1 sequence populations (n = 4632). Cells stably expressing the single-domain intrabody Nb(190) are protected against virus-induced cytopathogenic effect and display a selective survival advantage upon infection. In addition, Nb(190) suppresses the replication of a wide range of different HIV-1 subtypes. Large-scale sequence analysis reveals that the Nb(190) epitope positions in Rev are well conserved across major HIV-1

  8. Discovery of Small-Molecule Human Immunodeficiency Virus Type 1 Entry Inhibitors That Target the gp120-Binding Domain of CD4†

    PubMed Central

    Yang, Quan-en; Stephen, Andrew G.; Adelsberger, Joseph W.; Roberts, Paula E.; Zhu, Weimin; Currens, Michael J.; Feng, Yaxiong; Crise, Bruce J.; Gorelick, Robert J.; Rein, Alan R.; Fisher, Robert J.; Shoemaker, Robert H.; Sei, Shizuko

    2005-01-01

    The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4+ cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions. PMID:15857997

  9. Caspase recruitment domain of procaspase-2 could be a target for SUMO-1 modification through Ubc9

    SciTech Connect

    Shirakura, Hiromi; Hayashi, Naoko; Ogino, Shin-ichi; Tsuruma, Kazuhiro; Uehara, Takashi; Nomura, Yasuyuki . E-mail: nomura@pharm.hokudai.ac.jp

    2005-06-17

    To identify the binding proteins that regulate the function of procaspase-2, we screened for proteins using the yeast two-hybrid method and isolated human Ubc9 and SUMO-1 as the candidates. Ubc9 and SUMO-1 interacted with the caspase recruitment domain of procaspase-2 in its N-terminal. We elucidated the covalent modification of procaspase-2 by SUMO-1 in mammalian cells by immunoprecipitation followed by Western blot analysis. Procaspase-2 and SUMO-1 were co-localized by dot-like structures in the nucleus that are related to promyelocytic leukemia bodies. Interestingly, a conjugation-deficient mutant (K60R) procaspase-2 resulted in a delay of its enzyme maturation (appearance of p12 subunit) compared to that of wild-type. Thus, the modification with SUMO-1 may play a critical role in the nuclear localization and the activation (maturation) of procaspase-2.

  10. Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain

    SciTech Connect

    Mochizuki, Tomofumi; Hirai, Katsuyuki; Kanda, Ayami; Ohnishi, Jun; Ohki, Takehiro; Tsuda, Shinya

    2009-08-01

    The virulence factor of Melon necrotic spot virus (MNSV), a virus that induces systemic necrotic spot disease on melon plants, was investigated. When the replication protein p29 was expressed in N. benthamiana using a Cucumber mosaic virus vector, necrotic spots appeared on the leaf tissue. Transmission electron microscopy revealed abnormal mitochondrial aggregation in these tissues. Fractionation of tissues expressing p29 and confocal imaging using GFP-tagged p29 revealed that p29 associated with the mitochondrial membrane as an integral membrane protein. Expression analysis of p29 deletion fragments and prediction of hydrophobic transmembrane domains (TMDs) in p29 showed that deletion of the second putative TMD from p29 led to deficiencies in both the mitochondrial localization and virulence of p29. Taken together, these results indicated that MNSV p29 interacts with the mitochondrial membrane and that p29 may be a virulence factor causing the observed necrosis.

  11. A Novel Strategy for Controlling the Metastatic Phenotype: Targeting the SNAG Repression Domain in the SNAIL Zinc-Finger Protein

    DTIC Science & Technology

    2007-07-01

    Months 24-30) c . Examine target gene (from 3b.) expression patterns in the inducible cell lines by Northern analysis and validate gene function by...mediated gene silencing to spe - cific loci. KAP1 coordinates histone deacetylation via the recruitment of the NuRD complex (Schultz et al. 2001); histone H3...mammalian cells GENES & DEVELOPMENT 1859 on July 31, 2007 www.genesdev.orgDownloaded from results are presented in Figure 4. Using antibodies spe

  12. MicroRNA-181b inhibits thrombin-mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10.

    PubMed

    Lin, Jibin; He, Shaolin; Sun, Xinghui; Franck, Gregory; Deng, Yihuan; Yang, Dafeng; Haemmig, Stefan; Wara, A K M; Icli, Basak; Li, Dazhu; Feinberg, Mark W

    2016-09-01

    Thrombogenic and inflammatory mediators, such as thrombin, induce NF-κB-mediated endothelial cell (EC) activation and dysfunction, which contribute to pathogenesis of arterial thrombosis. The role of anti-inflammatory microRNA-181b (miR-181b) on thrombosis remains unknown. Our previous study demonstrated that miR-181b inhibits downstream NF-κB signaling in response to TNF-α. Here, we demonstrate that miR-181b uniquely inhibits upstream NF-κB signaling in response to thrombin. Overexpression of miR-181b inhibited thrombin-induced activation of NF-κB signaling, demonstrated by reduction of phospho-IKK-β, -IκB-α, and p65 nuclear translocation in ECs. MiR-181b also reduced expression of NF-κB target genes VCAM-1, intercellular adhesion molecule-1, E-selectin, and tissue factor. Mechanistically, miR-181b targets caspase recruitment domain family member 10 (Card10), an adaptor protein that participates in activation of the IKK complex in response to signals transduced from protease-activated receptor-1. miR-181b reduced expression of Card10 mRNA and protein, but not protease-activated receptor-1. 3'-Untranslated region reporter assays, argonaute-2 microribonucleoprotein immunoprecipitation studies, and Card10 rescue studies revealed that Card10 is a bona fide direct miR-181b target. Small interfering RNA-mediated knockdown of Card10 expression phenocopied effects of miR-181b on NF-κB signaling and targets. Card10 deficiency did not affect TNF-α-induced activation of NF-κB signaling, which suggested stimulus-specific regulation of NF-κB signaling and endothelial responses by miR-181b in ECs. Finally, in response to photochemical injury-induced arterial thrombosis, systemic delivery of miR-181b reduced thrombus formation by 73% in carotid arteries and prolonged time to occlusion by 1.6-fold, effects recapitulated by Card10 small interfering RNA. These data demonstrate that miR-181b and Card10 are important regulators of thrombin-induced EC activation and

  13. Monoclonal And Single Domain Antibodies Targeting β-Integrin Subunits Block Sexual Transmission of HIV-1 in in vitro and in vivo Model Systems

    PubMed Central

    Guedon, Janet Tai; Luo, Kun; Zhang, Hong; Markham, Richard B.

    2015-01-01

    Background Poor adherence to prevention regimens for gel-based anti-HIV-1 microbicides has been a major obstacle to more effective pre-exposure prophylaxis. Concern persists that the antiretroviral drug containing microbicides might promote development of antiretroviral resistance. Methods Using in vitro transwell systems and a humanized mouse model of HIV-1 sexual transmission, we examined, as candidate microbicides, antibodies targeting the heterodimeric leukocyte function associated antigen 1 (LFA-1), a non-virally encoded protein acquired by the virus that also plays a critical role cell movement across endothelial and epithelial barriers. LFA-1 specific single domain variable regions from alpaca heavy-chain only antibodies (VHH) were identified and evaluated for their ability to inhibit HIV-1 transmission in the in vitro transwell system. Results Monoclonal antibodies targeting the CD11a and CD18 components of LFA-1 significantly reduced cell-free and cell-associated HIV-1 transmission in the in vitro transwell culture system and prevented virus transmission in the humanized mouse model of vaginal transmission. The broadly neutralizing monoclonal antibody b12 was unable to block transmission of cell-free virus. CD11a-specific VHH were isolated and expressed and the purified variable region protein domains reduced in vitro transepithelial transmission with an efficacy comparable to that of the CD11a monoclonal antibody. Conclusions Targeting integrins acquired by HIV-1 during budding and which are critical to interactions between epithelial cells and lymphocytes can reduce viral movement across epithelial barriers and prevent transmission in a humanized mouse model of sexual transmission. VHH capable of being produced by transformed bacteria can significantly reduce transepithelial virus transmission in in vitro model systems. PMID:25828964

  14. PBK/TOPK interacts with the DBD domain of tumor suppressor p53 and modulates expression of transcriptional targets including p21.

    PubMed

    Hu, F; Gartenhaus, R B; Eichberg, D; Liu, Z; Fang, H-B; Rapoport, A P

    2010-10-07

    PBK/TOPK (PDZ-binding kinase, T-LAK-cell-originated protein kinase) is a serine-threonine kinase that is overexpressed in a variety of tumor cells but its role in oncogenesis remains unclear. Here we show, by co-immunoprecipitation experiments and yeast two-hybrid analysis, that PBK/TOPK physically interacts with the tumor suppressor p53 through its DNA-binding (DBD) domain in HCT116 colorectal carcinoma cells that express wild-type p53. PBK also binds to p53 mutants carrying five common point mutations in the DBD domain. The PBK-p53 interaction appears to downmodulate p53 transactivation function as indicated by PBK/TOPK knockdown experiments, which show upregulated expression of the key p53 target gene and cyclin-dependent kinase inhibitor p21 in HCT116 cells, particularly after genotoxic damage from doxorubicin. Furthermore, stable PBK/TOPK knockdown cell lines (derived from HCT116 and MCF-7 cells) showed increased apoptosis, G(2)/M arrest and slower growth as compared to stable empty vector-transfected control cell lines. Gene microarray studies identified additional p53 target genes involved in apoptosis or cell cycling, which were differentially regulated by PBK knockdown. Together, these data suggest that increased levels of PBK/TOPK may contribute to tumor cell development and progression through suppression of p53 function and consequent reductions in the cell-cycle regulatory proteins such as p21. PBK/TOPK may therefore be a valid target for antineoplastic kinase inhibitors to sensitize tumor cells to chemotherapy-induced apoptosis and growth suppression.

  15. Discovery of a novel azaindole class of antibacterial agents targeting the ATPase domains of DNA gyrase and Topoisomerase IV.

    PubMed

    Manchester, John I; Dussault, Daemian D; Rose, Jonathan A; Boriack-Sjodin, P Ann; Uria-Nickelsen, Maria; Ioannidis, Georgine; Bist, Shanta; Fleming, Paul; Hull, Kenneth G

    2012-08-01

    We present the discovery and optimization of a novel series of bacterial topoisomerase inhibitors. Starting from a virtual screening hit, activity was optimized through a combination of structure-based design and physical property optimization. Synthesis of fewer than a dozen compounds was required to achieve inhibition of the growth of methicillin-resistant Staphyloccus aureus (MRSA) at compound concentrations of 1.56 μM. These compounds simultaneously inhibit DNA gyrase and Topoisomerase IV at similar nanomolar concentrations, reducing the likelihood of the spontaneous occurrence of target-based mutations resulting in antibiotic resistance, an increasing threat in the treatment of serious infections.

  16. Characterization and target genes of nine human PRD-like homeobox domain genes expressed exclusively in early embryos

    PubMed Central

    Madissoon, Elo; Jouhilahti, Eeva-Mari; Vesterlund, Liselotte; Töhönen, Virpi; Krjutškov, Kaarel; Petropoulous, Sophie; Einarsdottir, Elisabet; Linnarsson, Sten; Lanner, Fredrik; Månsson, Robert; Hovatta, Outi; Bürglin, Thomas R.; Katayama, Shintaro; Kere, Juha

    2016-01-01

    PAIRED (PRD)-like homeobox genes belong to a class of predicted transcription factor genes. Several of these PRD-like homeobox genes have been predicted in silico from genomic sequence but until recently had no evidence of transcript expression. We found recently that nine PRD-like homeobox genes, ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB, NOBOX, TPRX1 and TPRX2, were expressed in human preimplantation embryos. In the current study we characterized these PRD-like homeobox genes in depth and studied their functions as transcription factors. We cloned multiple transcript variants from human embryos and showed that the expression of these genes is specific to embryos and pluripotent stem cells. Overexpression of the genes in human embryonic stem cells confirmed their roles as transcription factors as either activators (CPHX1, CPHX2, ARGFX) or repressors (DPRX, DUXA, TPRX2) with distinct targets that could be explained by the amino acid sequence in homeodomain. Some PRD-like homeodomain transcription factors had high concordance of target genes and showed enrichment for both developmentally important gene sets and a 36 bp DNA recognition motif implicated in Embryo Genome Activation (EGA). Our data implicate a role for these previously uncharacterized PRD-like homeodomain proteins in the regulation of human embryo genome activation and preimplantation embryo development. PMID:27412763

  17. The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain

    SciTech Connect

    Lechner, Mark S. . E-mail: msl27@drexel.edu; Schultz, David C.; Negorev, Dmitri; Maul, Gerd G.; Rauscher, Frank J.

    2005-06-17

    The HP1 proteins regulate epigenetic gene silencing by promoting and maintaining chromatin condensation. The HP1 chromodomain binds to methylated histone H3. More enigmatic is the chromoshadow domain (CSD), which mediates dimerization, transcription repression, and interaction with multiple nuclear proteins. Here we show that KAP-1, CAF-1 p150, and NIPBL carry a canonical amino acid motif, PxVxL, which binds directly to the CSD with high affinity. We also define a new class of variant PxVxL CSD-binding motifs in Sp100A, LBR, and ATRX. Both canonical and variant motifs recognize a similar surface of the CSD dimer as demonstrated by a panel of CSD mutants. These in vitro binding results were confirmed by the analysis of polypeptides found associated with nuclear HP1 complexes and we provide the first evidence of the NIPBL/delangin protein in human cells, a protein recently implicated in the developmental disorder, Cornelia de Lange syndrome. NIPBL is related to Nipped-B, a factor participating in gene activation by remote enhancers in Drosophila melanogaster. Thus, this spectrum of direct binding partners suggests an expanded role for HP1 as factor participating in promoter-enhancer communication, chromatin remodeling/assembly, and sub-nuclear compartmentalization.

  18. Cyclic di-AMP targets the cystathionine beta-synthase domain of the osmolyte transporter OpuC.

    PubMed

    Huynh, TuAnh Ngoc; Choi, Philip H; Sureka, Kamakshi; Ledvina, Hannah E; Campillo, Julian; Tong, Liang; Woodward, Joshua J

    2016-10-01

    Cellular turgor is of fundamental importance to bacterial growth and survival. Changes in external osmolarity as a consequence of fluctuating environmental conditions and colonization of diverse environments can significantly impact cytoplasmic water content, resulting in cellular lysis or plasmolysis. To ensure maintenance of appropriate cellular turgor, bacteria import ions and small organic osmolytes, deemed compatible solutes, to equilibrate cytoplasmic osmolarity with the extracellular environment. Here, we show that elevated levels of c-di-AMP, a ubiquitous second messenger among bacteria, result in significant susceptibility to elevated osmotic stress in the bacterial pathogen Listeria monocytogenes. We found that levels of import of the compatible solute carnitine show an inverse correlation with intracellular c-di-AMP content and that c-di-AMP directly binds to the CBS domain of the ATPase subunit of the carnitine importer OpuC. Biochemical and structural studies identify conserved residues required for this interaction and transport activity in bacterial cells. Overall, these studies reveal a role for c-di-AMP mediated regulation of compatible solute import and provide new insight into the molecular mechanisms by which this essential second messenger impacts bacterial physiology and adaptation to changing environmental conditions.

  19. A humanized neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein.

    PubMed

    Li, Yan; Wan, Yuhua; Liu, Peipei; Zhao, Jincun; Lu, Guangwen; Qi, Jianxun; Wang, Qihui; Lu, Xuancheng; Wu, Ying; Liu, Wenjun; Zhang, Buchang; Yuen, Kwok-Yung; Perlman, Stanley; Gao, George F; Yan, Jinghua

    2015-11-01

    The newly-emerging Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans. Despite global efforts, the potential for an associated pandemic in the future cannot be excluded. The development of effective counter-measures is urgent. MERS-CoV-specific anti-viral drugs or vaccines are not yet available. Using the spike receptor-binding domain of MERS-CoV (MERS-RBD) to immunize mice, we identified two neutralizing monoclonal antibodies (mAbs) 4C2 and 2E6. Both mAbs potently bind to MERS-RBD and block virus entry in vitro with high efficacy. We further investigated their mechanisms of neutralization by crystallizing the complex between the Fab fragments and the RBD, and solved the structure of the 4C2 Fab/MERS-RBD complex. The structure showed that 4C2 recognizes an epitope that partially overlaps the receptor-binding footprint in MERS-RBD, thereby interfering with the virus/receptor interactions by both steric hindrance and interface-residue competition. 2E6 also blocks receptor binding, and competes with 4C2 for binding to MERS-RBD. Based on the structure, we further humanized 4C2 by preserving only the paratope residues and substituting the remaining amino acids with the counterparts from human immunoglobulins. The humanized 4C2 (4C2h) antibody sustained similar neutralizing activity and biochemical characteristics to the parental mouse antibody. Finally, we showed that 4C2h can significantly abate the virus titers in lungs of Ad5-hCD26-transduced mice infected with MERS-CoV, therefore representing a promising agent for prophylaxis and therapy in clinical settings.

  20. Perspectives on the Two-Pore Domain Potassium Channel TREK-1 (TWIK-Related K(+) Channel 1). A Novel Therapeutic Target?

    PubMed

    Vivier, Delphine; Bennis, Khalil; Lesage, Florian; Ducki, Sylvie

    2016-06-09

    Potassium (K(+)) channels are membrane proteins expressed in most living cells that selectively control the flow of K(+) ions. More than 80 genes encode the K(+) channel subunits in the human genome. The TWIK-related K(+) channel (TREK-1) belongs to the two-pore domain K(+) channels (K2P) and displays various properties including sensitivity to physical (membrane stretch, acidosis, temperature) and chemical stimuli (signaling lipids, volatile anesthetics). The distribution of TREK-1 in the central nervous system, coupled with the physiological consequences of its opening and closing, leads to the emergence of this channel as an attractive therapeutic target. We review the TREK-1 channel, its structural and functional properties, and the pharmacological agents (agonists and antagonists) able to modulate its gating.

  1. A targeted mutation within the feline leukemia virus (FeLV) envelope protein immunosuppressive domain to improve a canarypox virus-vectored FeLV vaccine.

    PubMed

    Schlecht-Louf, Géraldine; Mangeney, Marianne; El-Garch, Hanane; Lacombe, Valérie; Poulet, Hervé; Heidmann, Thierry

    2014-01-01

    We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the "mechanical" function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be "switched off" by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.

  2. A Novel Domain Cassette Identifies Plasmodium falciparum PfEMP1 Proteins Binding ICAM-1 and Is a Target of Cross-Reactive, Adhesion-Inhibitory Antibodies

    PubMed Central

    Bengtsson, Anja; Joergensen, Louise; Rask, Thomas S.; Olsen, Rebecca W.; Andersen, Marianne A.; Turner, Louise; Theander, Thor G.; Higgins, Matthew K.; Craig, Alister; Brown, Alan

    2013-01-01

    Cerebral Plasmodium falciparum malaria is characterized by adhesion of infected erythrocytes (IEs) to the cerebral microvasculature. This has been linked to parasites expressing the structurally related group A subset of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of IE adhesion ligands and to IEs with affinity for ICAM-1. However, recent evidence has cast doubt on both these associations, tempering hopes of the feasibility of developing a vaccine based on ICAM-1–binding PfEMP1. In this study, we report the identification of a domain cassette (DC) present in group A var genes from six genetically distinct P. falciparum parasites. The three domains in the cassette, which we call DC4, had a high level of sequence identity and cluster together phylogenetically. Erythrocytes infected by these parasites and selected in vitro for expression of DC4 adhered specifically to ICAM-1. The ICAM-1–binding capacity of DC4 was mapped to the C-terminal third of its Duffy-binding–like β3 domain. DC4 was the target of broadly cross-reactive and adhesion-inhibitory IgG Abs, and levels of DC4-specific and adhesion-inhibitory IgG increased with age among P. falciparum–exposed children. Our study challenges earlier conclusions that group A PfEMP1 proteins are not central to ICAM-1–specific IE adhesion and support the feasibility of developing a vaccine preventing cerebral malaria by inhibiting cerebral IE sequestration. PMID:23209327

  3. A Targeted Mutation within the Feline Leukemia Virus (FeLV) Envelope Protein Immunosuppressive Domain To Improve a Canarypox Virus-Vectored FeLV Vaccine

    PubMed Central

    Schlecht-Louf, Géraldine; Mangeney, Marianne; El-Garch, Hanane; Lacombe, Valérie; Poulet, Hervé

    2014-01-01

    We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the “mechanical” function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be “switched off” by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation. PMID:24198407

  4. M.(phi)BssHII, a novel cytosine-C5-DNA-methyltransferase with target-recognizing domains at separated locations of the enzyme.

    PubMed Central

    Sethmann, S; Ceglowski, P; Willert, J; Iwanicka-Nowicka, R; Trautner, T A; Walter, J

    1999-01-01

    In all cytosine-C5-DNA-methyltransferases (MTases) from prokaryotes and eukaryotes, remarkably conserved amino acid sequence elements responsible for general enzymatic functions are arranged in the same canonical order. In addition, one variable region, which includes the target-recognizing domain(s) (TRDs) characteristic for each enzyme, has been localized in one region between the same blocks of these conserved elements. This conservation in the order of conserved and variable sequences suggests stringent structural constraints in the primary structure to obtain the correct folding of the enzymes. Here we report the characterization of a new type of a multispecific MTase, M.(phiphi)BssHII, which is expressed as two isoforms. Isoform I is an entirely novel type of MTase which has, in addition to the TRDs at the conventional location, one TRD located at a non-canonical position at its N-terminus. Isoform II is represented by the same MTase, but without the N-terminal TRD. The N-terminal TRD provides HaeII methylation specificity to isoform I. The TRD is fully functional when engineered into either the conventional variable region of M.(phiphi)BssHII or the related monospecific M.phi3TII MTase. The implications of this structural plasticity with respect to the evolution of MTases are discussed. PMID:10369689

  5. Targeting two-pore domain K+ channels TREK-1 and TASK-3 for the treatment of depression: a new therapeutic concept

    PubMed Central

    Borsotto, M; Veyssiere, J; Moha ou Maati, H; Devader, C; Mazella, J; Heurteaux, C

    2015-01-01

    Depression is a disease that is particularly frequent, affecting up to 20% of the population in Western countries. The origins of this pathology involve multiple genes as well as environmental and developmental factors leading to a disorder that remains difficult to treat. Several therapies for depression have been developed and these mainly target monoamine neurotransmitters. However, these treatments are not only associated with numerous adverse effects, but they are also ineffective for more than one-third of patients. Therefore, the need to develop new concepts to treat depression is crucial. Recently, studies using knockout mouse models have provided evidence for a crucial role of two members of the two-pore domain potassium channel (K2P) family, tandem P-domain weak inward rectifying K+ (TWIK)-related K+ channel 1 (TREK-1) and TWIK-related acid-sensitive K+ channel 3 (TASK-3) in the pathophysiology of depression. It is believed that TREK-1 and TASK-3 antagonists could lead to the development of new antidepressants. Herein, we describe the discovery of spadin, a natural peptide released from the maturation of the neurotensin receptor-3 (also known as sortilin), which specifically blocks the activity of the TREK-1 channel and displays particular antidepressant properties, with a rapid onset of action and the absence of adverse effects. The development of such molecules may open a new era in the field of psychiatry. PMID:25263033

  6. Site-Selective Monitoring of the Interaction of the SRA Domain of UHRF1 with Target DNA Sequences Labeled with 2-Aminopurine.

    PubMed

    Greiner, Vanille J; Kovalenko, Lesia; Humbert, Nicolas; Richert, Ludovic; Birck, Catherine; Ruff, Marc; Zaporozhets, Olga A; Dhe-Paganon, Sirano; Bronner, Christian; Mély, Yves

    2015-10-06

    UHRF1 plays a central role in the maintenance and transmission of epigenetic modifications by recruiting DNMT1 to hemimethylated CpG sites via its SET and RING-associated (SRA) domain, ensuring error-free duplication of methylation profiles. To characterize SRA-induced changes in the conformation and dynamics of a target 12 bp DNA duplex as a function of the methylation status, we labeled duplexes by the environment-sensitive probe 2-aminopurine (2-Ap) at various positions near or far from the central CpG recognition site containing either a nonmodified cytosine (NM duplex), a methylated cytosine (HM duplex), or methylated cytosines on both strands (BM duplex). Steady-state and time-resolved fluorescence indicated that binding of SRA induced modest conformational and dynamical changes in NM, HM, and BM duplexes, with only slight destabilization of base pairs, restriction of global duplex flexibility, and diminution of local nucleobase mobility. Moreover, significant restriction of the local motion of residues flanking the methylcytosine in the HM duplex suggested that these residues are more rigidly bound to SRA, in line with a slightly higher affinity of the HM duplex as compared to that of the NM or BM duplex. Our results are consistent with a "reader" role, in which the SRA domain scans DNA sequences for hemimethylated CpG sites without perturbation of the structure of contacted nucleotides.

  7. The extra domain A from fibronectin targets antigens to TLR4-expressing cells and induces cytotoxic T cell responses in vivo.

    PubMed

    Lasarte, Juan J; Casares, Noelia; Gorraiz, Marta; Hervás-Stubbs, Sandra; Arribillaga, Laura; Mansilla, Cristina; Durantez, Maika; Llopiz, Diana; Sarobe, Pablo; Borrás-Cuesta, Francisco; Prieto, Jesús; Leclerc, Claude

    2007-01-15

    Vaccination strategies based on the in vivo targeting of Ags to dendritic cells (DCs) are needed to improve the induction of specific T cell immunity against tumors and infectious agents. In this study, we have used a recombinant protein encompassing the extra domain A from fibronectin (EDA), an endogenous ligand for TLR4, to deliver Ags to TLR4-expressing DC. The purified EDA protein was shown to bind to TLR4-expressing HEK293 cells and to activate the TLR4 signaling pathway. EDA also stimulated the production by DC of proinflammatory cytokines such as IL-12 or TNF-alpha and induced their maturation in vitro and in vivo. A fusion protein between EDA and a cytotoxic T cell epitope from OVA efficiently presented this epitope to specific T cells and induced the in vivo activation of a strong and specific CTL response. Moreover, a fusion protein containing EDA and the full OVA also improved OVA presentation by DC and induced CTL responses in vivo. These EDA recombinant proteins protected mice from a challenge with tumor cells expressing OVA. These results strongly suggest that the fibronectin extra domain A may serve as a suitable Ag carrier for the development of antiviral or antitumoral vaccines.

  8. Targeting two-pore domain K(+) channels TREK-1 and TASK-3 for the treatment of depression: a new therapeutic concept.

    PubMed

    Borsotto, M; Veyssiere, J; Moha Ou Maati, H; Devader, C; Mazella, J; Heurteaux, C

    2015-02-01

    Depression is a disease that is particularly frequent, affecting up to 20% of the population in Western countries. The origins of this pathology involve multiple genes as well as environmental and developmental factors leading to a disorder that remains difficult to treat. Several therapies for depression have been developed and these mainly target monoamine neurotransmitters. However, these treatments are not only associated with numerous adverse effects, but they are also ineffective for more than one-third of patients. Therefore, the need to develop new concepts to treat depression is crucial. Recently, studies using knockout mouse models have provided evidence for a crucial role of two members of the two-pore domain potassium channel (K2P ) family, tandem P-domain weak inward rectifying K(+) (TWIK)-related K(+) channel 1 (TREK-1) and TWIK-related acid-sensitive K(+) channel 3 (TASK-3) in the pathophysiology of depression. It is believed that TREK-1 and TASK-3 antagonists could lead to the development of new antidepressants. Herein, we describe the discovery of spadin, a natural peptide released from the maturation of the neurotensin receptor-3 (also known as sortilin), which specifically blocks the activity of the TREK-1 channel and displays particular antidepressant properties, with a rapid onset of action and the absence of adverse effects. The development of such molecules may open a new era in the field of psychiatry.

  9. Targeting the Transient Receptor Potential Vanilloid Type 1 (TRPV1) Assembly Domain Attenuates Inflammation-induced Hypersensitivity*

    PubMed Central

    Flynn, Robyn; Chapman, Kevin; Iftinca, Mircea; Aboushousha, Reem; Varela, Diego; Altier, Christophe

    2014-01-01

    The transient receptor potential channel vanilloid type 1 (TRPV1) is a non-selective cation channel expressed in sensory neurons of the dorsal root and trigeminal ganglia. TRPV1 is a polymodal channel activated by noxious heat, capsaicin, and protons. As a sensor for noxious stimuli, TRPV1 channel has been described as a key contributor to pain signaling. To form a functional channel, TRPV1 subunits must assemble into tetramers, and several studies have identified the TRPV1 C terminus as an essential element in subunit association. Here we combined biochemical assays with electrophysiology and imaging-based bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) in live cells to identify a short motif in the C-terminal tail of the TRPV1 subunit that governs channel assembly. Removing this region through early truncation or targeted deletion results in loss of subunit association and channel function. Importantly, we found that interfering with TRPV1 subunit association using a plasma membrane-tethered peptide attenuated mechanical and thermal hypersensitivity in two mouse models of inflammatory hyperalgesia. This represents a novel mechanism to disrupt TRPV1 subunit assembly and hence may offer a new analgesic tool for pain relief. PMID:24808184

  10. Targeting the transient receptor potential vanilloid type 1 (TRPV1) assembly domain attenuates inflammation-induced hypersensitivity.

    PubMed

    Flynn, Robyn; Chapman, Kevin; Iftinca, Mircea; Aboushousha, Reem; Varela, Diego; Altier, Christophe

    2014-06-13

    The transient receptor potential channel vanilloid type 1 (TRPV1) is a non-selective cation channel expressed in sensory neurons of the dorsal root and trigeminal ganglia. TRPV1 is a polymodal channel activated by noxious heat, capsaicin, and protons. As a sensor for noxious stimuli, TRPV1 channel has been described as a key contributor to pain signaling. To form a functional channel, TRPV1 subunits must assemble into tetramers, and several studies have identified the TRPV1 C terminus as an essential element in subunit association. Here we combined biochemical assays with electrophysiology and imaging-based bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) in live cells to identify a short motif in the C-terminal tail of the TRPV1 subunit that governs channel assembly. Removing this region through early truncation or targeted deletion results in loss of subunit association and channel function. Importantly, we found that interfering with TRPV1 subunit association using a plasma membrane-tethered peptide attenuated mechanical and thermal hypersensitivity in two mouse models of inflammatory hyperalgesia. This represents a novel mechanism to disrupt TRPV1 subunit assembly and hence may offer a new analgesic tool for pain relief.

  11. Interaction of 14-3-3 proteins with the estrogen receptor alpha F domain provides a drug target interface.

    PubMed

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

    2013-05-28

    Estrogen receptor alpha (ERα) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ERα, either by blocking estrogen production through aromatase inhibitors or antiestrogens that compete for hormone binding. Due to resistance, new treatment modalities are needed and as ERα dimerization is essential for its activity, interference with receptor dimerization offers a new opportunity to exploit in drug design. Here we describe a unique mechanism of how ERα dimerization is negatively controlled by interaction with 14-3-3 proteins at the extreme C terminus of the receptor. Moreover, the small-molecule fusicoccin (FC) stabilizes this ERα/14-3-3 interaction. Cocrystallization of the trimeric ERα/14-3-3/FC complex provides the structural basis for this stabilization and shows the importance of phosphorylation of the penultimate Threonine (ERα-T(594)) for high-affinity interaction. We confirm that T(594) is a distinct ERα phosphorylation site in the breast cancer cell line MCF-7 using a phospho-T(594)-specific antibody and by mass spectrometry. In line with its ERα/14-3-3 interaction stabilizing effect, fusicoccin reduces the estradiol-stimulated ERα dimerization, inhibits ERα/chromatin interactions and downstream gene expression, resulting in decreased cell proliferation. Herewith, a unique functional phosphosite and an alternative regulation mechanism of ERα are provided, together with a small molecule that selectively targets this ERα/14-3-3 interface.

  12. Targeting RING domains of Mdm2-MdmX E3 complex activates apoptotic arm of the p53 pathway in leukemia/lymphoma cells.

    PubMed

    Wu, W; Xu, C; Ling, X; Fan, C; Buckley, B P; Chernov, M V; Ellis, L; Li, F; Muñoz, I G; Wang, X

    2015-12-31

    Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2-p53 interface or MdmX ((MDM4), mouse double minute 4)-p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2-MdmX really interesting new gene (RING)-RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2-MdmX RING-RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2-MdmX RING domain inhibitors)) that specifically inhibit Mdm2-MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2-MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2-MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.

  13. Nuclear localization domains of GATA activator Gln3 are required for transcription of target genes through dephosphorylation in Saccharomyces cerevisiae.

    PubMed

    Numamoto, Minori; Tagami, Shota; Ueda, Yusuke; Imabeppu, Yusuke; Sasano, Yu; Sugiyama, Minetaka; Maekawa, Hiromi; Harashima, Satoshi

    2015-08-01

    The GATA transcription activator Gln3 in the budding yeast (Saccharomyces cerevisiae) activates transcription of nitrogen catabolite repression (NCR)-sensitive genes. In cells grown in the presence of preferred nitrogen sources, Gln3 is phosphorylated in a TOR-dependent manner and localizes in the cytoplasm. In cells grown in non-preferred nitrogen medium or treated with rapamycin, Gln3 is dephosphorylated and is transported from the cytoplasm to the nucleus, thereby activating the transcription of NCR-sensitive genes. Caffeine treatment also induces dephosphorylation of Gln3 and its translocation to the nucleus and transcription of NCR-sensitive genes. However, the details of the mechanism by which phosphorylation controls Gln3 localization and transcriptional activity are unknown. Here, we focused on two regions of Gln3 with nuclear localization signal properties (NLS-K, and NLS-C) and one with nuclear export signal (NES). We constructed various mutants for our analyses: gln3 containing point mutations in all potential phosphoacceptor sites (Thr-339, Ser-344, Ser-347, Ser-355, Ser-391) in the NLS and NES regions to produce non-phosphorylatable (alanine) or mimic-phosphorylatable (aspartic acid) residues; and deletion mutants. We found that phosphorylation of Gln3 was impaired in all of these mutations and that the aspartic acid substitution mutants showed drastic reduction of Gln3-mediated transcriptional activity despite the fact that the mutations had no effect on nuclear localization of Gln3. Our observations suggest that these regions are required for transcription of target genes presumably through dephosphorylation.

  14. Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

    PubMed Central

    O'Herron, Philip; Shen, Zhiming; Lu, Zhongyang; Schramm, Adrien E.; Levy, Manuel; Kara, Prakash

    2012-01-01

    In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub

  15. Chromatin remodeling gene AT-rich interactive domain-containing protein 1A suppresses gastric cancer cell proliferation by targeting PIK3CA and PDK1

    PubMed Central

    Wang, Jie; Cui, Shu-Jian; Wang, Xiao-Qing; Jiang, Ying-Hua; Feng, Li; Yang, Peng-Yuan; Liu, Feng

    2016-01-01

    The tumor suppressor gene AT-rich interactive domain-containing protein 1A (ARID1A) was frequently mutated in cancers. The modulation mechanism of ARID1A for PI3K/AKT signaling in gastric cancer (GC) remains elusive. Here, we found that depletion of endogenous ARID1A enhanced the in vitro proliferation, colony formation, cellular growth, nutrient uptake and in vivo xenograft tumor growth of GC cells. PI3K/AKT activation by ARID1A-silencing was profiled using a phospho-protein antibody array. The phosphorylation of PDK1, AKT, GSK3β and 70S6K, and the protein and mRNA expressions of PI3K and PDK1, were upregulated by ARID1A-silencing. Chromatin immunoprecipitation and luciferase reporter assay revealed that ARID1A-involved SWI/SNF complex inhibited PIK3CA and PDK1 transcription by direct binding to their promoters. Serial deletion mutation analyses revealed that the ARID1A central region containing the HIC1-binding domain, but not the ARID DNA-binding domain and the C-terminal domain, was essential for the inhibition of GC cell growth, PI3K/AKT pathway phosphorylation and its transcriptional modulation activity of PIK3CA and PDK1. The proliferation, cellular growth and glucose consumption of ARID1A-deficient GC cells were efficiently prohibited by allosteric inhibitors mk2206 and LY294002, which targeting AKT and PI3K, respectively. Both inhibitors also downregulated the phosphorylation of PI3K/AKT pathway in ARID1A-deficient GC cells. Such cells were sensitized to the treatment of LY294002, and AT7867, another inhibitor of AKT and p70S6K. The administration of LY294002 alone inhibited the in vivo growth of ARID1A- deficient GC cells in mouse xenograft model. Our study provides a novel insight into the modulatory function and mechanism of ARID1A in PI3K/AKT signaling in GC. PMID:27323812

  16. Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell–Driven Protective Response

    PubMed Central

    Nico, Dirlei; Claser, Carla; Borja-Cabrera, Gulnara P.; Travassos, Luiz R.; Palatnik, Marcos; da Silva Soares, Irene; Rodrigues, Mauricio Martins; Palatnik-de-Sousa, Clarisa B.

    2010-01-01

    Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199–314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5–88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for

  17. Antibodies targeting dengue virus envelope domain III are not required for serotype-specific protection or prevention of enhancement in vivo.

    PubMed

    Williams, Katherine L; Wahala, Wahala M P B; Orozco, Susana; de Silva, Aravinda M; Harris, Eva

    2012-07-20

    The envelope (E) protein of dengue virus (DENV) is composed of three domains (EDI, EDII, EDIII) and is the main target of neutralizing antibodies. Many monoclonal antibodies that bind EDIII strongly neutralize DENV. However in vitro studies indicate that anti-EDIII antibodies contribute little to the neutralizing potency of human DENV-immune serum. In this study, we assess the role of anti-EDIII antibodies in mouse and human DENV-immune serum in neutralizing or enhancing DENV infection in mice. We demonstrate that EDIII-depleted human DENV-immune serum was protective against homologous DENV infection in vivo. Although EDIII-depleted DENV-immune mouse serum demonstrated decreased neutralization potency in vitro, reduced protection in some organs, and enhanced disease in vivo, administration of increased volumes of EDIII-depleted serum abrogated these effects. These data indicate that anti-EDIII antibodies contribute to protection and minimize enhancement when present, but can be replaced by neutralizing antibodies targeting other epitopes on the dengue virion.

  18. Distinct functional and conformational states of the human lymphoid tyrosine phosphatase catalytic domain can be targeted by choice of the inhibitor chemotype

    NASA Astrophysics Data System (ADS)

    Vidović, Dušica; Xie, Yuli; Rinderspacher, Alison; Deng, Shi-Xian; Landry, Donald W.; Chung, Caty; Smith, Deborah H.; Tautz, Lutz; Schürer, Stephan C.

    2011-09-01

    The lymphoid tyrosine phosphatase (LYP), encoded by the PTPN22 gene, has recently been identified as a promising drug target for human autoimmunity diseases. Like the majority of protein-tyrosine phosphatases LYP can adopt two functionally distinct forms determined by the conformation of the WPD-loop. The WPD-loop plays an important role in the catalytic dephosphorylation by protein-tyrosine phosphatases. Here we investigate the binding modes of two chemotypes of small molecule LYP inhibitors with respect to both protein conformations using computational modeling. To evaluate binding in the active form, we built a LYP protein structure model of high quality. Our results suggest that the two different compound classes investigated, bind to different conformations of the LYP phosphatase domain. Binding to the closed form is facilitated by an interaction with Asp195 in the WPD-loop, presumably stabilizing the active conformation. The analysis presented here is relevant for the design of inhibitors that specifically target either the closed or the open conformation of LYP in order to achieve better selectivity over phosphatases with similar binding sites.

  19. Inhibition of PlexA1-mediated brain tumor growth and tumor-associated angiogenesis using a transmembrane domain targeting peptide

    PubMed Central

    Jacob, Laurent; Goetz, Jacky; Vermot, Julien; Fernandez, Aurore; Baumlin, Nadège; Aci-Sèche, Samia; Orend, Gertraud; Roussel, Guy; Crémel, Gérard; Genest, Monique; Hubert, Pierre; Bagnard, Dominique

    2016-01-01

    The neuropilin-plexin receptor complex regulates tumor cell migration and proliferation and thus is an interesting therapeutic target. High expression of neuropilin-1 is indeed associated with a bad prognosis in glioma patients. Q-RTPCR and tissue-array analyses showed here that Plexin-A1 is highly expressed in glioblastoma and that the highest level of expression correlates with the worse survival of patients. We next identified a developmental and tumor-associated pro-angiogenic role of Plexin-A1. Hence, by using molecular simulations and a two-hybrid like assay in parallel with biochemical and cellular assays we developed a specific Plexin-A1 peptidic antagonist disrupting transmembrane domain-mediated oligomerization of the receptor and subsequent signaling and functional activity. We found that this peptide exhibits anti-tumor activity in vivo on different human glioblastoma models including glioma cancer stem cells. Thus, screening Plexin-A1 expression and targeting Plexin-A1 in glioblastoma patients exhibit diagnostic and therapeutic value. PMID:27506939

  20. Inhibition of PlexA1-mediated brain tumor growth and tumor-associated angiogenesis using a transmembrane domain targeting peptide.

    PubMed

    Jacob, Laurent; Sawma, Paul; Garnier, Norbert; Meyer, Lionel A T; Fritz, Justine; Hussenet, Thomas; Spenlé, Caroline; Goetz, Jacky; Vermot, Julien; Fernandez, Aurore; Baumlin, Nadège; Aci-Sèche, Samia; Orend, Gertraud; Roussel, Guy; Crémel, Gérard; Genest, Monique; Hubert, Pierre; Bagnard, Dominique

    2016-09-06

    The neuropilin-plexin receptor complex regulates tumor cell migration and proliferation and thus is an interesting therapeutic target. High expression of neuropilin-1 is indeed associated with a bad prognosis in glioma patients. Q-RTPCR and tissue-array analyses showed here that Plexin-A1 is highly expressed in glioblastoma and that the highest level of expression correlates with the worse survival of patients. We next identified a developmental and tumor-associated pro-angiogenic role of Plexin-A1. Hence, by using molecular simulations and a two-hybrid like assay in parallel with biochemical and cellular assays we developed a specific Plexin-A1 peptidic antagonist disrupting transmembrane domain-mediated oligomerization of the receptor and subsequent signaling and functional activity. We found that this peptide exhibits anti-tumor activity in vivo on different human glioblastoma models including glioma cancer stem cells. Thus, screening Plexin-A1 expression and targeting Plexin-A1 in glioblastoma patients exhibit diagnostic and therapeutic value.

  1. Targeting polyIC to EGFR over-expressing cells using a dsRNA binding protein domain tethered to EGF

    PubMed Central

    Edinger, Nufar; Lebendiker, Mario; Klein, Shoshana; Zigler, Maya; Langut, Yael; Levitzki, Alexander

    2016-01-01

    Selective delivery of drugs to tumor cells can increase potency and reduce toxicity. In this study, we describe a novel recombinant chimeric protein, dsRBEC, which can bind polyIC and deliver it selectively into EGFR over-expressing tumor cells. dsRBEC, comprises the dsRNA binding domain (dsRBD) of human PKR (hPKR), which serves as the polyIC binding moiety, fused to human EGF (hEGF), the targeting moiety. dsRBEC shows high affinity towards EGFR and triggers ligand-induced endocytosis of the receptor, thus leading to the selective internalization of polyIC into EGFR over-expressing tumor cells. The targeted delivery of polyIC by dsRBEC induced cellular apoptosis and the secretion of IFN-β and other pro-inflammatory cytokines. dsRBEC-delivered polyIC is much more potent than naked polyIC and is expected to reduce the toxicity caused by systemic delivery of polyIC. PMID:27598772

  2. Structure-based discovery of an inhibitor of Arf activation by Sec7 domains through targeting of protein-protein complexes.

    PubMed

    Viaud, Julien; Zeghouf, Mahel; Barelli, Hélène; Zeeh, Jean-Christophe; Padilla, André; Guibert, Bernard; Chardin, Pierre; Royer, Catherine A; Cherfils, Jacqueline; Chavanieu, Alain

    2007-06-19

    Small molecules that produce nonfunctional protein-protein complexes are an alternative to competitive inhibitors for the inhibition of protein functions. Here we target the activation of the small GTP-binding protein Arf1, a major regulator of membrane traffic, by the Sec7 catalytic domain of its guanine nucleotide exchange factor ARNO. The crystal structure of the Arf1-GDP/ARNO complex, which initiates the exchange reaction, was used to discover an inhibitor, LM11, using in silico screening of a flexible pocket near the Arf1/ARNO interface. Using fluorescence kinetics and anisotropy, NMR spectroscopy and mutagenesis, we show that LM11 acts following a noncompetitive mechanism in which the inhibitor targets both Arf1-GDP and the Arf1-GDP/ARNO complex and produces a nonfunctional Arf-GDP/ARNO complex whose affinity is similar to that of the native complex. In addition, LM11 recognizes features of both Arf and ARNO near the Arf/Sec7 interface, a characteristic reminiscent of the paradigm interfacial inhibitor Brefeldin A. We then show that LM11 is a cell-active inhibitor that impairs Arf-dependent trafficking structures at the Golgi. Furthermore, LM11 inhibits ARNO-dependent migration of Madin-Darby canine kidney (MDCK) cells, demonstrating that ARNO is a target of LM11 in cells. Remarkably, LM11 inhibits the activation of Arf1 but not Arf6 in vitro, pointing to a possible synergy between Arf1 and Arf6 activation by ARNO in cell migration. Our design method shows that flexible regions in protein-protein complexes provide drugable sites with the potential to develop novel tools for investigating and inhibiting signaling pathways.

  3. Identification of a Chromogranin A Domain That Mediates Binding to Secretogranin III and Targeting to Secretory Granules in Pituitary Cells and Pancreatic β-Cells

    PubMed Central

    Hosaka, Masahiro; Watanabe, Tsuyoshi; Sakai, Yuko; Uchiyama, Yasuo; Takeuchi, Toshiyuki

    2002-01-01

    Chromogranin A (CgA) is transported restrictedly to secretory granules in neuroendocrine cells. In addition to pH- and Ca2+-dependent aggregation, CgA is known to bind to a number of vesicle matrix proteins. Because the binding-prone property of CgA with secretory proteins may be essential for its targeting to secretory granules, we screened its binding partner proteins using a yeast two-hybrid system. We found that CgA bound to secretogranin III (SgIII) by specific interaction both in vitro and in endocrine cells. Localization analysis showed that CgA and SgIII were coexpressed in pituitary and pancreatic endocrine cell lines, whereas SgIII was not expressed in the adrenal glands and PC12 cells. Immunoelectron microscopy demonstrated that CgA and SgIII were specifically colocalized in large secretory granules in male rat gonadotropes, which possess large-type and small-type granules. An immunocytochemical analysis revealed that deletion of the binding domain (CgA 48–111) for SgIII missorted CgA to the constitutive pathway, whereas deletion of the binding domain (SgIII 214–373) for CgA did not affect the sorting of SgIII to the secretory granules in AtT-20 cells. These findings suggest that CgA localizes with SgIII by specific binding in secretory granules in SgIII-expressing pituitary and pancreatic endocrine cells, whereas other mechanisms are likely to be responsible for CgA localization in secretory granules of SgIII-lacking adrenal chromaffin cells and PC12 cells. PMID:12388744

  4. BTB-BACK Domain Protein POB1 Suppresses Immune Cell Death by Targeting Ubiquitin E3 ligase PUB17 for Degradation

    PubMed Central

    Mesmar, Joelle; McLellan, Hazel; Yang, Chengwei; Craig, Adam; Zhang, Cunjin; Moore, Jonathan David; Tian, Zhendong; Birch, Paul R. J.; Sadanandom, Ari

    2017-01-01

    Hypersensitive response programmed cell death (HR-PCD) is a critical feature in plant immunity required for pathogen restriction and prevention of disease development. The precise control of this process is paramount to cell survival and an effective immune response. The discovery of new components that function to suppress HR-PCD will be instrumental in understanding the regulation of this fundamental mechanism. Here we report the identification and characterisation of a BTB domain E3 ligase protein, POB1, that functions to suppress HR-PCD triggered by evolutionarily diverse pathogens. Nicotiana benthamiana and tobacco plants with reduced POB1 activity show accelerated HR-PCD whilst those with increased POB1 levels show attenuated HR-PCD. We demonstrate that POB1 dimerization and nuclear localization are vital for its function in HR-PCD suppression. Using protein-protein interaction assays, we identify the Plant U-Box E3 ligase PUB17, a well established positive regulator of plant innate immunity, as a target for POB1-mediated proteasomal degradation. Using confocal imaging and in planta immunoprecipitation assays we show that POB1 interacts with PUB17 in the nucleus and stimulates its degradation. Mutated versions of POB1 that show reduced interaction with PUB17 fail to suppress HR-PCD, indicating that POB1-mediated degradation of PUB17 U-box E3 ligase is an important step for negative regulation of specific immune pathways in plants. Our data reveals a new mechanism for BTB domain proteins in suppressing HR-PCD in plant innate immune responses. PMID:28056034

  5. Post-translational processing of surfactant protein-C proprotein: targeting motifs in the NH(2)-terminal flanking domain are cleaved in late compartments.

    PubMed

    Johnson, A L; Braidotti, P; Pietra, G G; Russo, S J; Kabore, A; Wang, W J; Beers, M F

    2001-03-01

    Rat surfactant protein (SP)-C is a 3.7-kD hydrophobic lung-specific protein generated from proteolytic processing of a 21-kD propeptide (SP-C(21)). We have demonstrated that initial post-translational processing of SP-C(21) involves two cleavages of the COOH-terminus (Beers and colleagues, J. Biol. Chem. 1994;269:20,318--20,328). The goal of the current study was to define processing and function of the NH(2)-terminal flanking domain. Epitope-specific antisera directed against spatially distinct regions of the NH(2) terminus, NPROSP-C(2-9) (epitope = D(2)-L(9)) and NPROSP-C(11-23) (= E(11)-Q(23)) were produced. By Western blotting, both antisera identified SP-C(21) in microsomes. A 6-kD form (SP-C(6)), enriched in lamellar bodies (LBs), was detected only by NPROSP-C(11-23) and not extractable with NaCO(3) treatment. Immunogold staining of ultrathin lung sections with NPROSP-C(11-23) identified proSP-C in both multivesicular bodies (mvb) and LBs whereas NPROSP-C(2-9) labeled only mvb. (35)S-pulse chase analysis demonstrated synthesis of SP-C(21) and three intermediate forms (SP-C(16), SP-C(7), and SP-C(6)). Complete processing involved four separate cleavages with a precursor- product relationship between the low molecular weight forms SP-C(7) and SP-C(6). Fluorescence microscopy of A549 cells expressing fusion proteins of enhanced green fluorescent protein (EGFP) and proSP-C NH(2)-terminal deletion mutants showed targeting of EGFP/SP-C(1-194) and EGFP/SP-C(10-194) to early endosomal antigen-1-negative, CD-63-positive cytoplasmic vesicles whereas EGFP/SP-C(19-194), EGFP/SP-C(Delta 10-18), and EGFP/SP-C(24-194) were restricted to the endoplasmic reticulum (ER). We conclude that synthetic processing includes a previously unrecognized cleavage of the proximal NH(2) terminus (M(1)-L(9)), which occurs after removal of COOH-flanking domains (H(59)-I(194)) but before packaging in LBs, and that the region M(10)-T(18) is required for targeting of proSP-C to post-ER vesicular

  6. Release of Nonmuscle Myosin II from the Cytosolic Domain of Tumor Necrosis Factor Receptor 2 Is Required for Target Gene Expression

    PubMed Central

    Chandrasekharan, Unni M.; Dechert, Lisa; Davidson, Uchechukwu I.; Waitkus, Matthew; Mavrakis, Lori; Lyons, Katherine; Beach, Jordan R.; Li, Xiaoxia; Egelhoff, Thomas T.; Fox, Paul L.; DiCorleto, Paul E.

    2013-01-01

    Tumor necrosis factor α (TNF-α) elicits its biological activities through activation of TNF receptor 1 (TNFR1, also known as p55) and TNFR2 (also known as p75). The activities of both receptors are required for the TNF-α–induced proinflammatory response. The adaptor protein TNFR-associated factor 2 (TRAF2) is critical for either p55- or p75-mediated activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, as well as for target gene expression. Here, we identified nonmuscle myosin II (myosin) as a binding partner of p75. TNF-α–dependent signaling by p75 and induction of target gene expression persisted for substantially longer in cells deficient in myosin regulatory light chain (MRLC, a component of myosin) than in cells replete in myosin. In resting endothelial cells, myosin was bound constitutively to the intracellular region of p75, a region that overlaps with the TRAF2-binding domain, and TNF-α caused the rapid dissociation of myosin from p75. At early time points after exposure to TNF-α, p75 activated Rho-associated kinase 1 (ROCK1). Inhibition of ROCK1 activity blocked TNF-α–dependent phosphorylation of MRLC and the dissociation of myosin from p75. ROCK1-dependent release of myosin was necessary for the TNF-α–dependent recruitment of TRAF2 to p75 and for p75-specific activation of NF-κB and MAPK signaling. Thus, our findings have revealed a previously uncharacterized, noncanonical regulatory function of myosin in cytokine signaling. PMID:23861542

  7. The Transmembrane Domain of HIV-1 gp41 Inhibits T-Cell Activation by Targeting Multiple T-Cell Receptor Complex Components through Its GxxxG Motif.

    PubMed

    Rotem, Etai; Reuven, Eliran Moshe; Klug, Yoel A; Shai, Yechiel

    2016-02-23

    To successfully infect and persist within its host, HIV-1 utilizes several immunosuppressive motifs within its gp41 envelope glycoprotein to manipulate and evade the immune system. The transmembrane domain (TMD) of gp41 downregulates T-cell receptor (TCR) signaling through a hitherto unknown mechanism. Interactions between TMDs within the membrane milieu have been shown to be typically mediated by particular amino acids, such as interactions between basic and acidic residues and dimerization motifs as GxxxG. The HIV-1 TMD exhibits both a polar arginine (Arg(696)) residue and a GxxxG motif, making them ideal candidates for mediators of TMD-TCR interaction. Using a primary T-cell activation assay and biochemical and biophysical methods, we demonstrate that the gp41 TMD directly interacts with TMDs of the TCR and the CD3 coreceptors (δ, γ, and ε) within the membrane, presumably leading to impairment of complex assembly. Additionally, we reveal that although Arg(696) does not affect TMD immunosuppression, the GxxxG motif is crucial in mediating gp41's TMD interaction with the CD3 coreceptors of the TCR. These findings suggest that compared with other gp41 immunosuppressive motifs, the gp41 TMD has multiple targets within the TCR complex, suggesting less susceptibility to evolutionary pressure and consequently being advantageous for the virus over the host immune response. Furthermore, as the GxxxG motif mediates interactions of the gp41 TMD with multiple receptors, it emerges as an attractive drug target. This multitarget inhibitory mechanism might be a strategy utilized by HIV to interfere with the function of additional host receptors.

  8. Eps15 Homology Domain-containing Protein 3 Regulates Cardiac T-type Ca2+ Channel Targeting and Function in the Atria*

    PubMed Central

    Curran, Jerry; Musa, Hassan; Kline, Crystal F.; Makara, Michael A.; Little, Sean C.; Higgins, John D.; Hund, Thomas J.; Band, Hamid; Mohler, Peter J.

    2015-01-01

    Proper trafficking of membrane-bound ion channels and transporters is requisite for normal cardiac function. Endosome-based protein trafficking of membrane-bound ion channels and transporters in the heart is poorly understood, particularly in vivo. In fact, for select cardiac cell types such as atrial myocytes, virtually nothing is known regarding endosomal transport. We previously linked the C-terminal Eps15 homology domain-containing protein 3 (EHD3) with endosome-based protein trafficking in ventricular cardiomyocytes. Here we sought to define the roles and membrane protein targets for EHD3 in atria. We identify the voltage-gated T-type Ca2+ channels (CaV3.1, CaV3.2) as substrates for EHD3-dependent trafficking in atria. Mice selectively lacking EHD3 in heart display reduced expression and targeting of both Cav3.1 and CaV3.2 in the atria. Furthermore, functional experiments identify a significant loss of T-type-mediated Ca2+ current in EHD3-deficient atrial myocytes. Moreover, EHD3 associates with both CaV3.1 and CaV3.2 in co-immunoprecipitation experiments. T-type Ca2+ channel function is critical for proper electrical conduction through the atria. Consistent with these roles, EHD3-deficient mice demonstrate heart rate variability, sinus pause, and atrioventricular conduction block. In summary, our findings identify CaV3.1 and CaV3.2 as substrates for EHD3-dependent protein trafficking in heart, provide in vivo data on endosome-based trafficking pathways in atria, and implicate EHD3 as a key player in the regulation of atrial myocyte excitability and cardiac conduction. PMID:25825486

  9. Eps15 Homology Domain-containing Protein 3 Regulates Cardiac T-type Ca2+ Channel Targeting and Function in the Atria.

    PubMed

    Curran, Jerry; Musa, Hassan; Kline, Crystal F; Makara, Michael A; Little, Sean C; Higgins, John D; Hund, Thomas J; Band, Hamid; Mohler, Peter J

    2015-05-08

    Proper trafficking of membrane-bound ion channels and transporters is requisite for normal cardiac function. Endosome-based protein trafficking of membrane-bound ion channels and transporters in the heart is poorly understood, particularly in vivo. In fact, for select cardiac cell types such as atrial myocytes, virtually nothing is known regarding endosomal transport. We previously linked the C-terminal Eps15 homology domain-containing protein 3 (EHD3) with endosome-based protein trafficking in ventricular cardiomyocytes. Here we sought to define the roles and membrane protein targets for EHD3 in atria. We identify the voltage-gated T-type Ca(2+) channels (CaV3.1, CaV3.2) as substrates for EHD3-dependent trafficking in atria. Mice selectively lacking EHD3 in heart display reduced expression and targeting of both Cav3.1 and CaV3.2 in the atria. Furthermore, functional experiments identify a significant loss of T-type-mediated Ca(2+) current in EHD3-deficient atrial myocytes. Moreover, EHD3 associates with both CaV3.1 and CaV3.2 in co-immunoprecipitation experiments. T-type Ca(2+) channel function is critical for proper electrical conduction through the atria. Consistent with these roles, EHD3-deficient mice demonstrate heart rate variability, sinus pause, and atrioventricular conduction block. In summary, our findings identify CaV3.1 and CaV3.2 as substrates for EHD3-dependent protein trafficking in heart, provide in vivo data on endosome-based trafficking pathways in atria, and implicate EHD3 as a key player in the regulation of atrial myocyte excitability and cardiac conduction.

  10. Straightforward selection of broadly neutralizing single-domain antibodies targeting the conserved CD4 and coreceptor binding sites of HIV-1 gp120.

    PubMed

    Matz, Julie; Kessler, Pascal; Bouchet, Jérôme; Combes, Olivier; Ramos, Oscar Henrique Pereira; Barin, Francis; Baty, Daniel; Martin, Loïc; Benichou, Serge; Chames, Patrick

    2013-01-01

    Few broadly neutralizing antibodies targeting determinants of the HIV-1 surface envelope glycoprotein (gp120) involved in sequential binding to host CD4 and chemokine receptors have been characterized. While these epitopes show low diversity among various isolates, HIV-1 employs many strategies to evade humoral immune response toward these sensitive sites, including a carbohydrate shield, low accessibility to these buried cavities, and conformational masking. Using trimeric gp140, free or bound to a CD4 mimic, as immunogens in llamas, we selected a panel of broadly neutralizing single-domain antibodies (sdAbs) that bind to either the CD4 or the coreceptor binding site (CD4BS and CoRBS, respectively). When analyzed as monomers or as homo- or heteromultimers, the best sdAb candidates could not only neutralize viruses carrying subtype B envelopes, corresponding to the Env molecule used for immunization and selection, but were also efficient in neutralizing a broad panel of envelopes from subtypes A, C, G, CRF01_AE, and CRF02_AG, including tier 3 viruses. Interestingly, sdAb multimers exhibited a broader neutralizing activity spectrum than the parental sdAb monomers. The extreme stability and high recombinant production yield combined with their broad neutralization capacity make these sdAbs new potential microbicide candidates for HIV-1 transmission prevention.

  11. A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1

    NASA Astrophysics Data System (ADS)

    Deyle, Kaycie M.; Farrow, Blake; Qiao Hee, Ying; Work, Jeremy; Wong, Michelle; Lai, Bert; Umeda, Aiko; Millward, Steven W.; Nag, Arundhati; Das, Samir; Heath, James R.

    2015-05-01

    Ligands that can bind selectively to proteins with single amino-acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wild type (WT). However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical synthetic epitope-targeting strategy that we used to discover a 5-mer peptide with selectivity for the E17K-transforming point mutation in the pleckstrin homology domain of the Akt1 oncoprotein. A fragment of Akt1 that contained the E17K mutation and an I19[propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that clicked covalently onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to the WT, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) substrate.

  12. A Novel Multivalent, Single-Domain Antibody Targeting TcdA and TcdB Prevents Fulminant Clostridium difficile Infection in Mice

    PubMed Central

    Yang, Zhiyong; Schmidt, Diane; Liu, Weilong; Li, Shan; Shi, Lianfa; Sheng, Jinliang; Chen, Kevin; Yu, Hua; Tremblay, Jacqueline M.; Chen, Xinhua; Piepenbrink, Kurt H.; Sundberg, Eric J.; Kelly, Ciaran P.; Bai, Guang; Shoemaker, Charles B.; Feng, Hanping

    2014-01-01

    The incidence of Clostridium difficile infection (CDI) and associated mortality have increased rapidly worldwide in recent years. Therefore, it is critical to develop new therapies for CDI. In this study, we generated a novel, potently neutralizing, tetravalent, and bispecific antibody composed of 2 heavy-chain-only VH (VHH) binding domains against both TcdA and TcdB (designated “ABA”) that reverses fulminant CDI in mice infected with an epidemic 027 strain after a single injection of the antibody. We demonstrated that ABA bound to both toxins simultaneously and displayed a significantly enhanced neutralizing activity both in vitro and in vivo. Additionally, ABA was able to broadly neutralize toxins from clinical C. difficile isolates that express both TcdA and TcdB but failed to neutralize the toxin from TcdA−TcdB+ C. difficile strains. This study thus provides a rationale for the development of multivalent VHHs that target both toxins and are broadly neutralizing for treating severe CDI. PMID:24683195

  13. Animal Protection and Structural Studies of a Consensus Sequence Vaccine Targeting the Receptor Binding Domain of the Type IV Pilus of Pseudomonas aeruginosa

    SciTech Connect

    Kao, Daniel J.; Churchill, Mair E.A.; Irvin, Randall T.; Hodges, Robert S.

    2008-09-23

    One of the main obstacles in the development of a vaccine against Pseudomonas aeruginosa is the requirement that it is protective against a wide range of virulent strains. We have developed a synthetic-peptide consensus-sequence vaccine (Cs1) that targets the host receptor-binding domain (RBD) of the type IV pilus of P. aeruginosa. Here, we show that this vaccine provides increased protection against challenge by the four piliated strains that we have examined (PAK, PAO, KB7 and P1) in the A.BY/SnJ mouse model of acute P. aeruginosa infection. To further characterize the consensus sequence, we engineered Cs1 into the PAK monomeric pilin protein and determined the crystal structure of the chimeric Cs1 pilin to 1.35 {angstrom} resolution. The substitutions (T130K and E135P) used to create Cs1 do not disrupt the conserved backbone conformation of the pilin RBD. In fact, based on the Cs1 pilin structure, we hypothesize that the E135P substitution bolsters the conserved backbone conformation and may partially explain the immunological activity of Cs1. Structural analysis of Cs1, PAK and K122-4 pilins reveal substitutions of non-conserved residues in the RBD are compensated for by complementary changes in the rest of the pilin monomer. Thus, the interactions between the RBD and the rest of the pilin can either be mediated by polar interactions of a hydrogen bond network in some strains or by hydrophobic interactions in others. Both configurations maintain a conserved backbone conformation of the RBD. Thus, the backbone conformation is critical in our consensus-sequence vaccine design and that cross-reactivity of the antibody response may be modulated by the composition of exposed side-chains on the surface of the RBD. This structure will guide our future vaccine design by focusing our investigation on the four variable residue positions that are exposed on the RBD surface.

  14. Epitope mapping of inhibitory antibodies targeting the C2 domain of coagulation factor VIII by hydrogen-deuterium exchange mass spectrometry

    PubMed Central

    Sevy, Alexander M.; Healey, John F.; Deng, Wei; Spiegel, P. Clint; Meeks, Shannon L.; Li, Renhao

    2014-01-01

    Summary Background The development of anti-factor VIII (fVIII) antibodies (inhibitors) is a significant complication in the management of patients with hemophilia A, leading to significant increases in morbidity and treatment cost. Using a panel of anti-fVIII monoclonal antibodies (MAbs) to different epitopes on fVIII, we recently have shown that epitope specificity, inhibitor kinetics, and time to maximum inhibition are more important than inhibitor titer in predicting response to fVIII and the combination of fVIII and recombinant factor VIIa. In particular, a subset of high-titer inhibitors responded to high dose fVIII, which would not be predicted based on their inhibitor titer alone. Thus the ability to quickly map the epitope spectrum of patient plasma using a clinically feasible assay may fundamentally change how clinicians approach the treatment of high-titer inhibitor patients. Objectives To map the epitopes of anti-fVIII MAbs, of which 3 are classical inhibitors and one non-classical, using hydrogen-deuterium exchange coupled with liquid chromatography-mass spectrometry (HDX-MS). Methods Binding epitopes of 4 MAbs targeting fVIII C2 domain were mapped using HDX-MS. Results The epitopes determined by HDX-MS are consistent with those obtained earlier through structural characterization and antibody competition assays. In addition classical and non-classical inhibitor epitopes could be distinguished using a limited subset of C2-derived peptic fragments. Conclusion Our results demonstrate the effectiveness and robustness of the HDX-MS method for epitope mapping and suggest a potential role of rapid mapping of fVIII inhibitor epitopes in facilitating individualized treatment of inhibitor patients. PMID:24152306

  15. Design, Synthesis and Characterization of A Potent, Non-Peptide, Cell-Permeable, Bivalent Smac Mimetic that Concurrently Targets both the BIR2 and BIR3 Domains in XIAP

    PubMed Central

    Sun, Haiying; Nikolovska-Coleska, Zaneta; Lu, Jianfeng; Meagher, Jennifer L.; Yang, Chao-Yie; Qiu, Su; Tomita, York; Ueda, Yumi; Jiang, Sheng; Krajewski, Krzysztof; Roller, Peter P.; Stuckey, Jeanne A.; Wang, Shaomeng

    2008-01-01

    XIAP is a central apoptosis regulator that inhibits apoptosis by binding to and inhibiting the effectors caspase-3/-7 and an initiator caspase-9 through its BIR2 and BIR3 domains, respectively. Smac protein in its dimeric form effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. We report the design, synthesis and characterization of a non-peptide, cell-permeable, bivalent small-molecule (SM-164) which mimics Smac protein for targeting XIAP. Our study shows that SM-164 binds to XIAP containing both BIR domains with an IC50 value of 1.39 nM, being 300 and 7000-times more potent than its monovalent counterparts and the natural Smac AVPI peptide, respectively. SM-164 concurrently interacts with both BIR domains in XIAP and functions as an ultra-potent antagonist of XIAP in both cell-free functional and cell-based assays. SM-164 targets cellular XIAP and effectively induces apoptosis at concentrations as low as 1 nM in leukemia cancer cells, while having a minimal toxicity to normal human primary cells at 10,000 nM. The potency of bivalent SM-164 in binding, functional and cellular assays is 2–3 orders of magnitude higher than its corresponding monovalent Smac mimetics. PMID:17999504

  16. A monoclonal antibody targeting ErbB2 domain III inhibits ErbB2 signaling and suppresses the growth of ErbB2-overexpressing breast tumors

    PubMed Central

    Meng, Y; Zheng, L; Yang, Y; Wang, H; Dong, J; Wang, C; Zhang, Y; Yu, X; Wang, L; Xia, T; Zhang, D; Guo, Y; Li, B

    2016-01-01

    The anti-ErbB2 antibodies trastuzumab and pertuzumab in combination have recently been approved for the treatment of patients with ErbB2-positive metastatic breast cancer. Pertuzumab, which binds to ErbB2 near the center of domain II, and trastuzumab, which binds to the juxtamembrane region of ErbB2 domain IV, directly interfere with domain II- and domain IV-mediated heterodimerization contacts, respectively. In this study, we report a novel anti-ErbB2 antibody, 3E10, which binds to an epitope in domain III that appears to be located opposite to the dimerization interfaces in domain II and domain IV of ErbB2. Our data show that the 3E10 antibody inhibits ErbB2 heterodimerization via a mechanism that strikingly differs from trastuzumab and pertuzumab. It could be speculated that the 3E10 antibody may affect ErbB2 heterodimerization by causing major conformational changes of ErbB2. Furthermore, 3E10 provides synergistic inhibition of ErbB2 heterodimerization and signaling in combination with either trastuzumab or pertuzumab. The combination of these three anti-ErbB2 antibodies that have complementary mechanisms of action appears to be an extremely potent ErbB2 heterodimerization blocker. Compared with trastuzumab plus pertuzumab, the combination of trastuzumab, pertuzumab and 3E10 provides a more potent blockade of ErbB2 signaling. Consistent with this, trastuzumab plus pertuzumab plus 3E10 results in greater in vitro and in vivo antitumor activity in ErbB2-overexpressing breast tumor models, suggesting its potential use for treating ErbB2-overexpressing breast cancer. PMID:26999718

  17. A monoclonal antibody targeting ErbB2 domain III inhibits ErbB2 signaling and suppresses the growth of ErbB2-overexpressing breast tumors.

    PubMed

    Meng, Y; Zheng, L; Yang, Y; Wang, H; Dong, J; Wang, C; Zhang, Y; Yu, X; Wang, L; Xia, T; Zhang, D; Guo, Y; Li, B

    2016-03-21

    The anti-ErbB2 antibodies trastuzumab and pertuzumab in combination have recently been approved for the treatment of patients with ErbB2-positive metastatic breast cancer. Pertuzumab, which binds to ErbB2 near the center of domain II, and trastuzumab, which binds to the juxtamembrane region of ErbB2 domain IV, directly interfere with domain II- and domain IV-mediated heterodimerization contacts, respectively. In this study, we report a novel anti-ErbB2 antibody, 3E10, which binds to an epitope in domain III that appears to be located opposite to the dimerization interfaces in domain II and domain IV of ErbB2. Our data show that the 3E10 antibody inhibits ErbB2 heterodimerization via a mechanism that strikingly differs from trastuzumab and pertuzumab. It could be speculated that the 3E10 antibody may affect ErbB2 heterodimerization by causing major conformational changes of ErbB2. Furthermore, 3E10 provides synergistic inhibition of ErbB2 heterodimerization and signaling in combination with either trastuzumab or pertuzumab. The combination of these three anti-ErbB2 antibodies that have complementary mechanisms of action appears to be an extremely potent ErbB2 heterodimerization blocker. Compared with trastuzumab plus pertuzumab, the combination of trastuzumab, pertuzumab and 3E10 provides a more potent blockade of ErbB2 signaling. Consistent with this, trastuzumab plus pertuzumab plus 3E10 results in greater in vitro and in vivo antitumor activity in ErbB2-overexpressing breast tumor models, suggesting its potential use for treating ErbB2-overexpressing breast cancer.

  18. A conserved acidic patch in the Myb domain is required for activation of an endogenous target gene and for chromatin binding

    PubMed Central

    Ko, Emily Ray; Ko, Dennis; Chen, Carolyn; Lipsick, Joseph S

    2008-01-01

    The c-Myb protein is a transcriptional regulator initially identified by homology to the v-Myb oncoprotein, and has since been implicated in human cancer. The most highly conserved portion of the c-Myb protein is the DNA-binding domain which consists of three imperfect repeats. Many other proteins contain one or more Myb-related domains, including a number of proteins that do not bind directly to DNA. We performed a phylogenetic analysis of diverse classes of Myb-related domains and discovered a highly conserved patch of acidic residues common to all Myb-related domains. These acidic residues are positioned in the first of three alpha-helices within each of the three repeats that comprise the c-Myb DNA-binding domain. Interestingly, these conserved acidic residues are present on a surface of the protein which is distinct from that which binds to DNA. Alanine mutagenesis revealed that the acidic patch of the third c-Myb repeat is essential for transcriptional activity, but neither for nuclear localization nor DNA-binding. Instead, these acidic residues are required for efficient chromatin binding and interaction with the histone H4 N-terminal tail. PMID:18840288

  19. Perspectives for the use of structural information and chemical genetics to develop inhibitors of Janus kinases

    PubMed Central

    Haan, Claude; Behrmann, Iris; Haan, Serge

    2010-01-01

    Abstract Gain-of-function mutations in the genes encoding Janus kinases have been discovered in various haematologic diseases. Jaks are composed of a FERM domain, an SH2 domain, a pseudokinase domain and a kinase domain, and a complex interplay of the Jak domains is involved in regulation of catalytic activity and association to cytokine receptors. Most activating mutations are found in the pseudokinase domain. Here we present recently discovered mutations in the context of our structural models of the respective domains. We describe two structural hotspots in the pseudokinase domain of Jak2 that seem to be associated either to myeloproliferation or to lymphoblastic leukaemia, pointing at the involvement of distinct signalling complexes in these disease settings. The different domains of Jaks are discussed as potential drug targets. We present currently available inhibitors targeting Jaks and indicate structural differences in the kinase domains of the different Jaks that may be exploited in the development of specific inhibitors. Moreover, we discuss recent chemical genetic approaches which can be applied to Jaks to better understand the role of these kinases in their biological settings and as drug targets. PMID:20132407

  20. beta2-chimaerin is a novel target for diacylglycerol: binding properties and changes in subcellular localization mediated by ligand binding to its C1 domain.

    PubMed

    Caloca, M J; Garcia-Bermejo, M L; Blumberg, P M; Lewin, N E; Kremmer, E; Mischak, H; Wang, S; Nacro, K; Bienfait, B; Marquez, V E; Kazanietz, M G

    1999-10-12

    The members of the chimaerin family of Rac-GTPase-activating proteins possess a single C1 domain with high homology to those present in protein kinase C (PKC) isozymes. This domain in PKCs is involved in phorbol ester and diacylglycerol (DAG) binding. We previously have demonstrated that one of the chimaerin isoforms, beta2-chimaerin, binds phorbol esters with high affinity. In this study we analyzed the properties of beta2-chimaerin as a DAG receptor by using a series of conformationally constrained cyclic DAG analogues (DAG lactones) as probes. We identified analogs that bind to beta2-chimaerin with more than 100-fold higher affinity than 1-oleoyl-2-acetylglycerol. The potencies of these analogs approach those of the potent phorbol ester tumor promoters. The different DAG lactones show some selectivity for this novel receptor compared with PKCalpha. Cellular studies revealed that these DAG analogs induce translocation of beta2-chimaerin from cytosolic (soluble) to particulate fractions. Using green fluorescent protein-fusion proteins for beta2-chimaerin we determined that this novel receptor translocates to the perinuclear region after treatment with DAG lactones. Binding and translocation were prevented by mutation of the conserved Cys-246 in the C1 domain. The structural homology between the C1 domain of beta2-chimaerin and the C1b domain of PKCdelta also was confirmed by modeling analysis. Our results demonstrate that beta2-chimaerin is a high affinity receptor for DAG through binding to its C1 domain and supports the emerging concept that multiple pathways transduce signaling through DAG and the phorbol esters.

  1. FBI-1 can stimulate HIV-1 Tat activity and is targeted to a novel subnuclear domain that includes the Tat-P-TEFb-containing nuclear speckles.

    PubMed

    Pendergrast, P Shannon; Wang, Chen; Hernandez, Nouria; Huang, Sui

    2002-03-01

    FBI-1 is a cellular POZ-domain-containing protein that binds to the HIV-1 LTR and associates with the HIV-1 transactivator protein Tat. Here we show that elevated levels of FBI-1 specifically stimulate Tat activity and that this effect is dependent on the same domain of FBI-1 that mediates Tat-FBI-1 association in vivo. FBI-1 also partially colocalizes with Tat and Tat's cellular cofactor, P-TEFb (Cdk9 and cyclin T1), at the splicing-factor-rich nuclear speckle domain. Further, a less-soluble population of FBI-1 distributes in a novel peripheral-speckle pattern of localization as well as in other nuclear regions. This distribution pattern is dependent on the FBI-1 DNA binding domain, on the presence of cellular DNA, and on active transcription. Taken together, these results suggest that FBI-1 is a cellular factor that preferentially associates with active chromatin and that can specifically stimulate Tat-activated HIV-1 transcription.

  2. Structure of the second RRM domain of Nrd1, a fission yeast MAPK target RNA binding protein, and implication for its RNA recognition and regulation

    SciTech Connect

    Kobayashi, Ayaho; Kanaba, Teppei; Satoh, Ryosuke; Fujiwara, Toshinobu; Ito, Yutaka; Sugiura, Reiko; Mishima, Masaki

    2013-07-19

    Highlights: •Solution structure of the second RRM of Nrd1 was determined. •RNA binding site of the second RRM was estimated. •Regulatory mechanism of RNA binding by phosphorylation is discussed. -- Abstract: Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and thereby suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed.

  3. Structural basis for membrane targeting by the MVB12-associated [beta]-prism domain of the human ESCRT-I MVB12 subunit

    SciTech Connect

    Boura, Evzen; Hurley, James H.

    2012-03-15

    MVB12-associated {beta}-prism (MABP) domains are predicted to occur in a diverse set of membrane-associated bacterial and eukaryotic proteins, but their existence, structure, and biochemical properties have not been characterized experimentally. Here, we find that the MABP domains of the MVB12A and B subunits of ESCRT-I are functional modules that bind in vitro to liposomes containing acidic lipids depending on negative charge density. The MABP domain is capable of autonomously localizing to subcellular puncta and to the plasma membrane. The 1.3-{angstrom} atomic resolution crystal structure of the MVB12B MABP domain reveals a {beta}-prism fold, a hydrophobic membrane-anchoring loop, and an electropositive phosphoinositide-binding patch. The basic patch is open, which explains how it senses negative charge density but lacks stereoselectivity. These observations show how ESCRT-I could act as a coincidence detector for acidic phospholipids and protein ligands, enabling it to function both in protein transport at endosomes and in cytokinesis and viral budding at the plasma membrane.

  4. A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels, an arachidonate-regulated store-independent Orai channel.

    PubMed

    Thompson, Jill L; Shuttleworth, Trevor J

    2012-01-01

    The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.

  5. D120 and K152 within the PH Domain of T Cell Adapter SKAP55 Regulate Plasma Membrane Targeting of SKAP55 and LFA-1 Affinity Modulation in Human T Lymphocytes.

    PubMed

    Witte, Amelie; Meineke, Bernhard; Sticht, Jana; Philipsen, Lars; Kuropka, Benno; Müller, Andreas J; Freund, Christian; Schraven, Burkhart; Kliche, Stefanie

    2017-04-01

    The β2-integrin lymphocyte function-associated antigen 1 (LFA-1) is needed for the T cell receptor (TCR)-induced activation of LFA-1 to promote T cell adhesion and interaction with antigen-presenting cells (APCs). LFA-1-mediated cell-cell interactions are critical for proper T cell differentiation and proliferation. The Src kinase-associated phosphoprotein of 55 kDa (SKAP55) is a key regulator of TCR-mediated LFA-1 signaling (inside-out/outside-in signaling). To gain an understanding of how SKAP55 controls TCR-mediated LFA-1 activation, we assessed the functional role of its pleckstrin homology (PH) domain. We identified two critical amino acid residues within the PH domain of SKAP55, aspartic acid 120 (D120) and lysine 152 (K152). D120 facilitates the retention of SKAP55 in the cytoplasm of nonstimulated T cells, while K152 promotes SKAP55 membrane recruitment via actin binding upon TCR triggering. Importantly, the K152-dependent interaction of the PH domain with actin promotes the binding of talin to LFA-1, thus facilitating LFA-1 activation. These data suggest that K152 and D120 within the PH domain of SKAP55 regulate plasma membrane targeting and TCR-mediated activation of LFA-1.

  6. TRIM14 inhibits hepatitis C virus infection by SPRY domain-dependent targeted degradation of the viral NS5A protein

    PubMed Central

    Wang, Shanshan; Chen, Yongzhi; Li, Chunfeng; Wu, Yaoxing; Guo, Lei; Peng, Changwei; Huang, Yueping; Cheng, Genhong; Qin, F. Xiao-Feng

    2016-01-01

    Tripartite motif 14 (TRIM14) was reported to function as a mitochondrial signaling adaptor in mediating innate immune responses. However, the involvement of TRIM14 in host defense against viral infection and molecular mechanisms remain unclear. Here, we demonstrated that enforced expression of TRIM14 could potently inhibit the infection and replication of HCV in hepatocytes, whereas TRIM14 knockout cells became more susceptible to HCV infection. Interestingly, further experiments revealed that such anti-HCV activity was independent of activating the NF-κB or interferon pathways but required the C-terminal SPRY domain of no signaling capacity. In searching for mechanisms how TRIM14 exerts its antiviral function we found that TRIM14 interacted with HCV encoded non-structural protein NS5A and could strongly induce its degradation dependent on the NS5A1 subdomain. Interestingly extensive domain mapping analyses revealed that NS5A degradation was mediated by the highly conserved SPRY domain of TRIM14, which might involve the K48 ubiquitination pathway. Collectively, our work uncovered a new mechanism responsible for host defense against HCV infection, and could potentially aid the development of novel anti-HCV therapeutics. PMID:27578425

  7. Single-domain antibody-based and linker-free bispecific antibodies targeting FcγRIII induce potent antitumor activity without recruiting regulatory T cells.

    PubMed

    Rozan, Caroline; Cornillon, Amélie; Pétiard, Corinne; Chartier, Martine; Behar, Ghislaine; Boix, Charlotte; Kerfelec, Brigitte; Robert, Bruno; Pèlegrin, André; Chames, Patrick; Teillaud, Jean-Luc; Baty, Daniel

    2013-08-01

    Antibody-dependent cell-mediated cytotoxicity, one of the most prominent modes of action of antitumor antibodies, suffers from important limitations due to the need for optimal interactions with Fcγ receptors. In this work, we report the design of a new bispecific antibody format, compact and linker-free, based on the use of llama single-domain antibodies that are capable of circumventing most of these limitations. This bispecific antibody format was created by fusing single-domain antibodies directed against the carcinoembryonic antigen and the activating FcγRIIIa receptor to human Cκ and CH1 immunoglobulin G1 domains, acting as a natural dimerization motif. In vitro and in vivo characterization of these Fab-like bispecific molecules revealed favorable features for further development as a therapeutic molecule. They are easy to produce in Escherichia coli, very stable, and elicit potent lysis of tumor cells by human natural killer cells at picomolar concentrations. Unlike conventional antibodies, they do not engage inhibitory FcγRIIb receptor, do not compete with serum immunoglobulins G for receptor binding, and their cytotoxic activity is independent of Fc glycosylation and FcγRIIIa polymorphism. As opposed to anti-CD3 bispecific antitumor antibodies, they do not engage regulatory T cells as these latter cells do not express FcγRIII. Studies in nonobese diabetic/severe combined immunodeficient gamma mice xenografted with carcinoembryonic antigen-positive tumor cells showed that Fab-like bispecific molecules in the presence of human peripheral blood mononuclear cells significantly slow down tumor growth. This new compact, linker-free bispecific antibody format offers a promising approach for optimizing antibody-based therapies.

  8. H3K4/H3K9me3 Bivalent Chromatin Domains Targeted by Lineage-Specific DNA Methylation Pauses Adipocyte Differentiation.

    PubMed

    Matsumura, Yoshihiro; Nakaki, Ryo; Inagaki, Takeshi; Yoshida, Ayano; Kano, Yuka; Kimura, Hiroshi; Tanaka, Toshiya; Tsutsumi, Shuichi; Nakao, Mitsuyoshi; Doi, Takefumi; Fukami, Kiyoko; Osborne, Timothy F; Kodama, Tatsuhiko; Aburatani, Hiroyuki; Sakai, Juro

    2015-11-19

    Bivalent H3K4me3 and H3K27me3 chromatin domains in embryonic stem cells keep active developmental regulatory genes expressed at very low levels and poised for activation. Here, we show an alternative and previously unknown bivalent modified histone signature in lineage-committed mesenchymal stem cells and preadipocytes that pairs H3K4me3 with H3K9me3 to maintain adipogenic master regulatory genes (Cebpa and Pparg) expressed at low levels yet poised for activation when differentiation is required. We show lineage-specific gene-body DNA methylation recruits H3K9 methyltransferase SETDB1, which methylates H3K9 immediately downstream of transcription start sites marked with H3K4me3 to establish the bivalent domain. At the Cebpa locus, this prevents transcription factor C/EBPβ binding, histone acetylation, and further H3K4me3 deposition and is associated with pausing of RNA polymerase II, which limits Cebpa gene expression and adipogenesis.

  9. Paring Down HIV Env: Design and Crystal Structure of a Stabilized Inner Domain of HIV-1 gp120 Displaying a Major ADCC Target of the A32 Region.

    PubMed

    Tolbert, William D; Gohain, Neelakshi; Veillette, Maxime; Chapleau, Jean-Philippe; Orlandi, Chiara; Visciano, Maria L; Ebadi, Maryam; DeVico, Anthony L; Fouts, Timothy R; Finzi, Andrés; Lewis, George K; Pazgier, Marzena

    2016-05-03

    Evidence supports a role of antibody-dependent cellular cytotoxicity (ADCC) toward transitional epitopes in the first and second constant (C1-C2) regions of gp120 (A32-like epitopes) in preventing HIV-1 infection and in vaccine-induced protection. Here, we describe the first successful attempt at isolating the inner domain (ID) of gp120 as an independent molecule that encapsulates the A32-like region within a minimal structural unit of the HIV-1 Env. Through structure-based design, we developed ID2, which consists of the ID expressed independently of the outer domain and stabilized in the CD4-bound conformation by an inter-layer disulfide bond. ID2 expresses C1-C2 epitopes in the context of CD4-triggered full-length gp120 but without any known neutralizing epitope present. Thus, ID2 represents a novel probe for the analysis and/or selective induction of antibody responses to the A32 epitope region. We also present the crystal structure of ID2 complexed with mAb A32, which defines its epitope.

  10. Tetratricopeptide repeat domain of Yarrowia lipolytica Pex5p is essential for recognition of the type 1 peroxisomal targeting signal but does not confer full biological activity on Pex5p.

    PubMed

    Szilard, R K; Rachubinski, R A

    2000-02-15

    Peroxins are proteins required for peroxisome assembly and are encoded by the PEX genes. The Yarrowia lipolytica pex5-1 mutant fails to import a subset of peroxisomal matrix proteins, including those with a type 1 peroxisomal targeting signal (PTS1). Pex5p family members interact with a PTS1 through their characteristic tetratricopeptide repeat (TPR) domain. We used binding assays in vitro to investigate the nature of the association of Y. lipolytica Pex5p (YlPex5p) with the PTS1 signal. A purified recombinant YlPex5p fusion protein interacted specifically, directly and autonomously with a protein terminating in a PTS1. Wild-type YlPex5p translated in vitro recognized functional PTS1s specifically. This activity is abrogated by the substitution of an aspartic residue for a conserved glycine residue in the TPR domain (G455D) of YlPex5p encoded by the pex5-1 allele. Deletion analysis demonstrated that an intact TPR domain of YlPex5p is necessary but not sufficient for both interaction with a PTS1 and functional complementation of a strain lacking YlPex5p.

  11. A PWWP Domain-Containing Protein Targets the NuA3 Acetyltransferase Complex via Histone H3 Lysine 36 trimethylation to Coordinate Transcriptional Elongation at Coding Regions*

    PubMed Central

    Gilbert, Tonya M.; McDaniel, Stephen L.; Byrum, Stephanie D.; Cades, Jessica A.; Dancy, Blair C. R.; Wade, Herschel; Tackett, Alan J.; Strahl, Brian D.; Taverna, Sean D.

    2014-01-01

    Post-translational modifications of histones, such as acetylation and methylation, are differentially positioned in chromatin with respect to gene organization. For example, although histone H3 is often trimethylated on lysine 4 (H3K4me3) and acetylated on lysine 14 (H3K14ac) at active promoter regions, histone H3 lysine 36 trimethylation (H3K36me3) occurs throughout the open reading frames of transcriptionally active genes. The conserved yeast histone acetyltransferase complex, NuA3, specifically binds H3K4me3 through a plant homeodomain (PHD) finger in the Yng1 subunit, and subsequently catalyzes the acetylation of H3K14 through the histone acetyltransferase domain of Sas3, leading to transcription initiation at a subset of genes. We previously found that Ylr455w (Pdp3), an uncharacterized proline-tryptophan-tryptophan-proline (PWWP) domain-containing protein, copurifies with stable members of NuA3. Here, we employ mass-spectrometric analysis of affinity purified Pdp3, biophysical binding assays, and genetic analyses to classify NuA3 into two functionally distinct forms: NuA3a and NuA3b. Although NuA3a uses the PHD finger of Yng1 to interact with H3K4me3 at the 5′-end of open reading frames, NuA3b contains the unique member, Pdp3, which regulates an interaction between NuA3b and H3K36me3 at the transcribed regions of genes through its PWWP domain. We find that deletion of PDP3 decreases NuA3-directed transcription and results in growth defects when combined with transcription elongation mutants, suggesting NuA3b acts as a positive elongation factor. Finally, we determine that NuA3a, but not NuA3b, is synthetically lethal in combination with a deletion of the histone acetyltransferase GCN5, indicating NuA3b has a specialized role at coding regions that is independent of Gcn5 activity. Collectively, these studies define a new form of the NuA3 complex that associates with H3K36me3 to effect transcriptional elongation. MS data are available via ProteomeXchange with

  12. Novel Injury Site Targeted Fusion Protein Comprising Annexin V and Kunitz Inhibitor Domains Ameliorates Ischemia-Reperfusion Injury and Promotes Survival of Ischemic Rat Abdominal Skin Flaps.

    PubMed

    Shyu, Victor Bong-Hang; Hsu, Chung En; Wen, Chih-Jen; Wun, Tze-Chein; Tang, Rui; Achilefu, Samuel; Wei, Fu-Chan; Cheng, Hui-Yun

    2017-03-01

    Appropriate antithrombotic therapy is critical for successful outcomes in reconstructive microsurgical procedures involving free tissue transfer. The annexin V-6L15 (ANV-6L15) fusion protein was developed as a targeted antithrombotic reagent. Annexin V specifically binds to exposed phosphatidylserine on apoptotic or injured cells, and prevents coagulation and cell adhesion, whereas 6L15 inhibits tissue factor-VIIa pathway within the coagulation cascade. The treatment efficacy of ANV-6L15 on rat island muscle and pedicled abdominal fasciocutaneous flaps following ischemic injury and ischemia-reperfusion injury (IRI) was evaluated.

  13. p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1.

    PubMed

    Ma-Lauer, Yue; Carbajo-Lozoya, Javier; Hein, Marco Y; Müller, Marcel A; Deng, Wen; Lei, Jian; Meyer, Benjamin; Kusov, Yuri; von Brunn, Brigitte; Bairad, Dev Raj; Hünten, Sabine; Drosten, Christian; Hermeking, Heiko; Leonhardt, Heinrich; Mann, Matthias; Hilgenfeld, Rolf; von Brunn, Albrecht

    2016-08-30

    Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PL(pro)), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95-144 of RCHY1 and 389-652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PL(pro)s from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD-PL(pro) fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PL(pro) alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes.

  14. p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1

    PubMed Central

    Ma-Lauer, Yue; Carbajo-Lozoya, Javier; Müller, Marcel A.; Deng, Wen; Lei, Jian; Meyer, Benjamin; Kusov, Yuri; von Brunn, Brigitte; Bairad, Dev Raj; Hünten, Sabine; Drosten, Christian; Hermeking, Heiko; Leonhardt, Heinrich; Mann, Matthias; Hilgenfeld, Rolf; von Brunn, Albrecht

    2016-01-01

    Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PLpro), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95–144 of RCHY1 and 389–652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PLpros from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD–PLpro fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PLpro alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes. PMID:27519799

  15. Identification of a Key Target Sequence To Block Human Immunodeficiency Virus Type 1 Replication within the gag-pol Transframe Domain

    PubMed Central

    Sei, Shizuko; Yang, Quan-en; O'Neill, Dennis; Yoshimura, Kazuhisa; Nagashima, Kunio; Mitsuya, Hiroaki

    2000-01-01

    Although the full sequence of the human immunodeficiency virus type 1 (HIV-1) genome has been known for more than a decade, effective genetic antivirals have yet to be developed. Here we show that, of 22 regions examined, one highly conserved sequence (ACTCTTTGGCAACGA) near the 3′ end of the HIV-1 gag-pol transframe region, encoding viral protease residues 4 to 8 and a C-terminal Vpr-binding motif of p6Gag protein in two different reading frames, can be successfully targeted by an antisense peptide nucleic acid oligomer named PNAPR2. A disrupted translation of gag-pol mRNA induced at the PNAPR2-annealing site resulted in a decreased synthesis of Pr160Gag-Pol polyprotein, hence the viral protease, a predominant expression of Pr55Gag devoid of a fully functional p6Gag protein, and the excessive intracellular cleavage of Gag precursor proteins, hindering the processes of virion assembly. Treatment with PNAPR2 abolished virion production by up to 99% in chronically HIV-1-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates with the multidrug-resistant phenotype. This particular segment of the gag-pol transframe gene appears to offer a distinctive advantage over other regions in invading viral structural genes and restraining HIV-1 replication in infected cells and may potentially be exploited as a novel antiviral genetic target. PMID:10775598

  16. Single-domain protein A-engineered magnetic nanoparticles: toward a universal strategy to site-specific labeling of antibodies for targeted detection of tumor cells.

    PubMed

    Mazzucchelli, Serena; Colombo, Miriam; De Palma, Clara; Salvadè, Agnese; Verderio, Paolo; Coghi, Maria D; Clementi, Emilio; Tortora, Paolo; Corsi, Fabio; Prosperi, Davide

    2010-10-26

    Highly monodisperse magnetite nanocrystals (MNC) were synthesized in organic media and transferred to the water phase by ultrasound-assisted ligand exchange with an iminodiacetic phosphonate. The resulting biocompatible magnetic nanoparticles were characterized by transmission electron microscopy, dynamic light scattering, and magnetorelaxometry, indicating that this method allowed us to obtain stable particle dispersions with narrow size distribution and unusually high magnetic resonance T(2) contrast power. These nanoparticles were conjugated to a newly designed recombinant monodomain protein A variant, which exhibited a convincingly strong affinity for human and rabbit IgG molecules. Owing to the nature of antibody-protein A binding, tight antibody immobilization occurred through the Fc fragment thus taking full advantage of the targeting potential of bound IgGs. If necessary, monoclonal antibodies could be removed under controlled conditions regenerating the original IgG-conjugatable MNC. As a proof of concept of the utility of our paramagnetic labeling system of human IgGs for biomedical applications, anti-HER-2 monoclonal antibody trastuzumab was immobilized on hybrid MNC (TMNC). TMNC were assessed by immunoprecipitation assay and confocal microscopy effected on HER-2-overexpressing MCF-7 breast cancer cells, demonstrating excellent recognition capability and selectivity for the target membrane receptor.

  17. TRAP150 interacts with the RNA-binding domain of PSF and antagonizes splicing of numerous PSF-target genes in T cells.

    PubMed

    Yarosh, Christopher A; Tapescu, Iulia; Thompson, Matthew G; Qiu, Jinsong; Mallory, Michael J; Fu, Xiang-Dong; Lynch, Kristen W

    2015-10-15

    PSF (a.k.a. SFPQ) is a ubiquitously expressed, essential nuclear protein with important roles in DNA damage repair and RNA biogenesis. In stimulated T cells, PSF binds to and suppresses the inclusion of CD45 exon 4 in the final mRNA; however, in resting cells, TRAP150 binds PSF and prevents access to the CD45 RNA, though the mechanism for this inhibition has remained unclear. Here, we show that TRAP150 binds a region encompassing the RNA recognition motifs (RRMs) of PSF using a previously uncharacterized, 70 residue region we have termed the PSF-interacting domain (PID). TRAP150's PID directly inhibits the interaction of PSF RRMs with RNA, which is mediated through RRM2. However, interaction of PSF with TRAP150 does not appear to inhibit the dimerization of PSF with other Drosophila Behavior, Human Splicing (DBHS) proteins, which is also dependent on RRM2. Finally, we use RASL-Seq to identify ∼40 T cell splicing events sensitive to PSF knockdown, and show that for the majority of these, PSF's effect is antagonized by TRAP150. Together these data suggest a model in which TRAP150 interacts with dimeric PSF to block access of RNA to RRM2, thereby regulating the activity of PSF toward a broad set of splicing events in T cells.

  18. The cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction

    PubMed Central

    Liu, Yanjie; Pei, Jimin; Grishin, Nick; Snell, William J.

    2015-01-01

    Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction. PMID:25655701

  19. The cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction.

    PubMed

    Liu, Yanjie; Pei, Jimin; Grishin, Nick; Snell, William J

    2015-03-01

    Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction.

  20. Tandem Mass Spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets

    SciTech Connect

    Alfaro, Joshua F.; Gong, Cheng-Xin; Monroe, Matthew E.; Aldrich, Joshua T.; Clauss, Therese RW; Purvine, Samuel O.; Wang, Zihao; Camp, David G.; Shabanowitz, Jeffrey; Stanley, Pamela; Hart, Gerald W.; Hunt, Donald F.; Yang, Feng; Smith, Richard D.

    2012-05-08

    O-Linked N-Acetylglucosamine (O-GlcNAc) is a reversible post-translational modification of Ser and Thr residues on cytosolic and nuclear proteins found in all higher eukaryotes. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing {approx}100 {micro}g of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites on Ser and Thr residues in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located in close proximity to known phosphorylation sites. These findings support the proposed regulatory crosstalk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-{beta}-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, thus representing the first evidence for extracellular O-GlcNAcylation in mammalian systems by the ER-resident O-GlcNAc transferase (EOGT). We also report a GlcNAc-{beta}-1,3-Fuc-{alpha}-1-O-Thr modification on the EGF-like repeat of the Versican core protein, a novel substrate of Fringe {beta}1,3-N-acetylglucosaminyltransferases.

  1. Review of the Third Domain Receptor Binding Fragment of Alpha-fetoprotein (AFP): Plausible Binding of AFP to Lysophospholipid Receptor Targets.

    PubMed

    Mizejewski, G J

    2016-01-31

    Alpha-fetoprotein (AFP) is a 69 kD fetal- and tumor-associated single-chain glycoprotein belonging to the albuminoid gene family. AFP functions as a carrier/transport molecule as well as a growth regulator and has been utilized as a clinical biomarker for both fetal defects and cancer growth. Lysophospholipids (LPLs) are plasma membrane-derived bioactive lipid signaling mediators composed of a small molecular weight single acyl carbon chain (palmitic, oleic acid) attached to a polar headgroup; they range in molecular mass from 250-750 daltons. The LPLs consist of either sphingosine-1-phosphate or lysophosphatidic acid, and mostly their choline, ethanolamine, serine or inositol derivatives. They are present only in vertebrates. These bioactive paracrine lipid mediators are ubiquitously distributed in tissues and are released from many different cell types (platelets, macrophages, monocytes, etc.) involved in developmental, physiological, and pathological processes. The LPLs bind to four different classes of G-protein coupled receptors described herein which transduce a multiple of cell effects encompassing activities such as morphogenesis, neural development, angiogenesis, and carcinogenesis. The identification of potential binding sites of LPL receptors on the AFP third domain receptor binding fragment were derived by computer modeling analysis. It is conceivable, but not proven, that AFP might bind not only to the LPL receptors, but also to LPLs themselves since AFP binds medium and long chain fatty acids. It is proposed that some of the activities ascribed to AFP in the past might be due in part to the presence of bound LPLs and/or their receptors.

  2. Target-specific cytotoxic effects on HER2-expressing cells by the tripartite fusion toxin ZHER2:2891-ABD-PE38X8, including a targeting affibody molecule and a half-life extension domain.

    PubMed

    Liu, Hao; Seijsing, Johan; Frejd, Fredrik Y; Tolmachev, Vladimir; Gräslund, Torbjörn

    2015-08-01

    Development of cancer treatment regimens including immunotoxins is partly hampered by their immunogenicity. Recently, deimmunized versions of toxins have been described, potentially being better suited for translation to the clinic. In this study, a recombinant tripartite fusion toxin consisting of a deimmunized version of exotoxin A from Pseudomonas aeruginosa (PE38) genetically fused to an affibody molecule specifically interacting with the human epidermal growth factor receptor 2 (HER2), and also an albumin binding domain (ABD) for half-life extension, has been produced and characterized in terms of functionality of the three moieties. Biosensor based assays showed that the fusion toxin was able to interact with human and mouse serum albumin, but not with bovine serum albumin and that it interacted with HER2 (KD=5 nM). Interestingly, a complex of the fusion toxin and human serum albumin also interacted with HER2 but with a somewhat weaker affinity (KD=12 nM). The IC50-values of the fusion toxin ranged from 6 to 300 pM on SKOV-3, SKBR-3 and A549 cells and was lower for cells with higher surface densities of HER2. The fusion toxin was found specific for HER2 as shown by blocking available HER2 receptors with free affibody molecule before subjecting the cells to the toxin. Analysis of contact time showed that 10 min was sufficient to kill 50% of the cells. In conclusion, all three regions of the fusion toxin were found to be functional.

  3. Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors

    PubMed Central

    Olivares, Simon; Mi, Tiejuan; Maiti, Sourindra; Deniger, Drew; Huls, Helen; Torikai, Hiroki; Singh, Harjeet; Champlin, Richard E.; Laskowski, Tamara; McNamara, George; Cooper, Laurence J. N.

    2016-01-01

    Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR’s in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies. PMID:27548616

  4. Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors.

    PubMed

    Thokala, Radhika; Olivares, Simon; Mi, Tiejuan; Maiti, Sourindra; Deniger, Drew; Huls, Helen; Torikai, Hiroki; Singh, Harjeet; Champlin, Richard E; Laskowski, Tamara; McNamara, George; Cooper, Laurence J N

    2016-01-01

    Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR's in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies.

  5. Chelation Motifs Affecting Metal-dependent Viral Enzymes: N′-acylhydrazone Ligands as Dual Target Inhibitors of HIV-1 Integrase and Reverse Transcriptase Ribonuclease H Domain

    PubMed Central

    Carcelli, Mauro; Rogolino, Dominga; Gatti, Anna; Pala, Nicolino; Corona, Angela; Caredda, Alessia; Tramontano, Enzo; Pannecouque, Christophe; Naesens, Lieve; Esposito, Francesca

    2017-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection, still represent a serious global health emergency. The chronic toxicity derived from the current anti-retroviral therapy limits the prolonged use of several antiretroviral agents, continuously requiring the discovery of new antiviral agents with innovative strategies of action. In particular, the development of single molecules targeting two proteins (dual inhibitors) is one of the current main goals in drug discovery. In this contest, metal-chelating molecules have been extensively explored as potential inhibitors of viral metal-dependent enzymes, resulting in some important classes of antiviral agents. Inhibition of HIV Integrase (IN) is, in this sense, paradigmatic. HIV-1 IN and Reverse Transcriptase-associated Ribonuclease H (RNase H) active sites show structural homologies, with the presence of two Mg(II) cofactors, hence it seems possible to inhibit both enzymes by means of chelating ligands with analogous structural features. Here we present a series of N′-acylhydrazone ligands with groups able to chelate the Mg(II) hard Lewis acid ions in the active sites of both the enzymes, resulting in dual inhibitors with micromolar and even nanomolar activities. The most interesting identified N′-acylhydrazone analog, compound 18, shows dual RNase H-IN inhibition and it is also able to inhibit viral replication in cell-based antiviral assays in the low micromolar range. Computational modeling studies were also conducted to explore the binding attitudes of some model ligands within the active site of both the enzymes. PMID:28373864

  6. Multi-domain short peptide molecules for in situ synthesis and biofunctionalization of gold nanoparticles for integrin-targeted cell uptake.

    PubMed

    Gulsuner, Hilal Unal; Ceylan, Hakan; Guler, Mustafa O; Tekinay, Ayse B

    2015-05-27

    We describe design and synthesis model of multidomain (modular) peptides (MDPs), which direct a reaction cascade coupling the synthesis and surface functionalization of gold nanoparticles (AuNPs) in a single step. The synthesis is achieved via simple mixing of the aqueous solutions of auric acid and MDPs at room temperature without the addition of any surfactants or toxic intermediate reagents. This method allows facile control over the nanoparticle size between ∼2-15 nm, which opens a practical window for biomedical applications. In contrast to the conventional citrate-mediated methods, peptide-mediated synthesis and stabilization provide increased colloidal stability to AuNPs. As a proof of this concept, we demonstrate active targeting of human breast adenocarcinoma cell line (MCF7) using the one-step-prepared engineered AuNPs. Overall, we propose a single-step, chemically greener, biologically safer method for the synthesis and surface functionalization of gold nanoparticles in a size-controlled manner. The chemical versatility of the MDP design broadens the applicability of this strategy, thereby emerging as a successful alternative for the currently available nanoparticle preparation technologies.

  7. Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1) enhance Gag-specific immune response.

    PubMed

    Godinho, Rodrigo Maciel da Costa; Matassoli, Flavio Lemos; Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J Thomas; Marques, Ernesto Torres de Azevedo; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

  8. Negative regulation of the Nrf1 transcription factor by its N-terminal domain is independent of Keap1: Nrf1, but not Nrf2, is targeted to the endoplasmic reticulum

    PubMed Central

    Zhang, Yiguo; Crouch, Dorothy H.; Yamamoto, Masayuki; Hayes, John D.

    2006-01-01

    Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) and Nrf2 regulate ARE (antioxidant response element)-driven genes. At its N-terminal end, Nrf1 contains 155 additional amino acids that are absent from Nrf2. This 155-amino-acid polypeptide includes the N-terminal domain (NTD, amino acids 1–124) and a region (amino acids 125–155) that is part of acidic domain 1 (amino acids 125–295). Within acidic domain 1, residues 156–242 share 43% identity with the Neh2 (Nrf2-ECH homology 2) degron of Nrf2 that serves to destabilize this latter transcription factor through an interaction with Keap1 (Kelch-like ECH-associated protein 1). We have examined the function of the 155-amino-acid N-terminal polypeptide in Nrf1, along with its adjacent Neh2-like subdomain. Activation of ARE-driven genes by Nrf1 was negatively controlled by the NTD (N-terminal domain) through its ability to direct Nrf1 to the endoplasmic reticulum. Ectopic expression of wild-type Nrf1 and mutants lacking either the NTD or portions of its Neh2-like subdomain into wild-type and mutant mouse embryonic fibroblasts indicated that Keap1 controls neither the activity of Nrf1 nor its subcellular distribution. Immunocytochemistry showed that whereas Nrf1 gave primarily cytoplasmic staining that was co-incident with that of an endoplasmic-reticulum marker, Nrf2 gave primarily nuclear staining. Attachment of the NTD from Nrf1 to the N-terminus of Nrf2 produced a fusion protein that was redirected from the nucleus to the endoplasmic reticulum. Although this NTD–Nrf2 fusion protein exhibited less transactivation activity than wild-type Nrf2, it was nevertheless still negatively regulated by Keap1. Thus Nrf1 and Nrf2 are targeted to different subcellular compartments and are negatively regulated by distinct mechanisms. PMID:16872277

  9. Substitution at aspartic acid 1128 in the SARS coronavirus spike glycoprotein mediates escape from a S2 domain-targeting neutralizing monoclonal antibody.

    PubMed

    Ng, Oi-Wing; Keng, Choong-Tat; Leung, Cynthia Sau-Wai; Peiris, J S Malik; Poon, Leo Lit Man; Tan, Yee-Joo

    2014-01-01

    The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2): 941-50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111-1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus.

  10. Substitution at Aspartic Acid 1128 in the SARS Coronavirus Spike Glycoprotein Mediates Escape from a S2 Domain-Targeting Neutralizing Monoclonal Antibody

    PubMed Central

    Ng, Oi-Wing; Keng, Choong-Tat; Leung, Cynthia Sau-Wai; Peiris, J. S. Malik; Poon, Leo Lit Man; Tan, Yee-Joo

    2014-01-01

    The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2): 941–50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111–1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus. PMID:25019613

  11. Prophylactic vaccination targeting ERBB3 decreases polyp burden in a mouse model of human colorectal cancer

    PubMed Central

    Bautz, David J.; Sherpa, Ang T.

    2017-01-01

    ABSTRACT Prophylactic vaccination is typically utilized for the prevention of communicable diseases such as measles and influenza but, with the exception of vaccines to prevent cervical cancer, is not widely used as a means of preventing or reducing the incidence of cancer. Here, we utilize a peptide-based immunotherapeutic approach targeting ERBB3, a pseudo-kinase member of the EGFR/ERBB family of receptor tyrosine kinases, as a means of preventing occurrence of colon polyps. Administration of the peptide resulted in a significant decrease in the development of intestinal polyps in C57BL/6J-ApcMin mice, a model of familial adenomatous polyposis (FAP). In addition, even though they were not vaccinated, ApcMin offspring born to vaccinated females developed significantly fewer polyps than offspring born to control females. Lastly, to validate ERBB as a valid target for vaccination, we found no overt toxicity, increases in apoptosis, or morphological changes in tissues where Erbb3 was ablated in adult mice. These results indicate that prophylactic vaccination targeting ERBB3 could prevent the development of colon polyps in an at-risk patient population. PMID:28197371

  12. High-Affinity DNA Aptamer Generation Targeting von Willebrand Factor A1-Domain by Genetic Alphabet Expansion for Systematic Evolution of Ligands by Exponential Enrichment Using Two Types of Libraries Composed of Five Different Bases.

    PubMed

    Matsunaga, Ken-Ichiro; Kimoto, Michiko; Hirao, Ichiro

    2017-01-11

    The novel evolutionary engineering method ExSELEX (genetic alphabet expansion for systematic evolution of ligands by exponential enrichment) provides high-affinity DNA aptamers that specifically bind to target molecules, by introducing an artificial hydrophobic base analogue as a fifth component into DNA aptamers. Here, we present a newer version of ExSELEX, using a library with completely randomized sequences consisting of five components: four natural bases and one unnatural hydrophobic base, 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). In contrast to the limited number of Ds-containing sequence combinations in our previous library, the increased complexity of the new randomized library could improve the success rates of high-affinity aptamer generation. To this end, we developed a sequencing method for each clone in the enriched library after several rounds of selection. Using the improved library, we generated a Ds-containing DNA aptamer targeting von Willebrand factor A1-domain (vWF) with significantly higher affinity (KD = 75 pM), relative to those generated by the initial version of ExSELEX, as well as that of the known DNA aptamer consisting of only the natural bases. In addition, the Ds-containing DNA aptamer was stabilized by introducing a mini-hairpin DNA resistant to nucleases, without any loss of affinity (KD = 61 pM). This new version is expected to consistently produce high-affinity DNA aptamers.

  13. Ligand binding by PDZ domains.

    PubMed

    Chi, Celestine N; Bach, Anders; Strømgaard, Kristian; Gianni, Stefano; Jemth, Per

    2012-01-01

    The postsynaptic density protein-95/disks large/zonula occludens-1 (PDZ) protein domain family is one of the most common protein-protein interaction modules in mammalian cells, with paralogs present in several hundred human proteins. PDZ domains are found in most cell types, but neuronal proteins, for example, are particularly rich in these domains. The general function of PDZ domains is to bring proteins together within the appropriate cellular compartment, thereby facilitating scaffolding, signaling, and trafficking events. The many functions of PDZ domains under normal physiological as well as pathological conditions have been reviewed recently. In this review, we focus on the molecular details of how PDZ domains bind their protein ligands and their potential as drug targets in this context.

  14. The Inhibitory Effect of α/β-Hydrolase Domain-Containing 6 (ABHD6) on the Surface Targeting of GluA2- and GluA3-Containing AMPA Receptors

    PubMed Central

    Wei, Mengping; Jia, Moye; Zhang, Jian; Yu, Lulu; Zhao, Yunzhi; Chen, Yingqi; Ma, Yimeng; Zhang, Wei; Shi, Yun S.; Zhang, Chen

    2017-01-01

    The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) are major excitatory receptors that mediate fast neurotransmission in the mammalian brain. The surface expression of functional AMPARs is crucial for synaptic transmission and plasticity. AMPAR auxiliary subunits control the biosynthesis, membrane trafficking, and synaptic targeting of AMPARs. Our previous report showed that α/β-hydrolase domain-containing 6 (ABHD6), an auxiliary subunit for AMPARs, suppresses the membrane delivery and function of GluA1-containing receptors in both heterologous cells and neurons. However, it remained unclear whether ABHD6 affects the membrane trafficking of glutamate receptor subunits, GluA2 and GluA3. Here, we examine the effects of ABHD6 overexpression in HEK293T cells expressing GluA1, GluA2, GluA3, and stargazin, either alone or in combination. The results show that ABHD6 suppresses the glutamate-induced currents and the membrane expression of AMPARs when expressing GluA2 or GluA3 in the HEK293T cells. We generated a series of GluA2 and GluA3 C-terminal deletion constructs and confirm that the C-terminus of GluAs is required for ABHD6’s inhibitory effects on glutamate-induced currents and surface expression of GluAs. Meanwhile, our pull-down experiments reveal that ABHD6 binds to GluA1–3, and deletion of the C-terminal domain of GluAs abolishes this binding. These findings demonstrate that ABHD6 inhibits the AMPAR-mediated currents and its surface expression, independent of the type of AMPAR subunits, and this inhibitor’s effects are mediated through the binding with the GluAs C-terminal regions. PMID:28303090

  15. Design of HIV-1 integrase inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75: a scaffold hopping approach using salicylate and catechol groups.

    PubMed

    Fan, Xing; Zhang, Feng-Hua; Al-Safi, Rasha I; Zeng, Li-Fan; Shabaik, Yumna; Debnath, Bikash; Sanchez, Tino W; Odde, Srinivas; Neamati, Nouri; Long, Ya-Qiu

    2011-08-15

    HIV-1 integrase (IN) is a validated therapeutic target for antiviral drug design. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands the discovery of novel inhibitors that are structurally as well mechanistically different. Herein, we describe the design and discovery of novel IN inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol, the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substituted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p, IC(50)=5 μM) with more than 40-fold selectivity for the strand transfer over the 3'-processing reaction. More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75 cofactor. The prototype example, N-(cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-ylsulfonyl) benzamide (5u) inhibited the IN-LEDGF/p75 interaction with an IC(50) value of 8 μM. Using molecular modeling, the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the IN active site. Furthermore, the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/p75 binding site on IN. This work provides a new and efficient approach to evolve novel HIV-1 IN inhibitors from rational integration and optimization of previously reported inhibitors.

  16. Design of HIV-1 Integrase Inhibitors Targeting the Catalytic Domain as Well as Its Interaction with LEDGF/p75: A Scaffold Hopping Approach Using Salicylate and Catechol Groups

    PubMed Central

    Fan, Xing; Zhang, Feng-Hua; Al-Safi, Rasha I.; Zeng, Li-Fan; Shabaik, Yumna; Debnath, Bikash; Sanchez, Tino W.; Odde, Srinivas; Neamati, Nouri; Long, Ya-Qiu

    2011-01-01

    HIV-1 integrase (IN) is a validated therapeutic target for antiviral agents. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands new structure and new mechanism IN inhibitors. Herein, we describe the design and discovery of novel IN inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol, the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substituted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p, IC50 = 5 μM) with more than 40-fold selectivity for the strand transfer over the 3′-processing reaction. More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75 cofactor. The prototype example, N-(cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-ylsulfonyl) benzamide (5u) inhibited the IN-LEDGF/p75 interaction with an IC50 value of 8 μM. Based on the molecular modeling, the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the IN active site. And the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/p75 binding site in IN protein. This work provided a new and efficient approach to evolve novel HIV-1 IN inhibitors from rational integration and optimization of previously reported inhibitors. PMID:21778063

  17. The adenoviral E1A N-terminal domain represses MYC transcription in human cancer cells by targeting both p300 and TRRAP and inhibiting MYC promoter acetylation of H3K18 and H4K16

    PubMed Central

    Zhao, Ling-Jun; Loewenstein, Paul M.; Green, Maurice

    2016-01-01

    Human cancers frequently arise from increased expression of proto-oncogenes, such as MYC and HER2. Understanding the cellular pathways regulating the transcription and expression of proto-oncogenes is important for targeted therapies for cancer treatment. Adenoviral (Ad) E1A 243R (243 aa residues) is a viral oncoprotein that interacts with key regulators of gene transcription and cell proliferation. We have shown previously that the 80 amino acid N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) can target the histone acetyltransferase (HAT) p300 and repress HER2 in the HER2-overexpressing human breast cancer cell line SKBR3. Expression of E1A 1-80 induces death of SKBR3 and other cancer cell lines. In this study, we performed total cell RNA sequence analysis and identified MYC as the regulatory gene for cellular proliferation most strongly repressed by E1A 1-80. By RT-quantitative PCR analysis we show that repression of MYC in SKBR3 cells occurs early after expression of E1A 1-80, suggesting that MYC may be an early responder of E1A 1-80-mediated transcriptional repression. Of interest, while E1A 1-80 repression of MYC occurs in all eight human cancer cell lines examined, repression of HER2 is cell-type dependent. We demonstrate by ChIP analysis that MYC transcriptional repression by E1A 1-80 is associated with inhibition of acetylation of H3K18 and H4K16 on the MYC promoter, as well as inhibition of RNA Pol II binding to the MYC promoter. Deletion mutant analysis of E1A 1-80 suggests that both p300/CBP and TRRAP are involved in E1A 1-80 repression of MYC transcription. Further, E1A 1-80 interaction with p300/CBP and TRRAP is correlated with inhibition of H3K18 and H4K16 acetylation on the MYC promoter, respectively. Our results indicate that E1A 1-80 may target two important pathways for histone modification to repress transcription in human cancer cells. PMID:27382434

  18. Structure of the S100A6 complex with a fragment from the C-terminal domain of Siah-1 interacting protein: A novel mode for S100 protein target recognition†

    PubMed Central

    Lee, Young-Tae; Dimitrova, Yoana N.; Schneider, Gabriela; Ridenour, Whitney B.; Bhattacharya, Shibani; Soss, Sarah E.; Caprioli, Richard M.; Filipek, Anna; Chazin, Walter J.

    2009-01-01

    S100A6 is a member of the S100 subfamily of Ca2+ binding EF-hand proteins that has been shown to interact with calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP; SIP), a subunit of an SCF-like E3 ligase complex (SCF-TBL1) formed under genotoxic stress. SIP serves as a scaffold in this complex, linking the E2-recruiting module Siah-1 to the substrate-recruiting module Skp1-TBL1. A cell-based functional assay suggests that S100A6 modulates the activity of SCF-TBL1. The results from the cell-based experiments could be enhanced if it were possible to selectively inhibit S100A6-SIP interactions without perturbing any other functions of the two proteins. To this end, the structure of the S100A6-SIP complex was determined in solution by NMR and the strength of the interaction was characterized by isothermal titration calorimetry. In an initial step, the minimal binding region in SIP for S100A6 was mapped to a 31 residue fragment (Ser189-Arg219) in the C-terminal domain. The structure of the S100A6-SIP(189–219) complex revealed that SIP(189–219) forms two helices, the first of which (Met193-Tyr200) interacts with S100A6 in a canonical binding mode. The second helix (Met207-Val216) lies over the S100A6 dimer interface, a mode of binding to S100A6 that has not previously been observed for any target bound to an S100 protein. A series of structure-based SIP mutations showed reduced S100A6 binding affinity, setting the stage for direct functional analysis of S100A6-SIP interactions. PMID:18803400

  19. Engineered autonomous human variable domains

    PubMed Central

    Nilvebrant, Johan; Tessier, Peter M.; Sidhu, Sachdev S.

    2017-01-01

    The complex multi-chain architecture of antibodies has spurred interest in smaller derivatives that retain specificity but can be more easily produced in bacteria. Domain antibodies consisting of single variable domains are the smallest antibody fragments and have been shown to possess enhanced ability to target epitopes that are difficult to access using multidomain antibodies. However, in contrast to natural camelid antibody domains, human variable domains typically suffer from low stability and high propensity to aggregate. This review summarizes strategies to improve the biophysical properties of heavy chain variable domains from human antibodies with an emphasis on aggregation resistance. Several protein engineering approaches have targeted antibody frameworks and complementarity determining regions to stabilize the native state and prevent aggregation of the denatured state. Recent findings enable the construction of highly diverse libraries enriched in aggregation-resistant variants that are expected to provide binders to diverse antigens. Engineered domain antibodies possess unique advantages in expression, epitope preference and flexibility of formatting over conventional immunoreagents and are a promising class of antibody fragments for biomedical development. PMID:27655414

  20. Single-domain antibodies for biomedical applications.

    PubMed

    Krah, Simon; Schröter, Christian; Zielonka, Stefan; Empting, Martin; Valldorf, Bernhard; Kolmar, Harald

    2016-01-01

    Single-domain antibodies are the smallest antigen-binding units of antibodies, consisting either only of one variable domain or one engineered constant domain that solely facilitates target binding. This class of antibody derivatives comprises naturally occurring variable domains derived from camelids and sharks as well as engineered human variable or constant antibody domains of the heavy or light chain. Because of their high affinity and specificity as well as stability, small size and benefit of multiple re-formatting opportunities, those molecules emerged as promising candidates for biomedical applications and some of these entities have already proven to be successful in clinical development.

  1. Domain Adaptation with Conditional Transferable Components

    PubMed Central

    Gong, Mingming; Zhang, Kun; Liu, Tongliang; Tao, Dacheng; Glymour, Clark; Schölkopf, Bernhard

    2017-01-01

    Domain adaptation arises in supervised learning when the training (source domain) and test (target domain) data have different distributions. Let X and Y denote the features and target, respectively, previous work on domain adaptation mainly considers the covariate shift situation where the distribution of the features P(X) changes across domains while the conditional distribution P(Y∣X) stays the same. To reduce domain discrepancy, recent methods try to find invariant components T(X) that have similar P(T(X)) on different domains by explicitly minimizing a distribution discrepancy measure. However, it is not clear if P(Y∣T(X)) in different domains is also similar when P(Y∣X) changes. Furthermore, transferable components do not necessarily have to be invariant. If the change in some components is identifiable, we can make use of such components for prediction in the target domain. In this paper, we focus on the case where P(X∣Y) and P(Y) both change in a causal system in which Y is the cause for X. Under appropriate assumptions, we aim to extract conditional transferable components whose conditional distribution P(T(X)∣Y) is invariant after proper location-scale (LS) transformations, and identify how P(Y) changes between domains simultaneously. We provide theoretical analysis and empirical evaluation on both synthetic and real-world data to show the effectiveness of our method. PMID:28239433

  2. Domain Adaptation with Conditional Transferable Components.

    PubMed

    Gong, Mingming; Zhang, Kun; Liu, Tongliang; Tao, Dacheng; Glymour, Clark; Schölkopf, Bernhard

    2016-06-01

    Domain adaptation arises in supervised learning when the training (source domain) and test (target domain) data have different distributions. Let X and Y denote the features and target, respectively, previous work on domain adaptation mainly considers the covariate shift situation where the distribution of the features P(X) changes across domains while the conditional distribution P(Y∣X) stays the same. To reduce domain discrepancy, recent methods try to find invariant components [Formula: see text] that have similar [Formula: see text] on different domains by explicitly minimizing a distribution discrepancy measure. However, it is not clear if [Formula: see text] in different domains is also similar when P(Y∣X) changes. Furthermore, transferable components do not necessarily have to be invariant. If the change in some components is identifiable, we can make use of such components for prediction in the target domain. In this paper, we focus on the case where P(X∣Y) and P(Y) both change in a causal system in which Y is the cause for X. Under appropriate assumptions, we aim to extract conditional transferable components whose conditional distribution [Formula: see text] is invariant after proper location-scale (LS) transformations, and identify how P(Y) changes between domains simultaneously. We provide theoretical analysis and empirical evaluation on both synthetic and real-world data to show the effectiveness of our method.

  3. IP3R2 levels dictate the apoptotic sensitivity of diffuse large B-cell lymphoma cells to an IP3R-derived peptide targeting the BH4 domain of Bcl-2.

    PubMed

    Akl, H; Monaco, G; La Rovere, R; Welkenhuyzen, K; Kiviluoto, S; Vervliet, T; Molgó, J; Distelhorst, C W; Missiaen, L; Mikoshiba, K; Parys, J B; De Smedt, H; Bultynck, G

    2013-05-16

    Disrupting inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)/B-cell lymphoma 2 (Bcl-2) complexes using a cell-permeable peptide (stabilized TAT-fused IP3R-derived peptide (TAT-IDP(S))) that selectively targets the BH4 domain of Bcl-2 but not that of B-cell lymphoma 2-extra large (Bcl-Xl) potentiated pro-apoptotic Ca(2+) signaling in chronic lymphocytic leukemia cells. However, the molecular mechanisms rendering cancer cells but not normal cells particularly sensitive to disrupting IP3R/Bcl-2 complexes are poorly understood. Therefore, we studied the effect of TAT-IDP(S) in a more heterogeneous Bcl-2-dependent cancer model using a set of 'primed to death' diffuse large B-cell lymphoma (DL-BCL) cell lines containing elevated Bcl-2 levels. We discovered a large heterogeneity in the apoptotic responses of these cells to TAT-IDP(S) with SU-DHL-4 being most sensitive and OCI-LY-1 being most resistant. This sensitivity strongly correlated with the ability of TAT-IDP(S) to promote IP3R-mediated Ca(2+) release. Although total IP3R-expression levels were very similar among SU-DHL-4 and OCI-LY-1, we discovered that the IP3R2-protein level was the highest for SU-DHL-4 and the lowest for OCI-LY-1. Strikingly, TAT-IDP(S)-induced Ca(2+) rise and apoptosis in the different DL-BCL cell lines strongly correlated with their IP3R2-protein level, but not with IP3R1-, IP3R3- or total IP3R-expression levels. Inhibiting or knocking down IP3R2 activity in SU-DHL-4-reduced TAT-IDP(S)-induced apoptosis, which is compatible with its ability to dissociate Bcl-2 from IP3R2 and to promote IP3-induced pro-apoptotic Ca(2+) signaling. Thus, certain chronically activated B-cell lymphoma cells are addicted to high Bcl-2 levels for their survival not only to neutralize pro-apoptotic Bcl-2-family members but also to suppress IP3R hyperactivity. In particular, cancer cells expressing high levels of IP3R2 are addicted to IP3R/Bcl-2 complex formation and disruption of these complexes using peptide tools

  4. Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway.

    PubMed

    Daleke, Maria H; Cascioferro, Alessandro; de Punder, Karin; Ummels, Roy; Abdallah, Abdallah M; van der Wel, Nicole; Peters, Peter J; Luirink, Joen; Manganelli, Riccardo; Bitter, Wilbert

    2011-05-27

    The type VII secretion system ESX-5 is a major pathway for export of PE and PPE proteins in pathogenic mycobacteria. These mycobacteria-specific protein families are characterized by conserved N-terminal domains of 100 and 180 amino acids, which contain the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) motifs after which they are named. Here we investigated secretion of the triacylglycerol lipase LipY, which in fast-growing mycobacteria contains a signal sequence, but in slow-growing species appears to have replaced the signal peptide with a PE or PPE domain. Selected LipY homologues were expressed in wild-type Mycobacterium marinum and its corresponding ESX-5 mutant, and localization of the proteins was investigated by immunoblotting and electron microscopy. Our study shows that Mycobacterium tuberculosis PE-LipY (LipY(tub)) and M. marinum PPE-LipY (LipY(mar)) are both secreted to the bacterial surface in an ESX-5-dependent fashion. After transport, the PE/PPE domains are removed by proteolytic cleavage. In contrast, Mycobacterium gilvum LipY, which has a signal sequence, is not transported to the cell surface. Furthermore, we show that LipY(tub) and LipY(mar) require their respective PE and PPE domains for ESX-5-dependent secretion. The role of the PE domain in ESX-5 secretion was confirmed in a whole cell lipase assay, in which wild-type bacteria expressing full-length LipY(tub), but not LipY(tub) lacking its PE domain, were shown to hydrolyze extracellular lipids. In conclusion, both PE and PPE domains contain a signal required for secretion of LipY by the ESX-5 system, and these domains are proteolytically removed upon translocation.

  5. ATP-Competitive MLKL Binders Have No Functional Impact on Necroptosis

    PubMed Central

    Ma, Bin; Marcotte, Doug; Paramasivam, Murugan; Michelsen, Klaus; Wang, Ti; Bertolotti-Ciarlet, Andrea; Jones, John Howard; Moree, Ben; Butko, Margaret; Salafsky, Joshua; Sun, Xin; McKee, Timothy

    2016-01-01

    MLKL is a pore forming pseudokinase involved in the final stage of necroptosis, a form of programmed cell death. Its phosphorylation by RIPK3 is necessary for triggering necroptosis but not for triggering apoptosis, which makes it a unique target for pharmacological inhibition to block necroptotic cell death. This mechanism has been described as playing a role in disease progression in neurodegenerative and inflammatory diseases. A type II kinase inhibitor (cpd 1) has been described that reportedly binds to the MLKL pseudokinase domain and prevents necroptosis. Here we describe five compounds that bind to the MLKL ATP-binding site, however the four MLKL-selective compounds have no activity in rescuing cells from necroptosis. We use kinase selectivity panels, crystallography and a new conformationally sensitive method of measuring protein conformational changes (SHG) to confirm that the one previously reported compound that can rescue cells (cpd 1) is a non-selective type II inhibitor that also inhibits the upstream kinase RIPK1. Although this compound can shift the GFE motif of the activation loop to an “out” position, the accessibility of the key residue Ser358 in the MLKL activation loop is not affected by compound binding to the MLKL active site. Our studies indicate that an ATP-pocket inhibitor of the MLKL pseudokinase domain does not have any impact on the necroptosis pathway, which is contrary to a previously reported study. PMID:27832137

  6. Flowing on Riemannian manifold: domain adaptation by shifting covariance.

    PubMed

    Cui, Zhen; Li, Wen; Xu, Dong; Shan, Shiguang; Chen, Xilin; Li, Xuelong

    2014-12-01

    Domain adaptation has shown promising results in computer vision applications. In this paper, we propose a new unsupervised domain adaptation method called domain adaptation by shifting covariance (DASC) for object recognition without requiring any labeled samples from the target domain. By characterizing samples from each domain as one covariance matrix, the source and target domain are represented into two distinct points residing on a Riemannian manifold. Along the geodesic constructed from the two points, we then interpolate some intermediate points (i.e., covariance matrices), which are used to bridge the two domains. By utilizing the principal components of each covariance matrix, samples from each domain are further projected into intermediate feature spaces, which finally leads to domain-invariant features after the concatenation of these features from intermediate points. In the multiple source domain adaptation task, we also need to effectively integrate different types of features between each pair of source and target domains. We additionally propose an SVM based method to simultaneously learn the optimal target classifier as well as the optimal weights for different source domains. Extensive experiments demonstrate the effectiveness of our method for both single source and multiple source domain adaptation tasks.

  7. Domain adaptive boosting method and its applications

    NASA Astrophysics Data System (ADS)

    Geng, Jie; Miao, Zhenjiang

    2015-03-01

    Differences of data distributions widely exist among datasets, i.e., domains. For many pattern recognition, nature language processing, and content-based analysis systems, a decrease in performance caused by the domain differences between the training and testing datasets is still a notable problem. We propose a domain adaptation method called domain adaptive boosting (DAB). It is based on the AdaBoost approach with extensions to cover the domain differences between the source and target domains. Two main stages are contained in this approach: source-domain clustering and source-domain sample selection. By iteratively adding the selected training samples from the source domain, the discrimination model is able to achieve better domain adaptation performance based on a small validation set. The DAB algorithm is suitable for the domains with large scale samples and easy to extend for multisource adaptation. We implement this method on three computer vision systems: the skin detection model in single images, the video concept detection model, and the object classification model. In the experiments, we compare the performances of several commonly used methods and the proposed DAB. Under most situations, the DAB is superior.

  8. 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.

  9. X-linked inhibitor of apoptosis protein (XIAP) lacking RING domain localizes to the nuclear and promotes cancer cell anchorage-independent growth by targeting the E2F1/Cyclin E axis

    PubMed Central

    Cao, Zipeng; Li, Xueyong; Li, Jingxia; Luo, Wenjing; Huang, Chuanshu; Chen, Jingyuan

    2014-01-01

    The inhibitor of apoptosis protein XIAP (X-linked inhibitor of apoptosis protein) is a well-documented protein that is located in cytoplasm acting as a potent regulator of cell apoptosis. Here, we showed that expressing XIAP with RING (Really Interesting New Gene) domain deletion (XIAPΔRING) in cancer cells promoted cancer cell anchorage-independent growth and G1/S phase transition companied with increasing cyclin e transcription activity and protein expression. Further studies revealed that XIAPΔRING was mainly localized in nuclear with increased binding with E2F1, whereas XIAP with BIR (Baculoviral IAP Repeat) domains deletion (XIAPΔBIRs) was entirely presented in cytoplasma with losing its binding with E2F1, suggesting that RING domain was able to inhibit BIR domains nuclear localization, by which impaired BIRs binding with E2F1 in cellular nucleus in intact cells. These studies identified a new function of XIAP protein in cellular nucleus is to regulate E2F1 transcriptional activity by binding with E2F1 in cancer cells. Our current finding of an effect of XIAPΔRING expression on cancer cell anchorage-independent growth suggests that overexpression of this protein may contribute to genetic instability associated with cell cycle and checkpoint perturbations, in addition to its impact on cellular apoptosis. PMID:25216527

  10. Understanding the Public Domain.

    ERIC Educational Resources Information Center

    Russell, Carrie

    2003-01-01

    This overview of the public domain covers: defining the public domain; figuring out if a work is protected by copyright; being sure a work is in the public domain; asserting the copyright protection and term; the Creative Commons initiative; building the Information Commons; when permission is needed for using a public domain work; and special…

  11. Domain-specific control of selective attention.

    PubMed

    Lin, Szu-Hung; Yeh, Yei-Yu

    2014-01-01

    Previous research has shown that loading information on working memory affects selective attention. However, whether the load effect on selective attention is domain-general or domain-specific remains unresolved. The domain-general effect refers to the findings that load in one content (e.g. phonological) domain in working memory influences processing in another content (e.g., visuospatial) domain. Attentional control supervises selection regardless of information domain. The domain-specific effect refers to the constraint of influence only when maintenance and processing operate in the same domain. Selective attention operates in a specific content domain. This study is designed to resolve this controversy. Across three experiments, we manipulated the type of representation maintained in working memory and the type of representation upon which the participants must exert control to resolve conflict and select a target into the focus of attention. In Experiments 1a and 1b, participants maintained digits and nonverbalized objects, respectively, in working memory while selecting a target in a letter array. In Experiment 2, we presented auditory digits with a letter flanker task to exclude the involvement of resource competition within the same input modality. In Experiments 3a and 3b, we replaced the letter flanker task with an object flanker task while manipulating the memory load on object and digit representation, respectively. The results consistently showed that memory load modulated distractibility only when the stimuli of the two tasks were represented in the same domain. The magnitude of distractor interference was larger under high load than under low load, reflecting a lower efficacy of information prioritization. When the stimuli of the two tasks were represented in different domains, memory load did not modulate distractibility. Control of processing priority in selective attention demands domain-specific resources.

  12. The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets

    PubMed Central

    Scheiba, Rafael M; de Opakua, Alain Ibáñez; Díaz-Quintana, Antonio; Cruz-Gallardo, Isabel; Martínez-Cruz, Luis A; Martínez-Chantar, María L; Blanco, Francisco J; Díaz-Moreno, Irene

    2014-01-01

    Human antigen R (HuR) is a 32 kDa protein with 3 RNA Recognition Motifs (RRMs), which bind to Adenylate and uridylate Rich Elements (AREs) of mRNAs. Whereas the N-terminal and central domains (RRM1 and RRM2) are essential for AREs recognition, little is known on the C-terminal RRM3 beyond its implication in HuR oligomerization and apoptotic signaling. We have developed a detergent-based strategy to produce soluble RRM3 for structural studies. We have found that it adopts the typical RRM fold, does not interact with the RRM1 and RRM2 modules, and forms dimers in solution. Our NMR measurements, combined with Molecular Dynamics simulations and Analytical Ultracentrifugation experiments, show that the protein dimerizes through a helical region that contains the conserved W261 residue. We found that HuR RRM3 binds to 5′-mer U-rich RNA stretches through the solvent exposed side of its β-sheet, located opposite to the dimerization site. Upon mimicking phosphorylation by the S318D replacement, RRM3 mutant shows less ability to recognize RNA due to an electrostatic repulsion effect with the phosphate groups. Our study brings new insights of HuR RRM3 as a domain involved in protein oligomerization and RNA interaction, both functions regulated by 2 surfaces on opposite sides of the RRM domain. PMID:25584704

  13. The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets.

    PubMed

    Scheiba, Rafael M; de Opakua, Alain Ibáñez; Díaz-Quintana, Antonio; Cruz-Gallardo, Isabel; Martínez-Cruz, Luis A; Martínez-Chantar, María L; Blanco, Francisco J; Díaz-Moreno, Irene

    2014-01-01

    Human antigen R (HuR) is a 32 kDa protein with 3 RNA Recognition Motifs (RRMs), which bind to Adenylate and uridylate Rich Elements (AREs) of mRNAs. Whereas the N-terminal and central domains (RRM1 and RRM2) are essential for AREs recognition, little is known on the C-terminal RRM3 beyond its implication in HuR oligomerization and apoptotic signaling. We have developed a detergent-based strategy to produce soluble RRM3 for structural studies. We have found that it adopts the typical RRM fold, does not interact with the RRM1 and RRM2 modules, and forms dimers in solution. Our NMR measurements, combined with Molecular Dynamics simulations and Analytical Ultracentrifugation experiments, show that the protein dimerizes through a helical region that contains the conserved W261 residue. We found that HuR RRM3 binds to 5'-mer U-rich RNA stretches through the solvent exposed side of its β-sheet, located opposite to the dimerization site. Upon mimicking phosphorylation by the S318D replacement, RRM3 mutant shows less ability to recognize RNA due to an electrostatic repulsion effect with the phosphate groups. Our study brings new insights of HuR RRM3 as a domain involved in protein oligomerization and RNA interaction, both functions regulated by 2 surfaces on opposite sides of the RRM domain.

  14. Time-Domain vs. Frequency-Domain CSEM: Implications for Marine Exploration

    NASA Astrophysics Data System (ADS)

    Connell, D. M.; Key, K. W.

    2010-12-01

    The frequency-domain marine controlled source electromagnetic (CSEM) method is now routinely applied to map resistive hydrocarbons buried beneath the seabed in deepwater. Alternatively, it has been suggested that time-domain CSEM methods may offer improved resolution of difficult targets such as deeply buried reservoirs. Furthermore, time-domain methods may overcome a sensitivity limitation imposed by the airwave saturation that is experienced for shallow-water frequency-domain CSEM. In order to examine and test these claims, we have developed a modeling code for computing time-domain responses for layered 1D models with arbitrarily located and oriented transmitters and receivers. Our code extends the open-source frequency domain code Dipole1D by efficiently computing the time-domain step-on and impulse responses by Fourier transformation of the frequency-domain kernels. By applying a realistic noise model to synthetic data generated from this code, we systematically examine the sensitivity and resolution of time-domain and frequency-domain CSEM to representative targets of interest for offshore hydrocarbon exploration and exploration surveys of seafloor volcanic and hydrothermal systems. These studies have practical implications for marine EM survey systems that use either towed or stationary transmitters and receivers.

  15. Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain

    SciTech Connect

    Suzuki, Nobuhiro; Yamazaki, Yasuo; Brown, R. Lane; Fujimoto, Zui; Morita, Takashi; Mizuno, Hiroshi

    2008-10-01

    The structures of pseudechetoxin and pseudecin suggest that both proteins bind to cyclic nucleotide-gated ion channels in a manner in which the concave surface occludes the pore entrance. Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction by retinal photoreceptors and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins belong to a cysteine-rich secretory protein (CRISP) family containing an N-terminal pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). PsTx and Pdc are highly homologous proteins, but their blocking affinities on CNG channels are different: PsTx blocks both the olfactory and retinal channels with ∼15–30-fold higher affinity than Pdc. To gain further insights into their structure and function, the crystal structures of PsTx, Pdc and Zn{sup 2+}-bound Pdc were determined. The structures revealed that most of the amino-acid-residue differences between PsTx and Pdc are located around the concave surface formed between the PR-1 domain and the CRD, suggesting that the concave surface is functionally important for CNG-channel binding and inhibition. A structural comparison in the presence and absence of Zn{sup 2+} ion demonstrated that the concave surface can open and close owing to movement of the CRD upon Zn{sup 2+} binding. The data suggest that PsTx and Pdc occlude the pore entrance and that the dynamic motion of the concave surface facilitates interaction with the CNG channels.

  16. Domain view: a web tool for protein domain visualization and analysis.

    PubMed

    Pan, Xiaokang; Bingman, Craig A; Wesenberg, Gary E; Sun, Zhaohui; Phillips, George N

    2010-12-01

    The identification of sequence-based protein domains and their boundaries is often a prelude to structure determination. An accurate prediction of disordered regions, secondary structures and low complexity segments of target protein sequences can improve the efficiency of selection in structural genomics and also aid in design of constructs for directed structural biology studies. At the Center for Eukaryotic Structural Genomics (CESG) we have developed DomainView, a web tool to visualize and analyze predicted protein domains, disordered regions, secondary structures and low complexity segments of target protein sequences for selection of experimental protein structure attempts. DomainView consists of a relational database and a web graphical-user interface. The database was developed based on MySQL, which stores data from target protein sequences and their domains, disordered regions, secondary structures and low complexity segments. The program of the web user interface is a Perl CGI script. When a user searches for a target protein sequence, the script displays the combinational information about the domains and other features of that target sequence graphically on a web page by querying the database. The graphical representation for each feature is linked to a web page showing more detailed annotation information or to a new window directly running the corresponding prediction program to show further information about that feature.

  17. Developing an Exploratory Response to Intervention Construct in the Behavior Domain: An Analysis of Outcome Measures and Targeted, School-Based Interventions for Elementary Students At-Risk for Severe Problem Behavior

    ERIC Educational Resources Information Center

    Mielenz, Christine R.

    2010-01-01

    Targeted, school-based behavioral interventions that are designed in the positive behavior support model and created for elementary students at-risk for severe problem behavior have used four approaches to measuring response to intervention: (1) office discipline referrals, (2) systematic direct observation, (3) standardized behavior rating…

  18. Extra domains in secondary transport carriers and channel proteins.

    PubMed

    Barabote, Ravi D; Tamang, Dorjee G; Abeywardena, Shannon N; Fallah, Neda S; Fu, Jeffrey Yu Chung; Lio, Jeffrey K; Mirhosseini, Pegah; Pezeshk, Ronnie; Podell, Sheila; Salampessy, Marnae L; Thever, Mark D; Saier, Milton H

    2006-10-01

    "Extra" domains in members of the families of secondary transport carrier and channel proteins provide secondary functions that expand, amplify or restrict the functional nature of these proteins. Domains in secondary carriers include TrkA and SPX domains in DASS family members, DedA domains in TRAP-T family members (both of the IT superfamily), Kazal-2 and PDZ domains in OAT family members (of the MF superfamily), USP, IIA(Fru) and TrkA domains in ABT family members (of the APC superfamily), ricin domains in OST family members, and TrkA domains in AAE family members. Some transporters contain highly hydrophilic domains consisting of multiple repeat units that can also be found in proteins of dissimilar function. Similarly, transmembrane alpha-helical channel-forming proteins contain unique, conserved, hydrophilic domains, most of which are not found in carriers. In some cases the functions of these domains are known. They may be ligand binding domains, phosphorylation domains, signal transduction domains, protein/protein interaction domains or complex carbohydrate-binding domains. These domains mediate regulation, subunit interactions, or subcellular targeting. Phylogenetic analyses show that while some of these domains are restricted to closely related proteins derived from specific organismal types, others are nearly ubiquitous within a particular family of transporters and occur in a tremendous diversity of organisms. The former probably became associated with the transporters late in the evolutionary process; the latter probably became associated with the carriers much earlier. These domains can be located at either end of the transporter or in a central region, depending on the domain and transporter family. These studies provide useful information about the evolution of extra domains in channels and secondary carriers and provide novel clues concerning function.

  19. A general strategy to characterize calmodulin-calcium complexes involved in CaM-target recognition: DAPK and EGFR calmodulin binding domains interact with different calmodulin-calcium complexes.

    PubMed

    Dagher, Rania; Peng, Shan; Gioria, Sophie; Fève, Marie; Zeniou, Maria; Zimmermann, Michael; Pigault, Claire; Haiech, Jacques; Kilhoffer, Marie-Claude

    2011-05-01

    Calmodulin (CaM) is a ubiquitous Ca(2+) sensor regulating many biochemical processes in eukaryotic cells. Its interaction with a great variety of different target proteins has led to the fundamental question of its mechanism of action. CaM exhibits four "EF hand" type Ca(2+) binding sites. One way to explain CaM functioning is to consider that the protein interacts differently with its target proteins depending on the number of Ca(2+) ions bound to it. To test this hypothesis, the binding properties of three entities known to interact with CaM (a fluorescent probe and two peptide analogs to the CaM binding sites of death associated protein kinase (DAPK) and of EGFR) were investigated using a quantitative approach based on fluorescence polarization (FP). Probe and peptide interactions with CaM were studied using a titration matrix in which both CaM and calcium concentrations were varied. Experiments were performed with SynCaM, a hybrid CaM able to activate CaM dependent enzymes from mammalian and plant cells. Results show that the interaction between CaM and its targets is regulated by the number of calcium ions bound to the protein, namely one for the DAPK peptide, two for the probe and four for the EGFR peptide. The approach used provides a new tool to elaborate a typology of CaM-targets, based on their recognition by the various CaM-Ca(n) (n=0-4) complexes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

  20. Identifying Targets from Filtering Effects

    DTIC Science & Technology

    2012-10-24

    Introduction Considering a radar (or sonar) target as more than a simple point scatterer brings up the possibility of identifying the target based on the...2000. [8] M. Vespe, C. J. Baker, and H. D. Griffiths , "Automatic target regognition using multi-diversity radar," Radar, Sonar \\& Navigation, IET, vol...A. Taflove and S. C. Hagness, Computational Electrodynamics : The Finite Difference Time Domain Method. Norwood, MA: Artech House, 2005.

  1. Multiple hypothesis tracking for the cyber domain

    NASA Astrophysics Data System (ADS)

    Schwoegler, Stefan; Blackman, Sam; Holsopple, Jared; Hirsch, Michael J.

    2011-09-01

    This paper discusses how methods used for conventional multiple hypothesis tracking (MHT) can be extended to domain-agnostic tracking of entities from non-kinematic constraints such as those imposed by cyber attacks in a potentially dense false alarm background. MHT is widely recognized as the premier method to avoid corrupting tracks with spurious data in the kinematic domain but it has not been extensively applied to other problem domains. The traditional approach is to tightly couple track maintenance (prediction, gating, filtering, probabilistic pruning, and target confirmation) with hypothesis management (clustering, incompatibility maintenance, hypothesis formation, and Nassociation pruning). However, by separating the domain specific track maintenance portion from the domain agnostic hypothesis management piece, we can begin to apply the wealth of knowledge gained from ground and air tracking solutions to the cyber (and other) domains. These realizations led to the creation of Raytheon's Multiple Hypothesis Extensible Tracking Architecture (MHETA). In this paper, we showcase MHETA for the cyber domain, plugging in a well established method, CUBRC's INFormation Engine for Real-time Decision making, (INFERD), for the association portion of the MHT. The result is a CyberMHT. We demonstrate the power of MHETA-INFERD using simulated data. Using metrics from both the tracking and cyber domains, we show that while no tracker is perfect, by applying MHETA-INFERD, advanced nonkinematic tracks can be captured in an automated way, perform better than non-MHT approaches, and decrease analyst response time to cyber threats.

  2. The cardiac L-type calcium channel alpha subunit is a target for direct redox modification during oxidative stress - the role of cysteine residues in the alpha interacting domain.

    PubMed

    Muralidharan, Padmapriya; Cserne Szappanos, Henrietta; Ingley, Evan; Hool, Livia C

    2017-03-17

    Cardiovascular disease is the leading cause of death in the Western world. The incidence of cardiovascular disease is predicted to further rise with the increase in obesity and diabetes and with the ageing population. Even though the survival rate from ischemic heart disease has improved over the past 30 years, many patients progress to a chronic pathological condition, known as cardiac hypertrophy that is associated with an increase in morbidity and mortality. Reactive oxygen species (ROS) and calcium play an essential role in mediating cardiac hypertrophy. The L-type calcium channel is the main route for calcium influx into cardiac myocytes. There is now good evidence for a direct role for the L-type calcium channel in the development of cardiac hypertrophy. Cysteines on the channel are targets for redox modification and glutathionylation of the channel can modulate the function of the channel protein leading to the onset of pathology. The cysteine responsible for modification of L-type calcium channel function has now been identified. Detailed understanding of the role of cysteines as possible targets during oxidative stress may assist in designing therapy to prevent the development of hypertrophy and heart failure. This article is protected by copyright. All rights reserved.

  3. The Apoptosis Repressor with a CARD Domain (ARC) Gene Is a Direct Hypoxia-Inducible Factor 1 Target Gene and Promotes Survival and Proliferation of VHL-Deficient Renal Cancer Cells

    PubMed Central

    Razorenova, Olga V.; Castellini, Laura; Colavitti, Renata; Edgington, Laura E.; Nicolau, Monica; Huang, Xin; Bedogni, Barbara; Mills, Edward M.; Bogyo, Matthew

    2014-01-01

    The induction of hypoxia-inducible factors (HIFs) is essential for the adaptation of tumor cells to a low-oxygen environment. We found that the expression of the apoptosis inhibitor ARC (apoptosis repressor with a CARD domain) was induced by hypoxia in a variety of cancer cell types, and its induction is primarily HIF1 dependent. Chromatin immunoprecipitation (ChIP) and reporter assays also indicate that the ARC gene is regulated by direct binding of HIF1 to a hypoxia response element (HRE) located at bp −190 upstream of the transcription start site. HIFs play an essential role in the pathogenesis of renal cell carcinoma (RCC) under normoxic conditions, through the loss of the Von Hippel-Lindau gene (VHL). Accordingly, our results show that ARC is not expressed in normal renal tissue but is highly expressed in 65% of RCC tumors, which also express high levels of carbonic anhydrase IX (CAIX), a HIF1-dependent protein. Compared to controls, ARC-deficient RCCs exhibited decreased colony formation and increased apoptosis in vitro. In addition, loss of ARC resulted in a dramatic reduction of RCC tumor growth in SCID mice in vivo. Thus, HIF-mediated increased expression of ARC in RCC can explain how loss of VHL can promote survival early in tumor formation. PMID:24344197

  4. Purification and Structural Analysis of LEM-Domain Proteins.

    PubMed

    Herrada, Isaline; Bourgeois, Benjamin; Samson, Camille; Buendia, Brigitte; Worman, Howard J; Zinn-Justin, Sophie

    2016-01-01

    LAP2-emerin-MAN1 (LEM)-domain proteins are modular proteins characterized by the presence of a conserved motif of about 50 residues. Most LEM-domain proteins localize at the inner nuclear membrane, but some are also found in the endoplasmic reticulum or nuclear interior. Their architecture has been analyzed by predicting the limits of their globular domains, determining the 3D structure of these domains and in a few cases calculating the 3D structure of specific domains bound to biological targets. The LEM domain adopts an α-helical fold also found in SAP and HeH domains of prokaryotes and unicellular eukaryotes. The LEM domain binds to BAF (barrier-to-autointegration factor; BANF1), which interacts with DNA and tethers chromatin to the nuclear envelope. LAP2 isoforms also share an N-terminal LEM-like domain, which binds DNA. The structure and function of other globular domains that distinguish LEM-domain proteins from each other have been characterized, including the C-terminal dimerization domain of LAP2α and C-terminal WH and UHM domains of MAN1. LEM-domain proteins also have large intrinsically disordered regions that are involved in intra- and intermolecular interactions and are highly regulated by posttranslational modifications in vivo.

  5. Protein function prediction using domain families

    PubMed Central

    2013-01-01

    Here we assessed the use of domain families for predicting the functions of whole proteins. These 'functional families' (FunFams) were derived using a protocol that combines sequence clustering with supervised cluster evaluation, relying on available high-quality Gene Ontology (GO) annotation data in the latter step. In essence, the protocol groups domain sequences belonging to the same superfamily into families based on the GO annotations of their parent proteins. An initial test based on enzyme sequences confirmed that the FunFams resemble enzyme (domain) families much better than do families produced by sequence clustering alone. For the CAFA 2011 experiment, we further associated the FunFams with GO terms probabilistically. All target proteins were first submitted to domain superfamily assignment, followed by FunFam assignment and, eventually, function assignment. The latter included an integration step for multi-domain target proteins. The CAFA results put our domain-based approach among the top ten of 31 competing groups and 56 prediction methods, confirming that it outperforms simple pairwise whole-protein sequence comparisons. PMID:23514456

  6. Blind Domain Adaptation With Augmented Extreme Learning Machine Features.

    PubMed

    Uzair, Muhammad; Mian, Ajmal

    2016-02-11

    In practical applications, the test data often have different distribution from the training data leading to suboptimal visual classification performance. Domain adaptation (DA) addresses this problem by designing classifiers that are robust to mismatched distributions. Existing DA algorithms use the unlabeled test data from target domain during training time in addition to the source domain data. However, target domain data may not always be available for training. We propose a blind DA algorithm that does not require target domain samples for training. For this purpose, we learn a global nonlinear extreme learning machine (ELM) model from the source domain data in an unsupervised fashion. The global ELM model is then used to initialize and learn class specific ELM models from the source domain data. During testing, the target domain features are augmented with the reconstructed features from the global ELM model. The resulting enriched features are then classified using the class specific ELM models based on minimum reconstruction error. Extensive experiments on 16 standard datasets show that despite blind learning, our method outperforms six existing state-of-the-art methods in cross domain visual recognition.

  7. Role of Electrostatic Interactions in Binding of Peptides and Intrinsically Disordered Proteins to Their Folded Targets: 2. The Model of Encounter Complex Involving the Double Mutant of the c-Crk N-SH3 Domain and Peptide Sos.

    PubMed

    Yuwen, Tairan; Xue, Yi; Skrynnikov, Nikolai R

    2016-03-29

    In the first part of this work (paper 1, Xue, Y. et al. Biochemistry 2014 , 53 , 6473 ), we have studied the complex between the 10-residue peptide Sos and N-terminal SH3 domain from adaptor protein c-Crk. In the second part (this paper), we designed the double mutant of the c-Crk N-SH3 domain, W169F/Y186L, with the intention to eliminate the interactions responsible for tight peptide-protein binding, while retaining the interactions that create the initial electrostatic encounter complex. The resulting system was characterized experimentally by measuring the backbone and side-chain (15)N relaxation rates, as well as binding shifts and (1)H(N) temperature coefficients. In addition, it was also modeled via a series of ∼5 μs molecular dynamics (MD) simulations recorded in a large water box under an Amber ff99SB*-ILDN force field. Similar to paper 1, we have found that the strength of arginine-aspartate and arginine-glutamate salt bridges is overestimated in the original force field. To address this problem we have applied the empirical force-field correction described in paper 1. Specifically, the Lennard-Jones equilibrium distance for the nitrogen-oxygen pair across Arg-to-Asp/Glu salt bridges has been increased by 3%. This modification led to MD models in good agreement with the experimental data. The emerging picture is that of a fuzzy complex, where the peptide "dances" over the surface of the protein, making transient contacts via salt-bridge interactions. Every once in a while the peptide assumes a certain more stable binding pose, assisted by a number of adventitious polar and nonpolar contacts. On the other hand, occasionally Sos flies off the protein surface; it is then guided by electrostatic steering to quickly reconnect with the protein. The dynamic interaction between Sos and the double mutant of c-Crk N-SH3 gives rise to only small binding shifts. The peptide retains a high degree of conformational mobility, although it is appreciably slowed down due

  8. CO2-cAMP-Responsive cis-Elements Targeted by a Transcription Factor with CREB/ATF-Like Basic Zipper Domain in the Marine Diatom Phaeodactylum tricornutum1[W][OA

    PubMed Central

    Ohno, Naoki; Inoue, Takuya; Yamashiki, Ryosuke; Nakajima, Kensuke; Kitahara, Yuhei; Ishibashi, Mikiko; Matsuda, Yusuke

    2012-01-01

    Expression controls of the carbon acquisition system in marine diatoms in response to environmental factors are an essential issue to understand the changes in marine primary productivity. A pyrenoidal β-carbonic anhydrase, PtCA1, is one of the most important candidates to investigate the control mechanisms of the CO2 acquisition system in the marine diatom Phaeodactylum tricornutum. A detailed functional assay was carried out on the putative core regulatory region of the ptca1 promoter using a β-glucuronidase reporter in P. tricornutum cells under changing CO2 conditions. A set of loss-of-function assays led to the identification of three CO2-responsive elements, TGACGT, ACGTCA, and TGACGC, at a region −86 to −42 relative to the transcription start site. Treatment with a cyclic (c)AMP analog, dibutyryl cAMP, revealed these three elements to be under the control of cAMP; thus, we designated them, from 5′ to 3′, as CO2-cAMP-Responsive Element1 (CCRE1), CCRE2, and CCRE3. Because the sequence TGACGT is known to be a typical target of human Activating Transcription Factor6 (ATF6), we searched for genes containing a basic zipper (bZIP) region homologous to that of ATF6 in the genome of P. tricornutum. Gel-shift assays using CCRE pentamers as labeled probes showed that at least one candidate of bZIP proteins, PtbZIP11, bound specifically to CCREs. A series of gain-of-function assays with CCREs fused to a minimal promoter strongly suggested that the alternative combination of CCRE1/2 or CCRE2/3 at proper distances from the minimal promoter is required as a potential target of PtbZIP11 for an effective CO2 response of the ptca1 gene. PMID:22095044

  9. Targeting the Cyclophilin Domain of Ran-binding Protein 2 (Ranbp2) with Novel Small Molecules to Control the Proteostasis of STAT3, hnRNPA2B1 and M-Opsin

    PubMed Central

    Cho, Kyoung-in; Orry, Andrew; Park, Se Eun; Ferreira, Paulo A.

    2015-01-01

    Cyclophilins are peptidyl cis-trans prolyl isomerases (PPIases), whose activity is typically inhibited by cyclosporine A (CsA), a potent immunosuppressor. Cyclophilins are also chaperones. Emerging evidence supports that cyclophilins present non-overlapping PPIase and chaperone activities. The proteostasis of the disease-relevant substrates, signal transducer and activator of transcription 3 and 5 (STAT3/STAT5), heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) and M-opsin, are regulated by non-overlapping chaperone and PPIase activities of the cyclophilin domain (CY) of Ranbp2, a multifunctional and modular scaffold which controls nucleocytoplasmic shuttling and proteostasis of selective substrates. Although highly homologous, CY and the archetypal cyclophilin A (CyPA) present distinct catalytic and CsA-binding activities owing to unique structural features between these cylophilins. We explored structural idiosyncrasies between CY and CyPA to screen in silico nearly 9 million small molecules (SM) against the CY PPIase pocket and identify SMs with selective bioactivity toward STAT3, hnRNPA2B1 and/or M-opsin proteostasis. We found three classes of SMs that enhance the cytokine-stimulated transcriptional activity of STAT3 without changing latent and activated STAT3 levels, down-regulate hnRNPA2B1 or M-opsin proteostasis, or a combination of these. Further, a SM which suppresses hnRNPA2B1 proteostasis also inhibits strongly and selectively the PPIase activity of CY. This study unravels chemical probes for multimodal regulation of CY of Ranbp2 and its substrates and this regulation likely results in the allosterism stemming from the interconversion of conformational substates of cyclophilins. The results also demonstrate the feasibility of CY in drug discovery against disease-relevant substrates controlled by Ranbp2 and they open new opportunities for therapeutic interventions. PMID:26030368

  10. Visualizing domain wall and reverse domain superconductivity.

    PubMed

    Iavarone, M; Moore, S A; Fedor, J; Ciocys, S T; Karapetrov, G; Pearson, J; Novosad, V; Bader, S D

    2014-08-28

    In magnetically coupled, planar ferromagnet-superconductor (F/S) hybrid structures, magnetic domain walls can be used to spatially confine the superconductivity. In contrast to a superconductor in a uniform applied magnetic field, the nucleation of the superconducting order parameter in F/S structures is governed by the inhomogeneous magnetic field distribution. The interplay between the superconductivity localized at the domain walls and far from the walls leads to effects such as re-entrant superconductivity and reverse domain superconductivity with the critical temperature depending upon the location. Here we use scanning tunnelling spectroscopy to directly image the nucleation of superconductivity at the domain wall in F/S structures realized with Co-Pd multilayers and Pb thin films. Our results demonstrate that such F/S structures are attractive model systems that offer the possibility to control the strength and the location of the superconducting nucleus by applying an external magnetic field, potentially useful to guide vortices for computing application.

  11. A Domain Analysis Bibliography

    DTIC Science & Technology

    1990-06-01

    Bauhaus , a prototype CASE workstation for D-SAPS development. [ARAN88A] Guillermo F. Arango. Domain Engineering for Software Reuse. PhD thesis...34 VITA90B: Domain Analysis within the ISEC Rapid Center 48 CMU/SEI-90-SR-3 Appendix III Alphabetical by Organization/Project BAUHAUS * ALLE87A

  12. Domain wall filters

    SciTech Connect

    Baer, Oliver; Narayanan, Rajamani; Neuberger, Herbert; Witzel, Oliver

    2007-03-15

    We propose using the extra dimension separating the domain walls carrying lattice quarks of opposite handedness to gradually filter out the ultraviolet fluctuations of the gauge fields that are felt by the fermionic excitations living in the bulk. This generalization of the homogeneous domain wall construction has some theoretical features that seem nontrivial.

  13. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  14. Causal Learning Across Domains

    ERIC Educational Resources Information Center

    Schulz, Laura E.; Gopnik, Alison

    2004-01-01

    Five studies investigated (a) children's ability to use the dependent and independent probabilities of events to make causal inferences and (b) the interaction between such inferences and domain-specific knowledge. In Experiment 1, preschoolers used patterns of dependence and independence to make accurate causal inferences in the domains of…

  15. Disruption of hypoxia-inducible transcription factor-prolyl hydroxylase domain-1 (PHD-1-/-) attenuates ex vivo myocardial ischemia/reperfusion injury through hypoxia-inducible factor-1α transcription factor and its target genes in mice.

    PubMed

    Adluri, Ram Sudheer; Thirunavukkarasu, Mahesh; Dunna, Nageswara Rao; Zhan, Lijun; Oriowo, Babatunde; Takeda, Kotaro; Sanchez, Juan A; Otani, Hajime; Maulik, Gautam; Fong, Guo-Hua; Maulik, Nilanjana

    2011-10-01

    Hypoxia-inducible transcription factor (HIF)-prolyl hydroxylases domain (PHD-1-3) are oxygen sensors that regulate the stability of the HIFs in an oxygen-dependent manner. Suppression of PHD enzymes leads to stabilization of HIFs and offers a potential treatment option for many ischemic disorders, such as peripheral artery occlusive disease, myocardial infarction, and stroke. Here, we show that homozygous disruption of PHD-1 (PHD-1(-/-)) could facilitate HIF-1α-mediated cardioprotection in ischemia/reperfused (I/R) myocardium. Wild-type (WT) and PHD-1(-/-) mice were randomized into WT time-matched control (TMC), PHD-1(-/-) TMC (PHD1TMC), WT I/R, and PHD-1(-/-) I/R (PHD1IR). Isolated hearts from each group were subjected to 30 min of global ischemia followed by 2 h of reperfusion. TMC hearts were perfused for 2 h 30 min without ischemia. Decreased infarct size (35%±0.6% vs. 49%±0.4%) and apoptotic cardiomyocytes (106±13 vs. 233±21 counts/100 high-power field) were observed in PHD1IR compared to wild-type ischemia/reperfusion (WTIR). Protein expression of HIF-1α was significantly increased in PHD1IR compared to WTIR. mRNA expression of β-catenin (1.9-fold), endothelial nitric oxide synthase (1.9-fold), p65 (1.9-fold), and Bcl-2 (2.7-fold) were upregulated in the PHD1IR compared with WTIR, which was studied by real-time quantitative polymerase chain reaction. Further, gel-shift analysis showed increased DNA binding activity of HIF-1α and nuclear factor-kappaB in PHD1IR compared to WTIR. In addition, nuclear translocation of β-catenin was increased in PHD1IR compared with WTIR. These findings indicated that silencing of PHD-1 attenuates myocardial I/R injury probably by enhancing HIF-1α/β-catenin/endothelial nitric oxide synthase/nuclear factor-kappaB and Bcl-2 signaling pathway.

  16. Sac phosphatase domain proteins.

    PubMed Central

    Hughes, W E; Cooke, F T; Parker, P J

    2000-01-01

    Advances in our understanding of the roles of phosphatidylinositol phosphates in controlling cellular functions such as endocytosis, exocytosis and the actin cytoskeleton have included new insights into the phosphatases that are responsible for the interconversion of these lipids. One of these is an entirely novel class of phosphatase domain found in a number of well characterized proteins. Proteins containing this Sac phosphatase domain include the yeast Saccharomyces cerevisiae proteins Sac1p and Fig4p. The Sac phosphatase domain is also found within the mammalian phosphoinositide 5-phosphatase synaptojanin and the yeast synaptojanin homologues Inp51p, Inp52p and Inp53p. These proteins therefore contain both Sac phosphatase and 5-phosphatase domains. This review describes the Sac phosphatase domain-containing proteins and their actions, with particular reference to the genetic and biochemical insights provided by study of the yeast Saccharomyces cerevisiae. PMID:10947947

  17. Design of protein function leaps by directed domain interface evolution

    PubMed Central

    Huang, Jin; Koide, Akiko; Makabe, Koki; Koide, Shohei

    2008-01-01

    Most natural proteins performing sophisticated tasks contain multiple domains where an active site is located at the domain interface. Comparative structural analyses suggest that major leaps in protein function occur through gene recombination events that connect two or more protein domains to generate a new active site, frequently occurring at the newly created domain interface. However, such functional leaps by combination of unrelated domains have not been directly demonstrated. Here we show that highly specific and complex protein functions can be generated by joining a low-affinity peptide-binding domain with a functionally inert second domain and subsequently optimizing the domain interface. These directed evolution processes dramatically enhanced both affinity and specificity to a level unattainable with a single domain, corresponding to >500-fold and >2,000-fold increases of affinity and specificity, respectively. An x-ray crystal structure revealed that the resulting “affinity clamp” had clamshell architecture as designed, with large additional binding surface contributed by the second domain. The affinity clamps having a single-nanomolar dissociation constant outperformed a monoclonal antibody in immunochemical applications. This work establishes evolutionary paths from isolated domains with primitive function to multidomain proteins with sophisticated function and introduces a new protein-engineering concept that allows for the generation of highly functional affinity reagents to a predefined target. The prevalence and variety of natural interaction domains suggest that numerous new functions can be designed by using directed domain interface evolution. PMID:18445649

  18. The Skap-hom dimerization and PH domains comprise a 3'-phosphoinositide-gated molecular switch.

    PubMed

    Swanson, Kenneth D; Tang, Yong; Ceccarelli, Derek F; Poy, Florence; Sliwa, Jan P; Neel, Benjamin G; Eck, Michael J

    2008-11-21

    PH domains, by binding to phosphoinositides, often serve as membrane-targeting modules. Using crystallographic, biochemical, and cell biological approaches, we have uncovered a mechanism that the integrin-signaling adaptor Skap-hom uses to mediate cytoskeletal interactions. Skap-hom is a homodimer containing an N-terminal four-helix bundle dimerization domain, against which its two PH domains pack in a conformation incompatible with phosphoinositide binding. The isolated PH domains bind PI[3,4,5]P(3), and mutations targeting the dimerization domain or the PH domain's PI[3,4,5]P(3)-binding pocket prevent Skap-hom localization to ruffles. Targeting is retained when the PH domain is deleted or by combined mutation of the PI[3,4,5]P(3)-binding pocket and the PH/dimerization domain interface. Thus, the dimerization and PH domain form a PI[3,4,5]P(3)-responsive molecular switch that controls Skap-hom function.

  19. Sputter target

    DOEpatents

    Gates, Willard G.; Hale, Gerald J.

    1980-01-01

    The disclosure relates to an improved sputter target for use in the deposition of hard coatings. An exemplary target is given wherein titanium diboride is brazed to a tantalum backing plate using a gold-palladium-nickel braze alloy.

  20. Targeting Epilepsy

    MedlinePlus

    ... National Center for Chronic Disease Prevention and Health Promotion Division of Population Health point in their lives. ... National Center for Chronic Disease Prevention and Health Promotion (NCCDPHP) works in four key areas or domains: ...

  1. Cross Domain Analogies for Learning Domain Theories

    DTIC Science & Technology

    2007-01-01

    Example Problem and Worked Solution All problems and worked solutions used in this work were taken from the same physics textbook ( Giancoli 1991...domain theory. We close with a discussion of related work and our plans for the future. Representations and Problem Solving Representing physics ...small compared to the 30,000+ concepts and 8,000+ predicates already defined in the KB. Thus, objects, relations, and events that appear in physics

  2. Compositional Dictionaries for Domain Adaptive Face Recognition.

    PubMed

    Qiang Qiu; Chellappa, Rama

    2015-12-01

    We present a dictionary learning approach to compensate for the transformation of faces due to the changes in view point, illumination, resolution, and so on. The key idea of our approach is to force domain-invariant sparse coding, i.e., designing a consistent sparse representation of the same face in different domains. In this way, the classifiers trained on the sparse codes in the source domain consisting of frontal faces can be applied to the target domain (consisting of faces in different poses, illumination conditions, and so on) without much loss in recognition accuracy. The approach is to first learn a domain base dictionary, and then describe each domain shift (identity, pose, and illumination) using a sparse representation over the base dictionary. The dictionary adapted to each domain is expressed as the sparse linear combinations of the base dictionary. In the context of face recognition, with the proposed compositional dictionary approach, a face image can be decomposed into sparse representations for a given subject, pose, and illumination. This approach has three advantages. First, the extracted sparse representation for a subject is consistent across domains, and enables pose and illumination insensitive face recognition. Second, sparse representations for pose and illumination can be subsequently used to estimate the pose and illumination condition of a face image. Last, by composing sparse representations for the subject and the different domains, we can also perform pose alignment and illumination normalization. Extensive experiments using two public face data sets are presented to demonstrate the effectiveness of the proposed approach for face recognition.

  3. The Promise of Domain Adaptation

    NASA Astrophysics Data System (ADS)

    Mahabal, Ashish A.; Li, Jingling; Vaijanapurkar, Samarth; Bue, Brian; Miller, Adam; Donalek, Ciro; Djorgovski, Stanislav G.; Drake, Andrew J.; Graham, Matthew; CRTS, iPTF

    2016-01-01

    Most new surveys spend an appreciable time in collecting data on which to train classifiers before they can be used on future observations from the same dataset. The result generating phase can start much earlier if the training could incorporate data accumulated from older surveys enhanced with a small set from the new survey. This is exactly what Domain Adaptation (DA) allows us to do. The main idea behind DAs can be summarized thus: if we have two classes of separable objects in some feature space of a Source survey (S), we can define a hyperplane to separate the two types. In a second Target survey (T), for the same features the hyperplane would be inclined differently. DA methods get the mapping between the two hyperplanes using a small fraction of data from the Target (T) survey and can then be used to predict the classes of the remaining majority of data in T. We discuss the parameters that need to be tuned, the difficulties involved, and ways to improve the results. As we move towards bigger, and deeper surveys, being able to use existing labelled information to conduct classification in future surveys will be more cost-effective and promote time efficiency as well. Starting with the light curve data of 50,000 periodic objects from Catalina Real-Time Transient Survey (CRTS), we have applied domain adaptation techniques such as Geodesic Flow Kernel (GFK) with Random forest classifier and Co-training for domain adaptation (CODA) to the CRTS data which has 35,000 points overlapping with Palomar Transient Factory (PTF), and 12,000 with Lincoln Near-Earth Asteroid Research (LINEAR). The results suggest that domain adaptation is an area worth exploring as the knowledge between these surveys is transferable and the approaches to find the mappings between these surveys can be applied to the remaining data as well as for near future surveys such as CRTS-II, Zwicky Transient Facility (ZTF) and the Large Synoptic Survey Telescope (LSST) to name a few at the optical

  4. Visualizing Knowledge Domains.

    ERIC Educational Resources Information Center

    Borner, Katy; Chen, Chaomei; Boyack, Kevin W.

    2003-01-01

    Reviews visualization techniques for scientific disciplines and information retrieval and classification. Highlights include historical background of scientometrics, bibliometrics, and citation analysis; map generation; process flow of visualizing knowledge domains; measures and similarity calculations; vector space model; factor analysis;…

  5. Software architecture design domain

    SciTech Connect

    White, S.A.

    1996-12-31

    Software architectures can provide a basis for the capture and subsequent reuse of design knowledge. The goal of software architecture is to allow the design of a system to take place at a higher level of abstraction; a level concerned with components, connections, constraints, rationale. This architectural view of software adds a new layer of abstraction to the traditional design phase of software development. It has resulted in a flurry of activity towards techniques, tools, and architectural design languages developed specifically to assist with this activity. An analysis of architectural descriptions, even though they differ in notation, shows a common set of key constructs that are present across widely varying domains. These common aspects form a core set of constructs that should belong to any ADL in order to for the language to offer the ability to specify software systems at the architectural level. This analysis also revealed a second set of constructs which served to expand the first set thereby improving the syntax and semantics. These constructs are classified according to whether they provide representation and analysis support for architectures belonging to many varying application domains (domain-independent construct class) or to a particular application domain (domain-dependent constructs). This paper presents the constructs of these two classes, their placement in the architecture design domain and shows how they may be used to classify, select, and analyze proclaimed architectural design languages (ADLs).

  6. Structured hints : extracting and abstracting domain expertise.

    SciTech Connect

    Hereld, M.; Stevens, R.; Sterling, T.; Gao, G. R.; Mathematics and Computer Science; California Inst. of Tech.; Louisiana State Univ.; Univ. of Delaware

    2009-03-16

    We propose a new framework for providing information to help optimize domain-specific application codes. Its design addresses problems that derive from the widening gap between the domain problem statement by domain experts and the architectural details of new and future high-end computing systems. The design is particularly well suited to program execution models that incorporate dynamic adaptive methodologies for live tuning of program performance and resource utilization. This new framework, which we call 'structured hints', couples a vocabulary of annotations to a suite of performance metrics. The immediate target is development of a process by which a domain expert describes characteristics of objects and methods in the application code that would not be readily apparent to the compiler; the domain expert provides further information about what quantities might provide the best indications of desirable effect; and the interactive preprocessor identifies potential opportunities for the domain expert to evaluate. Our development of these ideas is progressing in stages from case study, through manual implementation, to automatic or semi-automatic implementation. In this paper we discuss results from our case study, an examination of a large simulation of a neural network modeled after the neocortex.

  7. Joining RDC data from flexible protein domains

    NASA Astrophysics Data System (ADS)

    Sgheri, Luca

    2010-11-01

    We study the inverse problem of determining the conformational freedom of two protein domains from residual dipolar coupling (RDC) measurements. For each paramagnetic ion attached to one of the domains we obtain a magnetic susceptibility tensor χ from the RDC of couples of atoms of that domain, and a mean paramagnetic susceptibility tensor {\\bar{\\chi }} from the RDC of couples of atoms of the other domain. The latter is an integral average of rotations of χ which depends on the conformational freedom of the two domains. In this paper we consider the case when we have data from paramagnetic ions attached separately to each of the domains. We prove that in this case not all the elements of χ and {\\bar{\\chi }} are independent. We derive the mathematical equations for the compatibility of the measurements and show how these relations can be used in the presence of noisy data to determine a compatible set of χ and {\\bar{\\chi }} with an unconstrained minimization. If available, information about the shape of the noise can be included in the target function. We show that in this case the compatible set obtained has a reduced error with respect to the noisy data.

  8. Domains in Ferroelectric Nanostructures

    NASA Astrophysics Data System (ADS)

    Gregg, Marty

    2010-03-01

    Ferroelectric materials have great potential in influencing the future of small scale electronics. At a basic level, this is because ferroelectric surfaces are charged, and so interact strongly with charge-carrying metals and semiconductors - the building blocks for all electronic systems. Since the electrical polarity of the ferroelectric can be reversed, surfaces can both attract and repel charges in nearby materials, and can thereby exert complete control over both charge distribution and movement. It should be no surprise, therefore, that microelectronics industries have already looked very seriously at harnessing ferroelectric materials in a variety of applications, from solid state memory chips (FeRAMs) to field effect transistors (FeFETs). In all such applications, switching the direction of the polarity of the ferroelectric is a key aspect of functional behavior. The mechanism for switching involves the field-induced nucleation and growth of domains. Domain coarsening, through domain wall propagation, eventually causes the entire ferroelectric to switch its polar direction. It is thus the existence and behavior of domains that determine the switching response, and ultimately the performance of the ferroelectric device. A major issue, associated with the integration of ferroelectrics into microelectronic devices, has been that the fundamental properties associated with ferroelectrics, when in bulk form, appear to change quite dramatically and unpredictably when at the nanoscale: new modes of behaviour, and different functional characteristics from those seen in bulk appear. For domains, in particular, the proximity of surfaces and boundaries have a dramatic effect: surface tension and depolarizing fields both serve to increase the equilibrium density of domains, such that minor changes in scale or morphology can have major ramifications for domain redistribution. Given the importance of domains in dictating the overall switching characteristics of a device

  9. Multi-domain training enhances attentional control.

    PubMed

    Binder, Julia C; Martin, Mike; Zöllig, Jacqueline; Röcke, Christina; Mérillat, Susan; Eschen, Anne; Jäncke, Lutz; Shing, Yee Lee

    2016-06-01

    Multi-domain training potentially increases the likelihood of overlap in processing components with transfer tasks and everyday life, and hence is a promising training approach for older adults. To empirically test this, 84 healthy older adults aged 64 to 75 years were randomly assigned to one of three single-domain training conditions (inhibition, visuomotor function, spatial navigation) or to the simultaneous training of all three cognitive functions (multi-domain training condition). All participants trained on an iPad at home for 50 training sessions. Before and after the training, and at a 6-month follow-up measurement, cognitive functioning and training transfer were assessed with a neuropsychological test battery including tests targeting the trained functions (near transfer) and transfer to executive functions (far transfer: attentional control, working memory, speed). Participants in all four training groups showed a linear increase in training performance over the 50 training sessions. Using a latent difference score model, the multi-domain training group, compared with the single-domain training groups, showed more improvement on the far transfer attentional control composite. Individuals with initially lower baseline performance showed higher training-related improvements, indicating that training compensated for lower initial cognitive performance. At the 6-month follow-up, performance on the cognitive test battery remained stable. This is one of the first studies to investigate systematically multi-domain training including comparable single-domain training conditions. Our findings suggest that multi-domain training enhances attentional control involved in handling several different tasks at the same time, an aspect in everyday life that is particularly challenging for older people. (PsycINFO Database Record

  10. Iterative Re-Weighted Instance Transfer for Domain Adaptation

    NASA Astrophysics Data System (ADS)

    Paul, A.; Rottensteiner, F.; Heipke, C.

    2016-06-01

    Domain adaptation techniques in transfer learning try to reduce the amount of training data required for classification by adapting a classifier trained on samples from a source domain to a new data set (target domain) where the features may have different distributions. In this paper, we propose a new technique for domain adaptation based on logistic regression. Starting with a classifier trained on training data from the source domain, we iteratively include target domain samples for which class labels have been obtained from the current state of the classifier, while at the same time removing source domain samples. In each iteration the classifier is re-trained, so that the decision boundaries are slowly transferred to the distribution of the target features. To make the transfer procedure more robust we introduce weights as a function of distance from the decision boundary and a new way of regularisation. Our methodology is evaluated using a benchmark data set consisting of aerial images and digital surface models. The experimental results show that in the majority of cases our domain adaptation approach can lead to an improvement of the classification accuracy without additional training data, but also indicate remaining problems if the difference in the feature distributions becomes too large.

  11. LIQUID TARGET

    DOEpatents

    Martin, M.D.; Salsig, W.W. Jr.

    1959-01-13

    A liquid handling apparatus is presented for a liquid material which is to be irradiated. The apparatus consists essentially of a reservoir for the liquid, a target element, a drain tank and a drain lock chamber. The target is in the form of a looped tube, the upper end of which is adapted to be disposed in a beam of atomic particles. The lower end of the target tube is in communication with the liquid in the reservoir and a means is provided to continuously circulate the liquid material to be irradiated through the target tube. Means to heat the reservoir tank is provided in the event that a metal is to be used as the target material. The apparatus is provided with suitable valves and shielding to provide maximum safety in operation.

  12. 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.

  13. Extensions of PDZ domains as important structural and functional elements.

    PubMed

    Wang, Conan K; Pan, Lifeng; Chen, Jia; Zhang, Mingjie

    2010-08-01

    'Divide and conquer' has been the guiding strategy for the study of protein structure and function. Proteins are divided into domains with each domain having a canonical structural definition depending on its type. In this review, we push forward with the interesting observation that many domains have regions outside of their canonical definition that affect their structure and function; we call these regions 'extensions'. We focus on the highly abundant PDZ (PSD-95, DLG1 and ZO-1) domain. Using bioinformatics, we find that many PDZ domains have potential extensions and we developed an openly-accessible website to display our results ( http://bcz102.ust.hk/pdzex/ ). We propose, using well-studied PDZ domains as illustrative examples, that the roles of PDZ extensions can be classified into at least four categories: 1) protein dynamics-based modulation of target binding affinity, 2) provision of binding sites for macro-molecular assembly, 3) structural integration of multi-domain modules, and 4) expansion of the target ligand-binding pocket. Our review highlights the potential structural and functional importance of domain extensions, highlighting the significance of looking beyond the canonical boundaries of protein domains in general.

  14. Axion domain wall baryogenesis

    SciTech Connect

    Daido, Ryuji; Kitajima, Naoya; Takahashi, Fuminobu

    2015-07-28

    We propose a new scenario of baryogenesis, in which annihilation of axion domain walls generates a sizable baryon asymmetry. Successful baryogenesis is possible for a wide range of the axion mass and decay constant, m≃10{sup 8}–10{sup 13} GeV and f≃10{sup 13}–10{sup 16} GeV. Baryonic isocurvature perturbations are significantly suppressed in our model, in contrast to various spontaneous baryogenesis scenarios in the slow-roll regime. In particular, the axion domain wall baryogenesis is consistent with high-scale inflation which generates a large tensor-to-scalar ratio within the reach of future CMB B-mode experiments. We also discuss the gravitational waves produced by the domain wall annihilation and its implications for the future gravitational wave experiments.

  15. Optimal domain decomposition strategies

    NASA Technical Reports Server (NTRS)

    Yoon, Yonghyun; Soni, Bharat K.

    1995-01-01

    The primary interest of the authors is in the area of grid generation, in particular, optimal domain decomposition about realistic configurations. A grid generation procedure with optimal blocking strategies has been developed to generate multi-block grids for a circular-to-rectangular transition duct. The focus of this study is the domain decomposition which optimizes solution algorithm/block compatibility based on geometrical complexities as well as the physical characteristics of flow field. The progress realized in this study is summarized in this paper.

  16. Membrane targeting: what a difference a G makes.

    PubMed

    Cullen, P J; Chardin, P

    2000-11-30

    Pleckstrin homology domains are modular domains that direct membrane targeting of their host proteins by binding to polyphosphoinositides; recent results have increased our appreciation of how some of these domains actually bind 3-phosphoinositides, and along the way thrown up some unexpected observations.

  17. Domain adaptation for Alzheimer's disease diagnostics.

    PubMed

    Wachinger, Christian; Reuter, Martin

    2016-10-01

    With the increasing prevalence of Alzheimer's disease, research focuses on the early computer-aided diagnosis of dementia with the goal to understand the disease process, determine risk and preserving factors, and explore preventive therapies. By now, large amounts of data from multi-site studies have been made available for developing, training, and evaluating automated classifiers. Yet, their translation to the clinic remains challenging, in part due to their limited generalizability across different datasets. In this work, we describe a compact classification approach that mitigates overfitting by regularizing the multinomial regression with the mixed ℓ1/ℓ2 norm. We combine volume, thickness, and anatomical shape features from MRI scans to characterize neuroanatomy for the three-class classification of Alzheimer's disease, mild cognitive impairment and healthy controls. We demonstrate high classification accuracy via independent evaluation within the scope of the CADDementia challenge. We, furthermore, demonstrate that variations between source and target datasets can substantially influence classification accuracy. The main contribution of this work addresses this problem by proposing an approach for supervised domain adaptation based on instance weighting. Integration of this method into our classifier allows us to assess different strategies for domain adaptation. Our results demonstrate (i) that training on only the target training set yields better results than the naïve combination (union) of source and target training sets, and (ii) that domain adaptation with instance weighting yields the best classification results, especially if only a small training component of the target dataset is available. These insights imply that successful deployment of systems for computer-aided diagnostics to the clinic depends not only on accurate classifiers that avoid overfitting, but also on a dedicated domain adaptation strategy.

  18. Discoidin Domain Receptors in Disease

    PubMed Central

    Borza, Corina M; Pozzi, Ambra

    2014-01-01

    Discoidin domain receptors, DDR1 and DDR2, lie at the intersection of two large receptor families, namely the extracellular matrix and tyrosine kinase receptors. As such, DDRs are uniquely positioned to function as sensors for extracellular matrix and to regulate a wide range of cell functions from migration and proliferation to cytokine secretion and extracellular matrix homeostasis/remodeling. While activation of DDRs by extracellular matrix collagens is required for normal development and tissue homeostasis, aberrant activation of these receptors following injury or in disease is detrimental. The availability of mice lacking DDRs has enabled us to identify key roles played by these receptors in disease initiation and progression. DDR1 promotes inflammation in atherosclerosis, lung fibrosis and kidney injury, while DDR2 contributes to osteoarthritis. Furthermore, both DDRs have been implicated in cancer progression. Yet the mechanisms whereby DDRs contribute to diseases progression are poorly understood. In this review we highlight the mechanisms whereby DDRs regulate two important processes, namely inflammation and tissue fibrosis. In addition, we discuss the challenges of targeting DDRs in disease. Selective targeting of these receptors requires understanding of how they interact with and are activated by extracellular matrix, and whether their cellular function is dependent on or independent of receptor kinase activity. PMID:24361528

  19. Domain adaptation based on deep denoising auto-encoders for classification of remote sensing images

    NASA Astrophysics Data System (ADS)

    Riz, Emanuele; Demir, Begüm; Bruzzone, Lorenzo

    2016-10-01

    This paper investigates the effectiveness of deep learning (DL) for domain adaptation (DA) problems in the classification of remote sensing images to generate land-cover maps. To this end, we introduce two different DL architectures: 1) single-stage domain adaptation (SS-DA) architecture; and 2) hierarchal domain adaptation (H-DA) architecture. Both architectures require that a reliable training set is available only for one of the images (i.e., the source domain) from a previous analysis, whereas it is not for another image to be classified (i.e., the target domain). To classify the target domain image, the proposed architectures aim to learn a shared feature representation that is invariant across the source and target domains in a completely unsupervised fashion. To this end, both architectures are defined based on the stacked denoising auto-encoders (SDAEs) due to their high capability to define high-level feature representations. The SS-DA architecture leads to a common feature space by: 1) initially unifying the samples in source and target domains; and 2) then feeding them simultaneously into the SDAE. To further increase the robustness of the shared representations, the H-DA employs: 1) two SDAEs for learning independently the high level representations of source and target domains; and 2) a consensus SDAE to learn the domain invariant high-level features. After obtaining the domain invariant features through proposed architectures, the classifier is trained by the domain invariant labeled samples of the source domain, and then the domain invariant samples of the target domain are classified to generate the related classification map. Experimental results obtained for the classification of very high resolution images confirm the effectiveness of the proposed DL architectures.

  20. Cellulose binding domain proteins

    SciTech Connect

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  1. The Domains of TESOL.

    ERIC Educational Resources Information Center

    Robinett, Betty Wallace

    The domains of Teachers of English to Speakers of Other Languages (TESOL) are those spheres of concern involving persons who speak languages other than English or dialects of English other than the standard. This clientele has been classified traditionally in terms of programs in English as a foreign language, English as a second language, English…

  2. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

    1998-11-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  3. Domain Validity and Generalizability

    ERIC Educational Resources Information Center

    Kaiser, Henry F.; Michael, William B.

    1975-01-01

    An alternative derivation of Tryon's basic formula for the coefficient of domain validity or the coefficient of generalizability developed by Cronbach, Rajaratnam, and Glaser is provided. This derivation, which is also the generalized Kuder-Richardson coefficient, requires a relatively minimal number of assumptions compared with that in previously…

  4. Saccharomyces SRP RNA secondary structures: a conserved S-domain and extended Alu-domain.

    PubMed

    Van Nues, Rob W; Brown, Jeremy D

    2004-01-01

    The contribution made by the RNA component of signal recognition particle (SRP) to its function in protein targeting is poorly understood. We have generated a complete secondary structure for Saccharomyces cerevisiae SRP RNA, scR1. The structure conforms to that of other eukaryotic SRP RNAs. It is rod-shaped with, at opposite ends, binding sites for proteins required for the SRP functions of signal sequence recognition (S-domain) and translational elongation arrest (Alu-domain). Micrococcal nuclease digestion of purified S. cerevisiae SRP separated the S-domain of the RNA from the Alu-domain as a discrete fragment. The Alu-domain resolved into several stable fragments indicating a compact structure. Comparison of scR1 with SRP RNAs of five yeast species related to S. cerevisiae revealed the S-domain to be the most conserved region of the RNA. Extending data from nuclease digestion with phylogenetic comparison, we built the secondary structure model for scR1. The Alu-domain contains large extensions, including a sequence with hallmarks of an expansion segment. Evolutionarily conserved bases are placed in the Alu- and S-domains as in other SRP RNAs, the exception being an unusual GU(4)A loop closing the helix onto which the signal sequence binding Srp54p assembles (domain IV). Surprisingly, several mutations within the predicted Srp54p binding site failed to disrupt SRP function in vivo. However, the strength of the Srp54p-scR1 and, to a lesser extent, Sec65p-scR1 interaction was decreased in these mutant particles. The availability of a secondary structure for scR1 will facilitate interpretation of data from genetic analysis of the RNA.

  5. Recombinant spider silk genetically functionalized with affinity domains.

    PubMed

    Jansson, Ronnie; Thatikonda, Naresh; Lindberg, Diana; Rising, Anna; Johansson, Jan; Nygren, Per-Åke; Hedhammar, My

    2014-05-12

    Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

  6. Magnetic domain and domain wall in Co/Pt multilayer

    NASA Astrophysics Data System (ADS)

    Talapatra, A.; Mohanty, J.

    2016-05-01

    We report systematic micromagnetic investigation of formation of magnetic domains in perpendicularly magnetized Co/Pt multilayer with the variation in magnetic anisotropy and stack thickness. The lowering of anisotropy makes the domain wall broader and domain formation less efficient. Domain sizeincreases with increasing thickness of the stack to minimize the stray field energy.The minimization of energy of the system due to domain formation makes the M-H loop narrower whereas, lower stack thickness results in a wider loop. The magnetization reversalin this system occurs due tothe nucleation and growth of reverse domains.

  7. Tackling Targets.

    ERIC Educational Resources Information Center

    Further Education Unit, London (England).

    This document is designed to help British training and enterprise councils (TECs) and further education (FE) colleges develop and implement strategies for achieving the National Targets for Education and Training (NTET), which were developed by the Confederation of British Industry in 1992 and endorsed by the British government. The findings from…

  8. On Target.

    ERIC Educational Resources Information Center

    Barbalich, Andrea

    1991-01-01

    Campus public relations professionals offer advice for improving the effectiveness of public relations efforts by (1) setting behavioral goals; (2) targeting audiences carefully; (3) focusing appeals by making messages explicit; (4) connecting the public relations message with larger societal issues; and (5) reaching internal as well as external…

  9. WW domain-containing proteins: retrospectives and the future.

    PubMed

    Salah, Zaidoun; Alian, Akram; Aqeilan, Rami I

    2012-01-01

    WW domains are protein modules that mediate protein-protein interactions through recognition of proline-rich peptide motifs (PRM) and phosphorylated serine/threonine-proline sites. WW domains are found in many different structural and signaling proteins that are involved in a variety of cellular processes, including RNA transcription and processing, protein trafficking and stability, receptor signaling, and control of the cytoskeleton. WW domain-containing proteins and complexes have been implicated in major human diseases including cancer as well as in major signaling cascades such as the Hippo tumor suppressor pathway, making them targets for new diagnostics and therapeutics. In this review, we discuss how WW domains provide versatile platforms that link individual proteins into physiologically important networks and the indispensible role of WW domain-containing proteins in biology and pathology, especially tumorogenesis.

  10. Frequency domain nonlinear optics

    NASA Astrophysics Data System (ADS)

    Legare, Francois

    2016-05-01

    The universal dilemma of gain narrowing occurring in fs amplifiers prevents ultra-high power lasers from delivering few-cycle pulses. This problem is overcome by a new amplification concept: Frequency domain Optical Parametric Amplification - FOPA. It enables simultaneous up-scaling of peak power and amplified spectral bandwidth and can be performed at any wavelength range of conventional amplification schemes, however, with the capability to amplify single cycles of light. The key idea for amplification of octave-spanning spectra without loss of spectral bandwidth is to amplify the broad spectrum ``slice by slice'' in the frequency domain, i.e. in the Fourier plane of a 4f-setup. The striking advantages of this scheme, are its capability to amplify (more than) one octave of bandwidth without shorting the corresponding pulse duration. This is because ultrabroadband phase matching is not defined by the properties of the nonlinear crystal employed but the number of crystals employed. In the same manner, to increase the output energy one simply has to increase the spectral extension in the Fourier plane and to add one more crystal. Thus, increasing pulse energy and shortening its duration accompany each other. A proof of principle experiment was carried out at ALLS on the sub-two cycle IR beam line and yielded record breaking performance in the field of few-cycle IR lasers. 100 μJ two-cycle pulses from a hollow core fibre compression setup were amplified to 1.43mJ without distorting spatial or temporal properties. Pulse duration at the input of FOPA and after FOPA remains the same. Recently, we have started upgrading this system to be pumped by 250 mJ to reach 40 mJ two-cycle IR few-cycle pulses and latest results will be presented at the conference. Furthermore, the extension of the concept of FOPA to other nonlinear optical processes will be discussed. Frequency domain nonlinear optics.

  11. Target assembly

    DOEpatents

    Lewis, Richard A.

    1980-01-01

    A target for a proton beam which is capable of generating neutrons for absorption in a breeding blanket includes a plurality of solid pins formed of a neutron emissive target material disposed parallel to the path of the beam and which are arranged axially in a plurality of layers so that pins in each layer are offset with respect to pins in all other layers, enough layers being used so that each proton in the beam will strike at least one pin with means being provided to cool the pins. For a 300 mA, 1 GeV beam (300 MW), stainless steel pins, 12 inches long and 0.23 inches in diameter are arranged in triangular array in six layers with one sixth of the pins in each layer, the number of pins being such that the entire cross sectional area of the beam is covered by the pins with minimum overlap of pins.

  12. Field Evolution of Antiferromagnetic Domains and Domain Walls

    NASA Astrophysics Data System (ADS)

    Fullerton, Eric E.; Hellwig, Olav; Berger, Andreas K.

    2003-03-01

    We have used magnetron sputtered [Co(4Å)Pt(7Å)]X Co(4Å)Ru(9Å)N multiplayer films to create artificially layered antiferromagnets. In contrast to atomic antiferromagnets our model system has an antiferromagnetic (AF) exchange energy comparable to the Zeemann energy in moderate fields and allows to fine tune the relative magnitude of the different magnetic energy terms by varying the parameters X and N. With increasing X and N we observe a transition from traditionally observed sharp AF domain walls towards AF domain walls with a finite width which consist of ferromagnetic stripes, i.e. the AF domains have zero net moment whereas the domain walls carry a finite magnetic moment. Such AF domain walls have not been observed before and are a direct consequence of balancing out exchange and Zeeman energy. We also show that such domain walls are expected from theoretical energy calculations. In this contribution we study the nature and field evolution of the AF stripe domain walls by Magnetic Force Microscopy (MFM). The surface sensitivity of MFM and the finite moment of the AF domain walls allow us to image AF domains as well as domain walls. We are showing first experiments to study the AF domain wall evolution in real space while applying an external field. O.H. was supported by the Deutsche Forschungsgemeinschaft via a Forschungsstipendium under the contract number HE 3286/1-1.

  13. Space Domain Awareness

    DTIC Science & Technology

    2012-09-01

    information required to characterize a space object. Another key parameter to be considered is the frequency of observation. This sampling rate varies...useful to define the values of these parameters that approximate the current and future state of the space domain. The current catalog and network... Parameters used in estimating data needs for SDA Current Threshold Objective βmo , βimg 0.1, 10Kb 0.1, 10Kb 0.1, 10Kb Number of Objects (Na , Np

  14. Social cognitive conflict resolution: contributions of domain-general and domain-specific neural systems.

    PubMed

    Zaki, Jamil; Hennigan, Kelly; Weber, Jochen; Ochsner, Kevin N

    2010-06-23

    Cognitive control mechanisms allow individuals to behave adaptively in the face of complex and sometimes conflicting information. Although the neural bases of these control mechanisms have been examined in many contexts, almost no attention has been paid to their role in resolving conflicts between competing social cues, which is surprising given that cognitive conflicts are part of many social interactions. Evidence about the neural processing of social information suggests that two systems--the mirror neuron system (MNS) and mental state attribution system (MSAS)--are specialized for processing nonverbal and contextual social cues, respectively. This could support a model of social cognitive conflict resolution in which competition between social cues would recruit domain-general cognitive control mechanisms, which in turn would bias processing toward the MNS or MSAS. Such biasing could also alter social behaviors, such as inferences made about the internal states of others. We tested this model by scanning participants using functional magnetic resonance imaging while they drew inferences about the social targets' emotional states based on congruent or incongruent nonverbal and contextual social cues. Conflicts between social cues recruited the anterior cingulate and lateral prefrontal cortex, brain areas associated with domain-general control processes. This activation was accompanied by biasing of neural activity toward areas in the MNS or MSAS, which tracked, respectively, with perceivers' behavioral reliance on nonverbal or contextual cues when drawing inferences about targets' emotions. Together, these data provide evidence about both domain-general and domain-specific mechanisms involved in resolving social cognitive conflicts.

  15. Domain adaptation problems: a DASVM classification technique and a circular validation strategy.

    PubMed

    Bruzzone, Lorenzo; Marconcini, Mattia

    2010-05-01

    This paper addresses pattern classification in the framework of domain adaptation by considering methods that solve problems in which training data are assumed to be available only for a source domain different (even if related) from the target domain of (unlabeled) test data. Two main novel contributions are proposed: 1) a domain adaptation support vector machine (DASVM) technique which extends the formulation of support vector machines (SVMs) to the domain adaptation framework and 2) a circular indirect accuracy assessment strategy for validating the learning of domain adaptation classifiers when no true labels for the target--domain instances are available. Experimental results, obtained on a series of two-dimensional toy problems and on two real data sets related to brain computer interface and remote sensing applications, confirmed the effectiveness and the reliability of both the DASVM technique and the proposed circular validation strategy.

  16. Regulation of Adhesion and Migration by the Rsu1- and PINCH1-mediated Inhibition of Focal Adhesion Formation and Actin Polymerization

    DTIC Science & Technology

    2012-06-08

    terminal ankyrin (ANK) repeats , a plekstrin homology (PH) domain and a C-terminal pseudokinase domain. The ANK1 domain binds to the LIM1 domain of...Triphosphate ANK: Ankyrin ILK: Integrin Linked Kinase ATP...Complementary DNA Limk1: Lim kinase1 cGMP: Cyclic Guanosine Monophosphate LRR: Leucine Reach Repeat

  17. Accelerator target

    DOEpatents

    Schlyer, D.J.; Ferrieri, R.A.; Koehler, C.

    1999-06-29

    A target includes a body having a depression in a front side for holding a sample for irradiation by a particle beam to produce a radioisotope. Cooling fins are disposed on a backside of the body opposite the depression. A foil is joined to the body front side to cover the depression and sample therein. A perforate grid is joined to the body atop the foil for supporting the foil and for transmitting the particle beam therethrough. A coolant is circulated over the fins to cool the body during the particle beam irradiation of the sample in the depression. 5 figs.

  18. Accelerator target

    DOEpatents

    Schlyer, David J.; Ferrieri, Richard A.; Koehler, Conrad

    1999-01-01

    A target includes a body having a depression in a front side for holding a sample for irradiation by a particle beam to produce a radioisotope. Cooling fins are disposed on a backside of the body opposite the depression. A foil is joined to the body front side to cover the depression and sample therein. A perforate grid is joined to the body atop the foil for supporting the foil and for transmitting the particle beam therethrough. A coolant is circulated over the fins to cool the body during the particle beam irradiation of the sample in the depression.

  19. Targeting circuits

    PubMed Central

    Rajasethupathy, Priyamvada; Ferenczi, Emily; Deisseroth, Karl

    2017-01-01

    Current optogenetic methodology enables precise inhibition or excitation of neural circuits, spanning timescales as needed from the acute (milliseconds) to the chronic (many days or more), for experimental modulation of network activity and animal behavior. Such broad temporal versatility, unique to optogenetic control, is particularly powerful when combined with brain activity measurements that span both acute and chronic timescales as well. This enables, for instance, the study of adaptive circuit dynamics across the intact brain, and tuning interventions to match activity patterns naturally observed during behavior in the same individual. Although the impact of this approach has been greater on basic research than on clinical translation, it is natural to ask if specific neural circuit activity patterns discovered to be involved in controlling adaptive or maladaptive behaviors could become targets for treatment of neuropsychiatric diseases. Here we consider the landscape of such ideas related to therapeutic targeting of circuit dynamics, taking note of developments not only in optical but also in ultrasonic, magnetic, and thermal methods. We note the recent emergence of first-in-kind optogenetically-guided clinical outcomes, as well as opportunities related to the integration of interventions and readouts spanning diverse circuit-physiology, molecular, and behavioral modalities. PMID:27104976

  20. Swarming in bounded domains

    NASA Astrophysics Data System (ADS)

    Armbruster, Dieter; Motsch, Sébastien; Thatcher, Andrea

    2017-04-01

    The Vicsek model is a prototype for the emergence of collective motion. In free space, it is characterized by a swarm of particles all moving in the same direction. Since this dynamic does not include attraction among particles, the swarm, while aligning in velocity space, has no spatial coherence. Adding specular reflection at the boundaries generates global spatial coherence of the swarms while maintaining its velocity alignment. We investigate numerically how the geometry of the domain influences the Vicsek model using three type of geometry: a channel, a disk and a rectangle. Varying the parameters of the Vicsek model (e.g. noise levels and influence horizons), we discuss the mechanisms that generate spatial coherence and show how they create new dynamical solutions of the swarming motions in these geometries. Several observables are introduced to characterize the simulated patterns (e.g. mass profile, center of mass, connectivity of the swarm).

  1. Beyond the Number Domain

    PubMed Central

    Cantlon, Jessica F.; Platt, Michael L.; Brannon, Elizabeth M.

    2009-01-01

    In a world without numbers, we would be unable to build a skyscraper, hold a national election, plan a wedding, or pay for a chicken at the market. The numerical symbols used in all these behaviors build on the approximate number system (ANS) which represents the number of discrete objects or events as a continuous mental magnitude. In this review, we first discuss evidence that the ANS bears a set of behavioral and brain signatures that are universally displayed across animal species, human cultures, and development. We then turn to the question of whether the ANS constitutes a specialized cognitive and neural domain--a question central to understanding how this system works, the nature of its evolutionary and developmental trajectory, and its physical instantiation in the brain. PMID:19131268

  2. Receptor binding domain based HIV vaccines.

    PubMed

    Liu, Huan; Bi, Wenwen; Wang, Qian; Lu, Lu; Jiang, Shibo

    2015-01-01

    This paper analyzes the main trend of the development of acquired immunodeficiency syndrome (AIDS) vaccines in recent years. Designing an HIV-1 vaccine that provides robust protection from HIV-1 infection remains a challenge despite many years of effort. Therefore, we describe the receptor binding domain of gp120 as a target for developing AIDS vaccines. And we recommend some measures that could induce efficiently and produce cross-reactive neutralizing antibodies with high binding affinity. Those measures may offer a new way of the research and development of the potent and broad AIDS vaccines.

  3. Multifunctionalities driven by ferroic domains

    SciTech Connect

    Yang, J. C.; Huang, Y. L.; Chu, Y. H.; He, Q.

    2014-08-14

    Considerable attention has been paid to ferroic systems in pursuit of advanced applications in past decades. Most recently, the emergence and development of multiferroics, which exhibit the coexistence of different ferroic natures, has offered a new route to create functionalities in the system. In this manuscript, we step from domain engineering to explore a roadmap for discovering intriguing phenomena and multifunctionalities driven by periodic domain patters. As-grown periodic domains, offering exotic order parameters, periodic local perturbations and the capability of tailoring local spin, charge, orbital and lattice degrees of freedom, are introduced as modeling templates for fundamental studies and novel applications. We discuss related significant findings on ferroic domain, nanoscopic domain walls, and conjunct heterostructures based on the well-organized domain patterns, and end with future prospects and challenges in the field.

  4. Solution structure of Q388A3 PDZ domain from Trypanosoma brucei.

    PubMed

    Mei, Song; Dong, Yuanqiu; Zhang, Jiahai; Zhang, Xuecheng; Tu, Xiaoming

    2016-05-01

    PDZ domains are abundant protein interaction modules that often recognize short amino acid motifs at the C-termini of target proteins and regulate multiple biological processes. So far, no PDZ domain in Trypanosoma brucei, an eukaryotic parasite causing sleeping sickness, has been studied. Q388A3, conserved in the related kinetoplastid parasites, is a 1634-residue protein containing a PDZ domain at its C-terminus. In this work, the solution structure of Q388A3 PDZ domain was solved by NMR spectroscopy. Q388A3 PDZ domain adopts a PDZ-like fold composed by a five-stranded β-sheet capped by two α-helices, which is similar to the PDZ domains from HtrA family proteins. Meanwhile, Q388A3 PDZ domain shows some structural features quite different from HtrA PDZ domain.

  5. Mapping the Moral Domain

    PubMed Central

    Graham, Jesse; Nosek, Brian A.; Haidt, Jonathan; Iyer, Ravi; Koleva, Spassena; Ditto, Peter H.

    2010-01-01

    The moral domain is broader than the empathy and justice concerns assessed by existing measures of moral competence, and it is not just a subset of the values assessed by value inventories. To fill the need for reliable and theoretically-grounded measurement of the full range of moral concerns, we developed the Moral Foundations Questionnaire (MFQ) based on a theoretical model of five universally available (but variably developed) sets of moral intuitions: Harm/care, Fairness/reciprocity, Ingroup/loyalty, Authority/respect, and Purity/sanctity. We present evidence for the internal and external validity of the scale and the model, and in doing so present new findings about morality: 1. Comparative model fitting of confirmatory factor analyses provides empirical justification for a five-factor structure of moral concerns. 2. Convergent/discriminant validity evidence suggests that moral concerns predict personality features and social group attitudes not previously considered morally relevant. 3. We establish pragmatic validity of the measure in providing new knowledge and research opportunities concerning demographic and cultural differences in moral intuitions. These analyses provide evidence for the usefulness of Moral Foundations Theory in simultaneously increasing the scope and sharpening the resolution of psychological views of morality. PMID:21244182

  6. Discoidin Domain Receptor 1

    PubMed Central

    Song, Sunmi; Shackel, Nicholas A.; Wang, Xin M.; Ajami, Katerina; McCaughan, Geoffrey W.; Gorrell, Mark D.

    2011-01-01

    Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and is activated by collagens. Transcriptional profiling of cirrhosis in human liver using a DNA array and quantitative PCR detected elevated mRNA expression of DDR1 compared with that in nondiseased liver. The present study characterized DDR1 expression in cirrhotic and nondiseased human liver and examined the cellular effects of DDR1 expression. mRNA expression of all five isoforms of DDR1 was detected in human liver, whereas DDR1a demonstrated differential expression in liver with hepatitis C virus and primary biliary cirrhosis compared with nondiseased liver. In addition, immunoblot analysis detected shed fragments of DDR1 more readily in cirrhotic liver than in nondiseased liver. Inasmuch as DDR1 is subject to protease-mediated cleavage after prolonged interaction with collagen, this differential expression may indicate more intense activation of DDR1 protein in cirrhotic compared with nondiseased liver. In situ hybridization and immunofluorescence localized intense DDR1 mRNA and protein expression to epithelial cells including hepatocytes at the portal-parenchymal interface and the luminal aspect of the biliary epithelium. Overexpression of DDR1a altered hepatocyte behavior including increased adhesion and less migration on extracelular matrix substrates. DDR1a regulated extracellular expression of matrix metalloproteinases 1 and 2. These data elucidate DDR1 function pertinent to cirrhosis and indicate the importance of epithelial cell–collagen interactions in chronic liver injury. PMID:21356365

  7. Hydrophobic Compounds Reshape Membrane Domains

    PubMed Central

    Barnoud, Jonathan; Rossi, Giulia; Marrink, Siewert J.; Monticelli, Luca

    2014-01-01

    Cell membranes have a complex lateral organization featuring domains with distinct composition, also known as rafts, which play an essential role in cellular processes such as signal transduction and protein trafficking. In vivo, perturbations of membrane domains (e.g., by drugs or lipophilic compounds) have major effects on the activity of raft-associated proteins and on signaling pathways, but they are difficult to characterize because of the small size of the domains, typically below optical resolution. Model membranes, instead, can show macroscopic phase separation between liquid-ordered and liquid-disordered domains, and they are often used to investigate the driving forces of membrane lateral organization. Studies in model membranes have shown that some lipophilic compounds perturb membrane domains, but it is not clear which chemical and physical properties determine domain perturbation. The mechanisms of domain stabilization and destabilization are also unknown. Here we describe the effect of six simple hydrophobic compounds on the lateral organization of phase-separated model membranes consisting of saturated and unsaturated phospholipids and cholesterol. Using molecular simulations, we identify two groups of molecules with distinct behavior: aliphatic compounds promote lipid mixing by distributing at the interface between liquid-ordered and liquid-disordered domains; aromatic compounds, instead, stabilize phase separation by partitioning into liquid-disordered domains and excluding cholesterol from the disordered domains. We predict that relatively small concentrations of hydrophobic species can have a broad impact on domain stability in model systems, which suggests possible mechanisms of action for hydrophobic compounds in vivo. PMID:25299598

  8. Structural Dynamics of the Cereblon Ligand Binding Domain

    PubMed Central

    Hartmann, Marcus D.; Boichenko, Iuliia; Coles, Murray; Lupas, Andrei N.; Hernandez Alvarez, Birte

    2015-01-01

    Cereblon, a primary target of thalidomide and its derivatives, has been characterized structurally from both bacteria and animals. Especially well studied is the thalidomide binding domain, CULT, which shows an invariable structure across different organisms and in complex with different ligands. Here, based on a series of crystal structures of a bacterial representative, we reveal the conformational flexibility and structural dynamics of this domain. In particular, we follow the unfolding of large fractions of the domain upon release of thalidomide in the crystalline state. Our results imply that a third of the domain, including the thalidomide binding pocket, only folds upon ligand binding. We further characterize the structural effect of the C-terminal truncation resulting from the mental-retardation linked R419X nonsense mutation in vitro and offer a mechanistic hypothesis for its irresponsiveness to thalidomide. At 1.2Å resolution, our data provide a view of thalidomide binding at atomic resolution. PMID:26024445

  9. The structure of the Ca{sup 2+}-binding , glycosylated F-spondin domain of F-spondin- A C2-domain variant in an extracellular matrix protein.

    SciTech Connect

    Tan, K.; Lawler, J.

    2011-05-10

    F-spondin is a multi-domain extracellular matrix (ECM) protein and a contact-repellent molecule that directs axon outgrowth and cell migration during development. The reelin{_}N domain and the F-spondin domain (FS domain) comprise a proteolytic fragment that interacts with the cell membrane and guides the projection of commissural axons to floor plate. The FS domain is found in F-spondins, mindins, M-spondin and amphiF-spondin. We present the crystal structure of human F-spondin FS domain at 1.95{angstrom} resolution. The structure reveals a Ca{sup 2+}-binding C2 domain variant with an 8-stranded antiparallel {beta}-sandwich fold. Though the primary sequences of the FS domains of F-spondin and mindin are less than 36% identical, their overall structures are very similar. The unique feature of F-spondin FS domain is the presence of three disulfide bonds associated with the N- and C-termini of the domain and a highly conserved N-linked glycosylation site. The integrin-binding motif found in mindin is not conserved in the F-spondin FS domain. The structure of the F-spondin FS domain completes the structural studies of the multiple-domain ECM molecule. The homology of its core structure to a common Ca{sup 2+}- and lipid-binding C2 domain suggests that the F-spondin FS domain may be responsible for part of the membrane targeting of F-spondin in its regulation of axon development. The structural properties of the FS domain revealed in this study pave the way for further exploration into the functions of F-spondin.

  10. Wavelet-Based Signal and Image Processing for Target Recognition

    DTIC Science & Technology

    2002-01-01

    in target recognition applications. Classical spatial and frequency domain image processing algorithms were generalized to process discrete wavelet ... transform (DWT) data. Results include adaptation of classical filtering, smoothing and interpolation techniques to DWT. From 2003 the research

  11. Fractional diffusion on bounded domains

    DOE PAGES

    Defterli, Ozlem; D'Elia, Marta; Du, Qiang; ...

    2015-03-13

    We found that the mathematically correct specification of a fractional differential equation on a bounded domain requires specification of appropriate boundary conditions, or their fractional analogue. In this paper we discuss the application of nonlocal diffusion theory to specify well-posed fractional diffusion equations on bounded domains.

  12. Students' Target

    NASA Technical Reports Server (NTRS)

    2005-01-01

    [figure removed for brevity, see original site] Context image for PIA03648 Ascraeus Mons

    After examining numerous THEMIS images and using the JMars targeting software, eighth grade students from Charleston Middle School in Charleston, IL, selected the location of -8.37N and 276.66E for capture by the THEMIS visible camera during Mars Odyssey's sixth orbit of Mars on Nov. 22, 2005. The students are investigating relationships between channels, craters, and basins on Mars. The Charleston Middle School students participated in the Mars Student Imaging Project (MSIP) and submitted a proposal to use the THEMIS visible camera.

    Image information: VIS instrument. Latitude 8.8S, Longitude 279.6E. 17 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is led by Dr. Philip Christensen at Arizona State University. Lockheed Martin Astronautics, Denver, is the prime contractor for the Odyssey project, and developed and built the orbiter. Mission operations are conducted jointly from Lockheed Martin and from JPL, a division of the California Institute of Technology in Pasadena.

  13. 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

  14. Feature-level sentiment analysis by using comparative domain corpora

    NASA Astrophysics Data System (ADS)

    Quan, Changqin; Ren, Fuji

    2016-06-01

    Feature-level sentiment analysis (SA) is able to provide more fine-grained SA on certain opinion targets and has a wider range of applications on E-business. This study proposes an approach based on comparative domain corpora for feature-level SA. The proposed approach makes use of word associations for domain-specific feature extraction. First, we assign a similarity score for each candidate feature to denote its similarity extent to a domain. Then we identify domain features based on their similarity scores on different comparative domain corpora. After that, dependency grammar and a general sentiment lexicon are applied to extract and expand feature-oriented opinion words. Lastly, the semantic orientation of a domain-specific feature is determined based on the feature-oriented opinion lexicons. In evaluation, we compare the proposed method with several state-of-the-art methods (including unsupervised and semi-supervised) using a standard product review test collection. The experimental results demonstrate the effectiveness of using comparative domain corpora.

  15. Chemical synthesis and biotinylation of the thrombospondin domain TSR2

    PubMed Central

    Tiefenbrunn, Theresa K; Dawson, Philip E

    2009-01-01

    The type 1 repeat domain from thrombospondin has potent antiangiogenic activity and a structurally interesting fold, making it an attractive target for protein engineering. Chemical synthesis is an attractive approach for studying protein domains because it enables the use of unnatural amino acids for site-specific labeling and detailed structure-function analysis. Here, we demonstrate the first total chemical synthesis of the thrombospondin type 1 repeat domain by native chemical ligation. In addition to the natural domain, five sites for side chain modification were evaluated and two were found to be compatible with oxidative folding. Several challenges were encountered during peptide synthesis due to the functional complexity of the domain. These challenges were overcome by the use of new solid supports, scavengers, and the testing of multiple ligation sites. We also describe an unusual sequence-specific protecting group migration observed during cleavage resulting in +90 Da and +194 Da adducts. Synthetic access to this domain enables the synthesis of a number of variants that can be used to further our understanding of the biochemical interaction network of thrombospondin and provide insight into the structure and function of this important antitumorogenic protein domain. PMID:19384999

  16. NMR structure of the human Mediator MED25 ACID domain.

    PubMed

    Bontems, François; Verger, Alexis; Dewitte, Frédérique; Lens, Zoé; Baert, Jean-Luc; Ferreira, Elisabeth; de Launoit, Yvan; Sizun, Christina; Guittet, Eric; Villeret, Vincent; Monté, Didier

    2011-04-01

    MED25 (ARC92/ACID1) is a 747 residues subunit specific to higher eukaryote Mediator complex, an essential component of the RNA polymerase II general transcriptional machinery. MED25 is a target of the Herpes simplex virus transactivator protein VP16. MED25 interacts with VP16 through a central MED25 PTOV (Prostate tumour overexpressed)/ACID (Activator interacting domain) domain of unknown structure. As a first step towards understanding the mechanism of recruitment of transactivation domains by MED25, we report here the NMR structure of the MED25 ACID domain. The domain architecture consists of a closed β-barrel with seven strands (Β1-Β7) and three α-helices (H1-H3), an architecture showing similarities to that of the SPOC (Spen paralog and ortholog C-terminal domain) domain-like superfamily. Preliminary NMR chemical shift mapping showed that VP16 H2 (VP16C) interacts with MED25 ACID through one face of the β-barrel, defined by strands B4-B7-B6.

  17. Mechanistic insights into phosphoprotein-binding FHA domains.

    PubMed

    Liang, Xiangyang; Van Doren, Steven R

    2008-08-01

    [Structure: see text]. FHA domains are protein modules that switch signals in diverse biological pathways by monitoring the phosphorylation of threonine residues of target proteins. As part of the effort to gain insight into cellular avoidance of cancer, FHA domains involved in the cellular response to DNA damage have been especially well-characterized. The complete protein where the FHA domain resides and the interaction partners determine the nature of the signaling. Thus, a key biochemical question is how do FHA domains pick out their partners from among thousands of alternatives in the cell? This Account discusses the structure, affinity, and specificity of FHA domains and the formation of their functional structure. Although FHA domains share sequence identity at only five loop residues, they all fold into a beta-sandwich of two beta-sheets. The conserved arginine and serine of the recognition loops recognize the phosphorylation of the threonine targeted. Side chains emanating from loops that join beta-strand 4 with 5, 6 with 7, or 10 with 11 make specific contacts with amino acids of the ligand that tailor sequence preferences. Many FHA domains choose a partner in extended conformation, somewhat according to the residue three after the phosphothreonine in sequence (pT + 3 position). One group of FHA domains chooses a short carboxylate-containing side chain at pT + 3. Another group chooses a long, branched aliphatic side chain. A third group prefers other hydrophobic or uncharged polar side chains at pT + 3. However, another FHA domain instead chooses on the basis of pT - 2, pT - 3, and pT + 1 positions. An FHA domain from a marker of human cancer instead chooses a much longer protein fragment that adds a beta-strand to its beta-sheet and that presents hydrophobic residues from a novel helix to the usual recognition surface. This novel recognition site and more remote sites for the binding of other types of protein partners were predicted for the entire family

  18. Diversity in protein domain superfamilies

    PubMed Central

    Das, Sayoni; Dawson, Natalie L; Orengo, Christine A

    2015-01-01

    Whilst ∼93% of domain superfamilies appear to be relatively structurally and functionally conserved based on the available data from the CATH-Gene3D domain classification resource, the remainder are much more diverse. In this review, we consider how domains in some of the most ubiquitous and promiscuous superfamilies have evolved, in particular the plasticity in their functional sites and surfaces which expands the repertoire of molecules they interact with and actions performed on them. To what extent can we identify a core function for these superfamilies which would allow us to develop a ‘domain grammar of function’ whereby a protein's biological role can be proposed from its constituent domains? Clearly the first step is to understand the extent to which these components vary and how changes in their molecular make-up modifies function. PMID:26451979

  19. Microbial starch-binding domain.

    PubMed

    Rodríguez-Sanoja, Romina; Oviedo, Norma; Sánchez, Sergio

    2005-06-01

    Glucosidic bonds from different non-soluble polysaccharides such as starch, cellulose and xylan are hydrolyzed by amylases, cellulases and xylanases, respectively. These enzymes are produced by microorganisms. They have a modular structure that is composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. Starch-binding modules are present in microbial enzymes that are involved in starch metabolism; these are classified into several different families on the basis of their amino acid sequence similarities. Such binding domains promote attachment to the substrate and increase its concentration at the active site of the enzyme, which allows microorganisms to degrade non-soluble starch. Fold similarities are better conserved than sequences; nevertheless, it is possible to notice two evolutionary clusters of microbial starch-binding domains. These domains have enormous potential as tags for protein immobilization, as well as for the tailoring of enzymes that play a part in polysaccharide metabolism.

  20. Phase-domain photoacoustic sensing

    NASA Astrophysics Data System (ADS)

    Gao, Fei; Zhang, Ruochong; Feng, Xiaohua; Liu, Siyu; Ding, Ran; Kishor, Rahul; Qiu, Lei; Zheng, Yuanjin

    2017-01-01

    As one of the fastest-growing imaging modalities in recent years, photoacoustic imaging has attracted tremendous research interest for various applications including anatomical, functional, and molecular imaging. The majority of the photoacoustic imaging systems are based on the time-domain pulsed photoacoustic method, which utilizes a pulsed laser source to induce a wideband photoacoustic signal, revealing optical absorption contrast. An alternative way is the frequency-domain photoacoustic method utilizing the chirping modulation of laser intensity to achieve lower system cost. In this paper, we report another way of the photoacoustic method, called phase-domain photoacoustic sensing, which explores the phase difference between two consequent intensity-modulated laser pulse induced photoacoustic measurements to reveal the optical properties. The basic principle is introduced, modeled, and experimentally validated in this paper, which opens another potential pathway to perform photoacoustic sensing and imaging, eliminating acoustic detection variations beyond the conventional time-domain and frequency-domain photoacoustic methods.

  1. Specificity Profiling of Protein-Binding Domains Using One-Bead-One-Compound Peptide Libraries

    PubMed Central

    Kunys, Andrew R.; Lian, Wenlong; Pei, Dehua

    2013-01-01

    One-bead-one-compound (OBOC) libraries consist of structurally related compounds (e.g., peptides) covalently attached to a solid support, with each resin bead carrying a unique compound. OBOC libraries of high structural diversity can be rapidly synthesized and screened without the need of any special equipment and therefore can be employed in any chemical or biochemical laboratory. OBOC peptide libraries have been widely used to map the ligand specificity of proteins, to determine the substrate specificity of enzymes, and to develop inhibitors against macromolecular targets. They have proven particularly useful in profiling the binding specificity of protein modular domains (e.g., SH2 domains, BIR domains, and PDZ domains) and subsequently using the specificity information to predict the protein targets of these domains. The protocols outlined in this article describe the methodologies for synthesizing and screening OBOC peptide libraries against SH2 and PDZ domains and the related data analysis. PMID:23788558

  2. The monocyte binding domain(s) on human immunoglobulin G.

    PubMed

    Woof, J M; Nik Jaafar, M I; Jefferis, R; Burton, D R

    1984-06-01

    Monocyte binding has previously been assigned to the C gamma 3 domain of human immunoglobulin G (IgG) largely on the ability of the pFc' fragment to inhibit the monocyte-IgG interaction. This ability is markedly reduced compared to the intact parent IgG. We find this result with a conventional pFc' preparation but this preparation is found to contain trace contamination of parent IgG as demonstrated by reactivity with monoclonal antibodies directed against C gamma 2 domain and light-chain epitopes of human IgG. Extensive immunoaffinity purification of the pFc' preparation removes its inhibitory ability indicating that this originates in the trace contamination of parent IgG (or Fc). Neither of the human IgG1 paraproteins TIM, lacking the C gamma 2 domain, or SIZ, lacking the C gamma 3 domain, are found to inhibit the monocyte-IgG interaction. The hinge-deleted IgG1 Dob protein shows little or no inhibitory ability. Indirect evidence for the involvement of the C gamma 2 domain in monocyte binding is considered. We suggest finally that the site of interaction is found either on the C gamma 2 domain alone or between the C gamma 2 and C gamma 3 domains.

  3. Separated matter and antimatter domains with vanishing domain walls

    SciTech Connect

    Dolgov, A.D.; Godunov, S.I.; Rudenko, A.S.; Tkachev, I.I. E-mail: sgodunov@itep.ru E-mail: tkachev@ms2.inr.ac.ru

    2015-10-01

    We present a model of spontaneous (or dynamical) C and CP violation where it is possible to generate domains of matter and antimatter separated by cosmologically large distances. Such C(CP) violation existed only in the early universe and later it disappeared with the only trace of generated baryonic and/or antibaryonic domains. So the problem of domain walls in this model does not exist. These features are achieved through a postulated form of interaction between inflaton and a new scalar field, realizing short time C(CP) violation.

  4. Geometry-induced fluctuations of olfactory searches in bounded domains

    NASA Astrophysics Data System (ADS)

    Rodríguez, Juan Duque; Gómez-Ullate, David; Mejía-Monasterio, Carlos

    2014-04-01

    In olfactory search an immobile target emits chemical molecules at constant rate. The molecules are transported by the medium, which is assumed to be turbulent. Considering a searcher able to detect such chemical signals and whose motion follows the infotaxis strategy, we study the statistics of the first-passage time to the target when the searcher moves on a finite two-dimensional lattice of different geometries. Far from the target, where the concentration of chemicals is low, the direction of the searcher's first movement is determined by the geometry of the domain and the topology of the lattice, inducing strong fluctuations on the average search time with respect to the initial position of the searcher. The domain is partitioned in well-defined regions characterized by the direction of the first movement. If the search starts over the interface between two different regions, large fluctuations in the search time are observed.

  5. Geometry-induced fluctuations of olfactory searches in bounded domains.

    PubMed

    Rodríguez, Juan Duque; Gómez-Ullate, David; Mejía-Monasterio, Carlos

    2014-04-01

    In olfactory search an immobile target emits chemical molecules at constant rate. The molecules are transported by the medium, which is assumed to be turbulent. Considering a searcher able to detect such chemical signals and whose motion follows the infotaxis strategy, we study the statistics of the first-passage time to the target when the searcher moves on a finite two-dimensional lattice of different geometries. Far from the target, where the concentration of chemicals is low, the direction of the searcher's first movement is determined by the geometry of the domain and the topology of the lattice, inducing strong fluctuations on the average search time with respect to the initial position of the searcher. The domain is partitioned in well-defined regions characterized by the direction of the first movement. If the search starts over the interface between two different regions, large fluctuations in the search time are observed.

  6. Recent development of anticancer therapeutics targeting Akt.

    PubMed

    Morrow, John K; Du-Cuny, Lei; Chen, Lu; Meuillet, Emmanuelle J; Mash, Eugene A; Powis, Garth; Zhang, Shuxing

    2011-01-01

    The serine/threonine kinase Akt has proven to be a significant signaling target, involved in various biological functions. Because of its cardinal role in numerous cellular responses, Akt has been implicated in many human diseases, particularly cancer. It has been established that Akt is a viable and feasible target for anticancer therapeutics. Analysis of all Akt kinases reveals conserved homology for an N-terminal regulatory domain, which contains a pleckstrin-homology (PH) domain for cellular translocation, a kinase domain with serine/threonine specificity, and a C-terminal extension domain. These well defined regions have been targeted, and various approaches, including in silico methods, have been implemented to develop Akt inhibitors. In spite of unique techniques and a prolific body of knowledge surrounding Akt, no targeted Akt therapeutics have reached the market yet. Here we will highlight successes and challenges to date on the development of anticancer agents modulating the Akt pathway in recent patents as well as discuss the methods employed for this task. Special attention will be given to patents with focus on those discoveries using computer-aided drug design approaches.

  7. Channel gating pore: a new therapeutic target.

    PubMed

    Kornilov, Polina; Peretz, Asher; Attali, Bernard

    2013-09-01

    Each subunit of voltage-gated cation channels comprises a voltage-sensing domain and a pore region. In a paper recently published in Cell Research, Li et al. showed that the gating charge pathway of the voltage sensor of the KCNQ2 K+ channel can accommodate small opener molecules and offer a new target to treat hyperexcitability disorders.

  8. Model-based target and background characterization

    NASA Astrophysics Data System (ADS)

    Mueller, Markus; Krueger, Wolfgang; Heinze, Norbert

    2000-07-01

    Up to now most approaches of target and background characterization (and exploitation) concentrate solely on the information given by pixels. In many cases this is a complex and unprofitable task. During the development of automatic exploitation algorithms the main goal is the optimization of certain performance parameters. These parameters are measured during test runs while applying one algorithm with one parameter set to images that constitute of image domains with very different domain characteristics (targets and various types of background clutter). Model based geocoding and registration approaches provide means for utilizing the information stored in GIS (Geographical Information Systems). The geographical information stored in the various GIS layers can define ROE (Regions of Expectations) and may allow for dedicated algorithm parametrization and development. ROI (Region of Interest) detection algorithms (in most cases MMO (Man- Made Object) detection) use implicit target and/or background models. The detection algorithms of ROIs utilize gradient direction models that have to be matched with transformed image domain data. In most cases simple threshold calculations on the match results discriminate target object signatures from the background. The geocoding approaches extract line-like structures (street signatures) from the image domain and match the graph constellation against a vector model extracted from a GIS (Geographical Information System) data base. Apart from geo-coding the algorithms can be also used for image-to-image registration (multi sensor and data fusion) and may be used for creation and validation of geographical maps.

  9. Domain walls riding the wave.

    SciTech Connect

    Karapetrov, G.; Novosad, V.; Materials Science Division

    2010-11-01

    Recent years have witnessed a rapid proliferation of electronic gadgets around the world. These devices are used for both communication and entertainment, and it is a fact that they account for a growing portion of household energy consumption and overall world consumption of electricity. Increasing the energy efficiency of these devices could have a far greater and immediate impact than a gradual switch to renewable energy sources. The advances in the area of spintronics are therefore very important, as gadgets are mostly comprised of memory and logic elements. Recent developments in controlled manipulation of magnetic domains in ferromagnet nanostructures have opened opportunities for novel device architectures. This new class of memories and logic gates could soon power millions of consumer electronic devices. The attractiveness of using domain-wall motion in electronics is due to its inherent reliability (no mechanical moving parts), scalability (3D scalable architectures such as in racetrack memory), and nonvolatility (retains information in the absence of power). The remaining obstacles in widespread use of 'racetrack-type' elements are the speed and the energy dissipation during the manipulation of domain walls. In their recent contribution to Physical Review Letters, Oleg Tretiakov, Yang Liu, and Artem Abanov from Texas A&M University in College Station, provide a theoretical description of domain-wall motion in nanoscale ferromagnets due to the spin-polarized currents. They find exact conditions for time-dependent resonant domain-wall movement, which could speed up the motion of domain walls while minimizing Ohmic losses. Movement of domain walls in ferromagnetic nanowires can be achieved by application of external magnetic fields or by passing a spin-polarized current through the nanowire itself. On the other hand, the readout of the domain state is done by measuring the resistance of the wire. Therefore, passing current through the ferromagnetic wire is

  10. In situ dissection of RNA functional subunits by domain-specific chromatin isolation by RNA purification (dChIRP).

    PubMed

    Quinn, Jeffrey J; Chang, Howard Y

    2015-01-01

    Here we describe domain-specific chromatin isolation by RNA purification (dChIRP), a technique for dissecting the functional domains of a target RNA in situ. For an RNA of interest, dChIRP can identify domain-level intramolecular and intermolecular RNA-RNA, RNA-protein, and RNA-DNA interactions and maps the RNA's genomic binding sites with higher precision than domain-agnostic methods. We illustrate how this technique has been applied to the roX1 lncRNA to resolve its domain-level architecture, discover its protein- and chromatin-interacting domains, and map its occupancy on the X chromosome.

  11. Mutation of domain III and domain VI in L gene conserved domain of Nipah virus

    NASA Astrophysics Data System (ADS)

    Jalani, Siti Aishah; Ibrahim, Nazlina

    2016-11-01

    Nipah virus (NiV) is the etiologic agent responsible for the respiratory illness and causes fatal encephalitis in human. NiV L protein subunit is thought to be responsible for the majority of enzymatic activities involved in viral transcription and replication. The L protein which is the viral RNA dependent RNA polymerase has high sequence homology among negative sense RNA viruses. In negative stranded RNA viruses, based on sequence alignment six conserved domain (domain I-IV) have been determined. Each domain is separated on variable regions that suggest the structure to consist concatenated functional domain. To directly address the roles of domains III and VI, site-directed mutations were constructed by the substitution of bases at sequences 2497, 2500, 5528 and 5532. Each mutated L gene can be used in future studies to test the ability for expression on in vitro translation.

  12. Modeling software systems by domains

    NASA Technical Reports Server (NTRS)

    Dippolito, Richard; Lee, Kenneth

    1992-01-01

    The Software Architectures Engineering (SAE) Project at the Software Engineering Institute (SEI) has developed engineering modeling techniques that both reduce the complexity of software for domain-specific computer systems and result in systems that are easier to build and maintain. These techniques allow maximum freedom for system developers to apply their domain expertise to software. We have applied these techniques to several types of applications, including training simulators operating in real time, engineering simulators operating in non-real time, and real-time embedded computer systems. Our modeling techniques result in software that mirrors both the complexity of the application and the domain knowledge requirements. We submit that the proper measure of software complexity reflects neither the number of software component units nor the code count, but the locus of and amount of domain knowledge. As a result of using these techniques, domain knowledge is isolated by fields of engineering expertise and removed from the concern of the software engineer. In this paper, we will describe kinds of domain expertise, describe engineering by domains, and provide relevant examples of software developed for simulator applications using the techniques.

  13. Domain adaptation for semantic role labeling of clinical text

    PubMed Central

    Zhang, Yaoyun; Tang, Buzhou; Jiang, Min; Wang, Jingqi

    2015-01-01

    Objective Semantic role labeling (SRL), which extracts a shallow semantic relation representation from different surface textual forms of free text sentences, is important for understanding natural language. Few studies in SRL have been conducted in the medical domain, primarily due to lack of annotated clinical SRL corpora, which are time-consuming and costly to build. The goal of this study is to investigate domain adaptation techniques for clinical SRL leveraging resources built from newswire and biomedical literature to improve performance and save annotation costs. Materials and Methods Multisource Integrated Platform for Answering Clinical Questions (MiPACQ), a manually annotated SRL clinical corpus, was used as the target domain dataset. PropBank and NomBank from newswire and BioProp from biomedical literature were used as source domain datasets. Three state-of-the-art domain adaptation algorithms were employed: instance pruning, transfer self-training, and feature augmentation. The SRL performance using different domain adaptation algorithms was evaluated by using 10-fold cross-validation on the MiPACQ corpus. Learning curves for the different methods were generated to assess the effect of sample size. Results and Conclusion When all three source domain corpora were used, the feature augmentation algorithm achieved statistically significant higher F-measure (83.18%), compared to the baseline with MiPACQ dataset alone (F-measure, 81.53%), indicating that domain adaptation algorithms may improve SRL performance on clinical text. To achieve a comparable performance to the baseline method that used 90% of MiPACQ training samples, the feature augmentation algorithm required <50% of training samples in MiPACQ, demonstrating that annotation costs of clinical SRL can be reduced significantly by leveraging existing SRL resources from other domains. PMID:26063745

  14. Protein domain connectivity and essentiality

    NASA Astrophysics Data System (ADS)

    da F. Costa, L.; Rodrigues, F. A.; Travieso, G.

    2006-10-01

    Protein-protein interactions can be properly modeled as scale-free complex networks, while the lethality of proteins has been correlated with the node degrees, therefore defining a lethality-centrality rule. In this work the authors revisit this relevant problem by focusing attention not on proteins as a whole, but on their functional domains, which are ultimately responsible for their binding potential. Four networks are considered: the original protein-protein interaction network, its randomized version, and two domain networks assuming different lethality hypotheses. By using formal statistical analysis, they show that the correlation between connectivity and essentiality is higher for domains than for proteins.

  15. 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

  16. Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

    PubMed Central

    Li, Fuyang; Papworth, Monika; Minczuk, Michal; Rohde, Christian; Zhang, Yingying; Ragozin, Sergei; Jeltsch, Albert

    2007-01-01

    Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation. PMID:17151075

  17. A two-dimensional time domain near zone to far zone transformation

    NASA Technical Reports Server (NTRS)

    Luebbers, Raymond J.; Ryan, Deirdre; Beggs, John H.; Kunz, Karl S.

    1991-01-01

    A time domain transformation useful for extrapolating three dimensional near zone finite difference time domain (FDTD) results to the far zone was presented. Here, the corresponding two dimensional transform is outlined. While the three dimensional transformation produced a physically observable far zone time domain field, this is not convenient to do directly in two dimensions, since a convolution would be required. However, a representative two dimensional far zone time domain result can be obtained directly. This result can then be transformed to the frequency domain using a Fast Fourier Transform, corrected with a simple multiplicative factor, and used, for example, to calculate the complex wideband scattering width of a target. If an actual time domain far zone result is required, it can be obtained by inverse Fourier transform of the final frequency domain result.

  18. A two-dimensional time domain near zone to far zone transformation

    NASA Technical Reports Server (NTRS)

    Luebbers, Raymond J.; Ryan, Deirdre; Beggs, John H.; Kunz, Karl S.

    1991-01-01

    In a previous paper, a time domain transformation useful for extrapolating 3-D near zone finite difference time domain (FDTD) results to the far zone was presented. In this paper, the corresponding 2-D transform is outlined. While the 3-D transformation produced a physically observable far zone time domain field, this is not convenient to do directly in 2-D, since a convolution would be required. However, a representative 2-D far zone time domain result can be obtained directly. This result can then be transformed to the frequency domain using a Fast Fourier Transform, corrected with a simple multiplicative factor, and used, for example, to calculate the complex wideband scattering width of a target. If an actual time domain far zone result is required it can be obtained by inverse Fourier transform of the final frequency domain result.

  19. Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.

    PubMed

    Reinhardt, H Christian; Yaffe, Michael B

    2013-09-01

    Coordinated progression through the cell cycle is a complex challenge for eukaryotic cells. Following genotoxic stress, diverse molecular signals must be integrated to establish checkpoints specific for each cell cycle stage, allowing time for various types of DNA repair. Phospho-Ser/Thr-binding domains have emerged as crucial regulators of cell cycle progression and DNA damage signalling. Such domains include 14-3-3 proteins, WW domains, Polo-box domains (in PLK1), WD40 repeats (including those in the E3 ligase SCF(βTrCP)), BRCT domains (including those in BRCA1) and FHA domains (such as in CHK2 and MDC1). Progress has been made in our understanding of the motif (or motifs) that these phospho-Ser/Thr-binding domains connect with on their targets and how these interactions influence the cell cycle and DNA damage response.

  20. Structure of NS1A effector domain from the influenza A/Udorn/72 virus

    SciTech Connect

    Xia, Shuangluo; Monzingo, Arthur F.; Robertus, Jon D.

    2009-01-01

    The structure of the effector domain of the influenza protein NS1, a validated antiviral drug target, has been solved in two space groups. The nonstructural protein NS1A from influenza virus is a multifunctional virulence factor and a potent inhibitor of host immunity. It has two functional domains: an N-terminal 73-amino-acid RNA-binding domain and a C-terminal effector domain. Here, the crystallographic structure of the NS1A effector domain of influenza A/Udorn/72 virus is presented. Structure comparison with the NS1 effector domain from mouse-adapted influenza A/Puerto Rico/8/34 (PR8) virus strain reveals a similar monomer conformation but a different dimer interface. Further analysis and evaluation shows that the dimer interface observed in the structure of the PR8 NS1 effector domain is likely to be a crystallographic packing effect. A hypothetical model of the intact NS1 dimer is presented.

  1. WW or WoW: the WW domains in a union of bliss.

    PubMed

    Sudol, Marius; Recinos, Claudia C; Abraczinskas, Jennifer; Humbert, Jasper; Farooq, Amjad

    2005-12-01

    WW domains are small protein modules that recognize proline-rich peptide motifs or phosphorylated-serine/threonine proline sites in cognate proteins. Within host proteins these modules are joined to other protein domains or to a variety of catalytic domains acting together as adaptors or targeting anchors of enzymes. An important aspect of signaling by WW domains is their ability to recognize their cognate ligands in tandem. Tandem WW domains not only act in a synergistic manner but also appear to chaperone the function of each other. In this review, we focus on structure, function, and mechanism of the tandem WW domains co-operativity as well as independent actions. We emphasize here the implications of tandem arrangement and cooperative function of the domains for signaling pathways.

  2. Molecular insights into regulation of JAK2 in myeloproliferative neoplasms

    PubMed Central

    Hubbard, Stevan R.

    2015-01-01

    The critical role of Janus kinase-2 (JAK2) in regulation of myelopoiesis was established 2 decades ago, but identification of mutations in the pseudokinase domain of JAK2 in myeloproliferative neoplasms (MPNs) and in other hematologic malignancies highlighted the role of JAK2 in human disease. These findings have revolutionized the diagnostics of MPNs and led to development of novel JAK2 therapeutics. However, the molecular mechanisms by which mutations in the pseudokinase domain lead to hyperactivation of JAK2 and clinical disease have been unclear. Here, we describe recent advances in the molecular characterization of the JAK2 pseudokinase domain and how pathogenic mutations lead to constitutive activation of JAK2. PMID:25824690

  3. Functional domains of the floral regulator AGAMOUS: characterization of the DNA binding domain and analysis of dominant negative mutations.

    PubMed Central

    Mizukami, Y; Huang, H; Tudor, M; Hu, Y; Ma, H

    1996-01-01

    The Arabidopsis MADS box gene AGAMOUS (AG) controls reproductive organ identity and floral meristem determinacy. The AG protein binds in vitro to DNA sequences similar to the targets of known MADS domain transcription factors. Whereas most plant MADS domain proteins begin with the MADS domain, AG and its orthologs contain a region N-terminal to the MADS domain. All plant MADS domain proteins share another region with moderate sequence similarity called the K domain. Neither the region (I region) that lies between the MADS and K domains nor the C-terminal region is conserved. We show here that the AG MADS domain and the I region are necessary and sufficient for DNA binding in vitro and that AG binds to DNA as a dimer. To investigate the in vivo function of the regions of AG not required for in vitro DNA binding, we introduced several AG constructs into wild-type plants and characterized their floral phenotypes. We show that transgenic Arabidopsis plants with a 35S-AG construct encoding an AG protein lacking the N-terminal region produced apetala 2 (ap2)-like flowers similar to those ectopically expressing AG proteins retaining the N-terminal region. This result suggests that the N-terminal region is not required to produce the ap2-like phenotype. In addition, transformants with a 35S-AG construct encoding an AG protein lacking the C-terminal region produced ag-like flowers, indicating that this truncated AG protein inhibits normal AG function. Finally, transformants with a 35S-AG construct encoding an AG protein lacking both K and C regions produced flowers with more stamens and carpels. The phenotypes of the AG transformants demonstrate that both the K domain and the C-terminal region have important and distinct in vivo functions. We discuss possible mechanisms through which AG may regulate downstream genes. PMID:8672883

  4. Structure of axionic domain walls

    NASA Astrophysics Data System (ADS)

    Huang, M. C.; Sikivie, P.

    1985-09-01

    The structure of axionic domain walls is investigated using the low-energy effective theory of axions and pions. We derive the spatial dependence of the phases of the Peccei-Quinn scalar field and the QCD quark-antiquark condensates inside an axionic domain wall. Thence an accurate estimate of the wall surface energy density is obtained. The equations of motion for axions, photons, leptons, and baryons in the neighborhood of axionic domain walls are written down and estimates are given for the wall reflection and transmission coefficients of these particles. Finally, we discuss the energy dissipation by axionic domain walls oscillating in the early universe due to the reflection of particles in the primordial soup.

  5. Current Domain Challenges in the Emergency Response Community

    SciTech Connect

    Barr, Jonathan L.; Peddicord, Annie M Boe; Burtner, Edwin R.; Mahy, Heidi A.

    2011-05-08

    This paper describes the development of a framework targeted to technology providers in order to better understand the grand domain challenges of the emergency response and management community (EM). In developing this framework, Pacific Northwest National Laboratory researchers interviewed subject matter experts (SMEs) across the EM domain and corroborated these findings with current literature. We are currently examining relationships and dependencies within the framework. A thorough understanding of these gaps and dependencies will allow for a more informed approach prioritizing research, developing tools, and applying technology to enhance performance in the EM community.

  6. Advances in targeted genome editing.

    PubMed

    Perez-Pinera, Pablo; Ousterout, David G; Gersbach, Charles A

    2012-08-01

    New technologies have recently emerged that enable targeted editing of genomes in diverse systems. This includes precise manipulation of gene sequences in their natural chromosomal context and addition of transgenes to specific genomic loci. This progress has been facilitated by advances in engineering targeted nucleases with programmable, site-specific DNA-binding domains, including zinc finger proteins and transcription activator-like effectors (TALEs). Recent improvements have enhanced nuclease performance, accelerated nuclease assembly, and lowered the cost of genome editing. These advances are driving new approaches to many areas of biotechnology, including biopharmaceutical production, agriculture, creation of transgenic organisms and cell lines, and studies of genome structure, regulation, and function. Genome editing is also being investigated in preclinical and clinical gene therapies for many diseases.

  7. Prevalence of the F-type lectin domain.

    PubMed

    Bishnoi, Ritika; Khatri, Indu; Subramanian, Srikrishna; Ramya, T N C

    2015-08-01

    F-type lectins are fucolectins with characteristic fucose and calcium-binding sequence motifs and a unique lectin fold (the "F-type" fold). F-type lectins are phylogenetically widespread with selective distribution. Several eukaryotic F-type lectins have been biochemically and structurally characterized, and the F-type lectin domain (FLD) has also been studied in the bacterial proteins, Streptococcus mitis lectinolysin and Streptococcus pneumoniae SP2159. However, there is little knowledge about the extent of occurrence of FLDs and their domain organization, especially, in bacteria. We have now mined the extensive genomic sequence information available in the public databases with sensitive sequence search techniques in order to exhaustively survey prokaryotic and eukaryotic FLDs. We report 437 FLD sequence clusters (clustered at 80% sequence identity) from eukaryotic, eubacterial and viral proteins. Domain architectures are diverse but mostly conserved in closely related organisms, and domain organizations of bacterial FLD-containing proteins are very different from their eukaryotic counterparts, suggesting unique specialization of FLDs to suit different requirements. Several atypical phylogenetic associations hint at lateral transfer. Among eukaryotes, we observe an expansion of FLDs in terms of occurrence and domain organization diversity in the taxa Mollusca, Hemichordata and Branchiostomi, perhaps coinciding with greater emphasis on innate immune strategies in these organisms. The naturally occurring FLDs with diverse domain organizations that we have identified here will be useful for future studies aimed at creating designer molecular platforms for directing desired biological activities to fucosylated glycoconjugates in target niches.

  8. Selective Target Protein Degradation via Phthalimide Conjugation

    PubMed Central

    Winter, Georg E.; Buckley, Dennis L.; Paulk, Joshiawa; Roberts, Justin M.; Souza, Amanda; Dhe-Paganon, Sirano; Bradner, James E.

    2016-01-01

    Small-molecule antagonists disable discrete biochemical properties of protein targets. For multi-domain protein targets, the pharmacologic consequence of drug action is limited by selective disruption of one domain-specific activity. More broadly, target inhibition is kinetically limited by the durability and degree of target engagement. These features of traditional drug molecules are challenging to the development of inhibitors targeting transcription factors and chromatin-associated epigenetic proteins, which function as multi-domain biomolecular scaffolds and generally feature rapid association and dissociation kinetics. We therefore devised a chemical strategy to prompt ligand-dependent target protein degradation, via chemical conjugation with derivatized phthalimides that hijack the function of the Cereblon E3 ubiquitin ligase complex. Using this approach, we converted an acetyl-lysine competitive antagonist that displaces BET bromodomains from chromatin (JQ1) to a phthalimide-conjugated ligand that prompts immediate Cereblon-dependent BET protein degradation (dBET1). Expression proteomics confirms high specificity for BET family members BRD2, BRD3 and BRD4 among 7429 proteins detected. Degradation of BET bromodomains is associated with a more rapid and robust apoptotic response compared to bromodomain inhibition in primary human leukemic blasts, and dBET1 exhibits in vivo efficacy in a human leukemia xenograft. The reach of this approach is illustrated by a second series of probes that degrade the cytosolic signaling protein, FKBP12. Together, these findings identify a facile and general new strategy to control target protein stability, with implications for approaching previously intractable protein targets. PMID:25999370

  9. Transcriptional activation by the acidic domain of Vmw65 requires the integrity of the domain and involves additional determinants distinct from those necessary for TFIIB binding.

    PubMed

    Walker, S; Greaves, R; O'Hare, P

    1993-09-01

    In this work we have examined the requirements for activity of the acidic domain of Vmw65 (VP16) by deletion and site-directed mutagenesis of the region in the context of GAL4 fusion proteins. The results indicate that the present interpretation of what actually constitutes the activation domain is not correct. We demonstrate, using a promoter with one target site which is efficiently activated by the wild-type (wt) fusion protein, that amino acids distal to residue 453 are critical for activity. Truncation of the domain or substitution of residues in the distal region almost completely abrogate activity. However, inactivating mutations within the distal region are complemented by using a promoter containing multiple target sites. Moreover, duplication of the proximal region, but not the distal region, restores the ability to activate a promoter with a single target site. These results indicate some distinct qualitative difference between the proximal and distal regions. We have also examined the binding of nuclear proteins to the wt domain and to a variant with the distal region inactivated by mutation. The lack of activity of this variant is not explained by a lack of binding of TFIIB, a protein previously reported to be the likely target of the acidic domain. Therefore some additional function is involved in transcriptional activation by the acid domain, and determinants distinct from those involved in TFIIB binding are required for this function. Analysis of the total protein profiles binding to the wt and mutant domains has demonstrated the selective binding to the wt domain of a 135-kDa polypeptide, which is therefore a candidate component involved in this additional function. This is the first report to provide evidence for the proposal of a multiplicity of interactions within the acidic domain, by uncoupling requirements for one function from those for another.

  10. Domain and Specification Models for Software Engineering

    NASA Technical Reports Server (NTRS)

    Iscoe, Neil; Liu, Zheng-Yang; Feng, Guohui

    1992-01-01

    This paper discusses our approach to representing application domain knowledge for specific software engineering tasks. Application domain knowledge is embodied in a domain model. Domain models are used to assist in the creation of specification models. Although many different specification models can be created from any particular domain model, each specification model is consistent and correct with respect to the domain model. One aspect of the system-hierarchical organization is described in detail.

  11. A Carboxy-Terminal Trimerization Domain Stabilizes Conformational Epitopes on the Stalk Domain of Soluble Recombinant Hemagglutinin Substrates

    PubMed Central

    Krammer, Florian; Margine, Irina; Tan, Gene S.; Pica, Natalie; Krause, Jens C.; Palese, Peter

    2012-01-01

    Recently, a new class of broadly neutralizing anti-influenza virus antibodies that target the stalk domain of the viral hemagglutinin was discovered. As such, induction, isolation, characterization, and quantification of these novel antibodies has become an area of intense research and great interest. Since most of these antibodies bind to conformational epitopes, the structural integrity of hemagglutinin substrates for the detection and quantification of these antibodies is of high importance. Here we evaluate the binding of these antibodies to soluble, secreted hemagglutinins with or without a carboxy-terminal trimerization domain based on the natural trimerization domain of T4 phage fibritin. The lack of such a domain completely abolishes binding to group 1 hemagglutinins and also affects binding to group 2 hemagglutinins. Additionally, the presence of a trimerization domain positively influences soluble hemagglutinin stability during expression and purification. Our findings suggest that a carboxy-terminal trimerization domain is a necessary requirement for the structural integrity of stalk epitopes on recombinant soluble influenza virus hemagglutinin. PMID:22928001

  12. Targeting DNA double-strand breaks with TAL effector nucleases.

    PubMed

    Christian, Michelle; Cermak, Tomas; Doyle, Erin L; Schmidt, Clarice; Zhang, Feng; Hummel, Aaron; Bogdanove, Adam J; Voytas, Daniel F

    2010-10-01

    Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.

  13. BAR domain proteins regulate Rho GTPase signaling

    PubMed Central

    Aspenström, Pontus

    2014-01-01

    BAR proteins comprise a heterogeneous group of multi-domain proteins with diverse biological functions. The common denominator is the Bin-Amphiphysin-Rvs (BAR) domain that not only confers targeting to lipid bilayers, but also provides scaffolding to mold lipid membranes into concave or convex surfaces. This function of BAR proteins is an important determinant in the dynamic reconstruction of membrane vesicles, as well as of the plasma membrane. Several BAR proteins function as linkers between cytoskeletal regulation and membrane dynamics. These links are provided by direct interactions between BAR proteins and actin-nucleation-promoting factors of the Wiskott-Aldrich syndrome protein family and the Diaphanous-related formins. The Rho GTPases are key factors for orchestration of this intricate interplay. This review describes how BAR proteins regulate the activity of Rho GTPases, as well as how Rho GTPases regulate the function of BAR proteins. This mutual collaboration is a central factor in the regulation of vital cellular processes, such as cell migration, cytokinesis, intracellular transport, endocytosis, and exocytosis. PMID:25483303

  14. Localization of resistive domains in inhomogeneous superconductors

    SciTech Connect

    Gurevich, A.V.; Mints, R.G.

    1981-01-01

    The properties of resistive domains due to the Joule heating in inhomogeneous superconductors with transport currents are studied. The equilibrium of a domain at an inhomogeneity of arbitrary type and with dimensions much smaller than the dimensions of the domain is investigated. It is shown that resistive domains can become localized at inhomogeneities. The temperature distribution in a domain and the current--voltage characteristic of the domain are determined. The stability of localized domains is discussed. It is shown that such domains give rise to a hysteresis in the destruction (recovery) of the superconductivity by the transport current.

  15. The HARP domain dictates the annealing helicase activity of HARP/SMARCAL1.

    PubMed

    Ghosal, Gargi; Yuan, Jingsong; Chen, Junjie

    2011-06-01

    Mutations in HepA-related protein (HARP, or SMARCAL1) cause Schimke immunoosseous dysplasia (SIOD). HARP has ATP-dependent annealing helicase activity, which helps to stabilize stalled replication forks and facilitate DNA repair during replication. Here, we show that the conserved tandem HARP (2HP) domain dictates this annealing helicase activity. Furthermore, chimeric proteins generated by fusing the 2HP domain of HARP with the SNF2 domain of BRG1 or HELLS show annealing helicase activity in vitro and, when targeted to replication forks, mimic the functions of HARP in vivo. We propose that the HARP domain endows HARP with this ATP-driven annealing helicase activity.

  16. Cytokinin Response Factor 5 has transcriptional activity governed by its C-terminal domain.

    PubMed

    Striberny, Bernd; Melton, Anthony E; Schwacke, Rainer; Krause, Kirsten; Fischer, Karsten; Goertzen, Leslie R; Rashotte, Aaron M

    2017-02-01

    Cytokinin Response Factors (CRFs) are AP2/ERF transcription factors involved in cytokinin signal transduction. CRF proteins consist of a N-terminal dimerization domain (CRF domain), an AP2 DNA-binding domain, and a clade-specific C-terminal region of unknown function. Using a series of sequential deletions in yeast-2-hybrid assays, we provide evidence that the C-terminal region of Arabidopsis CRF5 can confer transactivation activity. Although comparative analyses identified evolutionarily conserved protein sequence within the C-terminal region, deletion experiments suggest that this transactivation domain has a partially redundant modular structure required for activation of target gene transcription.

  17. Unfolding-resistant Translocase Targeting

    PubMed Central

    Dagda, Ruben K.; Barwacz, Chris A.; Cribbs, J. Thomas; Strack, Stefan

    2015-01-01

    Heterotrimeric serine/threonine protein phosphatase 2A (PP2A) consists of scaffolding (A), catalytic (C), and variable (B, B’, and B”) subunits. Variable subunits dictate subcellular localization and substrate specificity of the PP2A holoenzyme. The Bβ regulatory subunit gene is mutated in spinocerebellar ataxia type 12, and one of its splice variants, Bβ2, targets PP2A to mitochondria to promote apoptosis in PC12 cells. Here, we report that Bβ2 is localized to the outer mitochondrial membrane by a novel mechanism, combining a cryptic mitochondrial import signal with a structural arrest domain. Scanning mutagenesis demonstrates that basic and hydrophobic residues mediate mitochondrial association and the proapoptotic activity of Bβ2. When fused to green fluorescent protein, the N terminus of Bβ2 acts as a cleavable mitochondrial import signal. Surprisingly, full-length Bβ2 is not detectably cleaved and is retained at the outer mitochondrial membrane, even though it interacts with the TOM22 import receptor, as shown by luciferase complementation in intact cells. Mutations that open the C-terminal β-propeller of Bβ2 facilitate mitochondrial import, indicating that this rigid fold acts as a stop-transfer domain by resisting the partial unfolding step prerequisite for matrix translocation. Because hybrids of prototypical import and β-propeller domains recapitulate this behavior, we predict the existence of other similarly localized proteins and a selection against highly stable protein folds in the mitochondrial matrix. This unfolding-resistant targeting to the mitochondrial translocase is necessary but not sufficient for the proapoptotic activity of Bβ2, which also requires association with the rest of the PP2A holoenzyme. PMID:15923182

  18. Functional domain walls in multiferroics.

    PubMed

    Meier, Dennis

    2015-11-25

    During the last decade a wide variety of novel and fascinating correlation phenomena has been discovered at domain walls in multiferroic bulk systems, ranging from unusual electronic conductance to inseparably entangled spin and charge degrees of freedom. The domain walls represent quasi-2D functional objects that can be induced, positioned, and erased on demand, bearing considerable technological potential for future nanoelectronics. Most of the challenges that remain to be solved before turning related device paradigms into reality, however, still fall in the field of fundamental condensed matter physics and materials science. In this topical review seminal experimental findings gained on electric and magnetic domain walls in multiferroic bulk materials are addressed. A special focus is put on the physical properties that emerge at so-called charged domain walls and the added functionality that arises from coexisting magnetic order. The research presented in this review highlights that we are just entering a whole new world of intriguing nanoscale physics that is yet to be explored in all its details. The goal is to draw attention to the persistent challenges and identify future key directions for the research on functional domain walls in multiferroics.

  19. 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.

  20. Variable buoyancy system for unmanned multi-domain vehicles

    NASA Astrophysics Data System (ADS)

    MacLeod, Marc; Bryant, Matthew

    2016-04-01

    This paper presents the system design, construction, and testing of an active variable buoyancy system (VBS) actuator with applications to unmanned multi-domain vehicles. Unmanned multi-domain vehicles require nontraditional VBS designs because of their unique operation requirements. We present a VBS actuator design that targets multi-domain vehicle design objectives of high endurance, stealth, and underwater loitering. The design features a rigid ballast tank with an inner elastic bladder connected to a hydraulic pump and a proportionally controlled vent valve. The system working fluid is obtained from the ambient surrounding water and the elastic bladder separates the water from pressurized gas, thus preventing any gas from escaping during a venting operation. An analytic model of the VBS characterizing the system dynamics is derived. Ballast tank prototype design and construction is discussed. A VBS test platform vehicle is presented, featuring two ballast tanks, motor, pump, and RF receiver for control.

  1. Identification and Analysis of the SET-Domain Family in Silkworm, Bombyx mori.

    PubMed

    Zhao, Hailong; Zheng, Chunqin; Cui, Hongjuan

    2015-01-01

    As an important economic insect, Bombyx mori is also a useful model organism for lepidopteran insect. SET-domain-containing proteins belong to a group of enzymes named after a common domain that utilizes the cofactor S-adenosyl-L-methionine (SAM) to achieve methylation of its substrates. Many SET-domain-containing proteins have been shown to display catalytic activity towards particular lysine residues on histones, but emerging evidence also indicates that various nonhistone proteins are specifically targeted by this clade of enzymes. To explore their diverse functions of SET-domain superfamily in insect, we identified, cloned, and analyzed the SET-domains proteins in silkworm, Bombyx mori. Firstly, 24 genes containing SET domain from silkworm genome were characterized and 17 of them belonged to six subfamilies of SUV39, SET1, SET2, SUV4-20, EZ, and SMYD. Secondly, SET domains of silkworm SET-domain family were intraspecifically and interspecifically conserved, especially for the catalytic core "NHSC" motif, substrate binding site, and catalytic site in the SET domain. Lastly, further analyses indicated that silkworm SET-domain gene BmSu(var)3-9 owned different characterization and expression profiles compared to other invertebrates. Overall, our results provide a new insight into the functional and evolutionary features of SET-domain family.

  2. Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies*

    PubMed Central

    Rouet, Romain; Dudgeon, Kip; Christie, Mary; Langley, David; Christ, Daniel

    2015-01-01

    Human VH single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human VH domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (VHH) domains. Naturally devoid of light chain partners, these domains are characterized by favorable biophysical properties and propensity for cleft binding, a highly desirable characteristic, allowing the targeting of cryptic epitopes. In contrast, previously reported structures of human VH single domains had failed to recapitulate this property. Here we report the engineering and characterization of phage display libraries of stable human VH domains and the selection of binders against a diverse set of antigens. Unlike “camelized” human domains, the domains do not rely on potentially immunogenic framework mutations and maintain the structure of the VH/VL interface. Structure determination in complex with hen egg white lysozyme revealed an extended VH binding interface, with complementarity-determining region 3 deeply penetrating into the active site cleft, highly reminiscent of what has been observed for camelid domains. Taken together, our results demonstrate that fully human VH domains can be constructed that are not only stable and well expressed but also rival the cleft binding properties of camelid antibodies. PMID:25737448

  3. Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies.

    PubMed

    Rouet, Romain; Dudgeon, Kip; Christie, Mary; Langley, David; Christ, Daniel

    2015-05-08

    Human VH single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human VH domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (VHH) domains. Naturally devoid of light chain partners, these domains are characterized by favorable biophysical properties and propensity for cleft binding, a highly desirable characteristic, allowing the targeting of cryptic epitopes. In contrast, previously reported structures of human VH single domains had failed to recapitulate this property. Here we report the engineering and characterization of phage display libraries of stable human VH domains and the selection of binders against a diverse set of antigens. Unlike "camelized" human domains, the domains do not rely on potentially immunogenic framework mutations and maintain the structure of the VH/VL interface. Structure determination in complex with hen egg white lysozyme revealed an extended VH binding interface, with complementarity-determining region 3 deeply penetrating into the active site cleft, highly reminiscent of what has been observed for camelid domains. Taken together, our results demonstrate that fully human VH domains can be constructed that are not only stable and well expressed but also rival the cleft binding properties of camelid antibodies.

  4. Structural basis of diverse membrane target recognitions by ankyrins.

    PubMed

    Wang, Chao; Wei, Zhiyi; Chen, Keyu; Ye, Fei; Yu, Cong; Bennett, Vann; Zhang, Mingjie

    2014-11-10

    Ankyrin adaptors together with their spectrin partners coordinate diverse ion channels and cell adhesion molecules within plasma membrane domains and thereby promote physiological activities including fast signaling in the heart and nervous system. Ankyrins specifically bind to numerous membrane targets through their 24 ankyrin repeats (ANK repeats), although the mechanism for the facile and independent evolution of these interactions has not been resolved. Here we report the structures of ANK repeats in complex with an inhibitory segment from the C-terminal regulatory domain and with a sodium channel Nav1.2 peptide, respectively, showing that the extended, extremely conserved inner groove spanning the entire ANK repeat solenoid contains multiple target binding sites capable of accommodating target proteins with very diverse sequences via combinatorial usage of these sites. These structures establish a framework for understanding the evolution of ankyrins' membrane targets, with implications for other proteins containing extended ANK repeat domains.

  5. Charge density influences C1 domain ligand affinity and membrane interactions

    PubMed Central

    Lewin, Nancy E.; Kedei, Noemi; Hill, Colin S.; Selezneva, Julia S.; Valle, Christopher J.; Woo, Wonhee; Gorshkova, Inna; Blumberg, Peter M.

    2014-01-01

    The C1 domain, which represents the recognition motif on protein kinase C for the lipophilic second messenger diacylglycerol and its ultrapotent analog the phorbol esters, has emerged as a promising therapeutic target for cancer and other indications. Potential target selectivity is markedly enhanced both because binding reflects ternary complex formation between ligand, the C1 domain, and phospholipid, and because binding drives membrane insertion of the C1 domain, permitting aspects of the C1 domain surface outside the binding site per se to influence binding energetics. Here, focusing on charged residues identified in atypical C1 domains which contribute to their loss of ligand binding activity, we show that increasing charge along the rim of the binding cleft of the protein kinase C δ C1b domain raises the requirement for anionic phospholipids. Correspondingly, it shifts the selectivity of C1 domain translocation to the plasma membrane, which is more negatively charged than internal membranes. This change in localization is most pronounced in the case of more hydrophilic ligands, which provide weaker membrane stabilization than do the more hydrophobic ligands, and thus contributes an element to the structure activity relations for C1 domain ligands. Co-expressing pairs of C1 containing constructs with differing charges each expressing a distinct fluorescent tag provided a powerful tool to demonstrate the effect of increasing charge in the C1 domain. PMID:24777910

  6. Faraday instability in deformable domains

    NASA Astrophysics Data System (ADS)

    Pucci, Giuseppe; Ben Amar, Martine; Couder, Yves

    2014-11-01

    We investigate the Faraday instability in floating liquid lenses, as an example of hydrodynamic instability that develops in a domain with flexible boundaries. We show that a mutual adaptation of the instability pattern and the domain shape occurs, as a result of the competition between the wave radiation pressure and the capillary response of the lens border. Two archetypes of behaviour are observed. In the first, stable shapes are obtained experimentally and predicted theoretically as the exact solutions of a Riccati equation, and they result from the equilibrium between wave radiation pressure and capillarity. In the second, the radiation pressure exceeds the capillary response of the lens border and leads to non-equilibrium behaviours, with breaking into smaller domains that have a complex dynamics including spontaneous propagation. The authors are grateful to Université Franco-Italienne (UFI) for financial support.

  7. Proteasomes and protein conjugation across domains of life.

    PubMed

    Maupin-Furlow, Julie

    2011-12-19

    Like other energy-dependent proteases, proteasomes, which are found across the three domains of life, are self-compartmentalized and important in the early steps of proteolysis. Proteasomes degrade improperly synthesized, damaged or misfolded proteins and hydrolyse regulatory proteins that must be specifically removed or cleaved for cell signalling. In eukaryotes, proteins are typically targeted for proteasome-mediated destruction through polyubiquitylation, although ubiquitin-independent pathways also exist. Interestingly, actinobacteria and archaea also covalently attach small proteins (prokaryotic ubiquitin-like protein (Pup) and small archaeal modifier proteins (Samps), respectively) to certain proteins, and this may serve to target the modified proteins for degradation by proteasomes.

  8. Sinh-domain complex integrators

    NASA Astrophysics Data System (ADS)

    Skotis, George-Drosos; Khanday, Farooq A.; Psychalinos, Costas

    2015-07-01

    The basic building blocks for performing complex signal processing in the Sinh-domain are introduced in this article. Attractive offered benefits are the capabilities for achieving resistorless realisations with electronic adjustment of their frequency characteristics, independent tuning of centre frequency and bandwidth and operating in a low-voltage environment. In addition, the inherent class-AB operation of Sinh-domain filters allows the handling of signals greater than the bias current, leading to a power saving. The aforementioned benefits have been evaluated through simulation results, using the Analog Design Environment of the Cadence software.

  9. Application of Celluspots peptide arrays for the analysis of the binding specificity of epigenetic reading domains to modified histone tails

    PubMed Central

    2011-01-01

    Background Epigenetic reading domains are involved in the regulation of gene expression and chromatin state by interacting with histones in a post-translational modification specific manner. A detailed knowledge of the target modifications of reading domains, including enhancing and inhibiting secondary modifications, will lead to a better understanding of the biological signaling processes mediated by reading domains. Results We describe the application of Celluspots peptide arrays which contain 384 histone peptides carrying 59 post translational modifications in different combinations as an inexpensive, reliable and fast method for initial screening for specific interactions of reading domains with modified histone peptides. To validate the method, we tested the binding specificities of seven known epigenetic reading domains on Celluspots peptide arrays, viz. the HP1ß and MPP8 Chromo domains, JMJD2A and 53BP1 Tudor domains, Dnmt3a PWWP domain, Rag2 PHD domain and BRD2 Bromo domain. In general, the binding results agreed with literature data with respect to the primary specificity of the reading domains, but in almost all cases we obtained additional new information concerning the influence of secondary modifications surrounding the target modification. Conclusions We conclude that Celluspots peptide arrays are powerful screening tools for studying the specificity of putative reading domains binding to modified histone peptides. PMID:21884582

  10. Targeted Therapy for Cancer

    Cancer.gov

    Targeted therapy is a type of cancer treatment that targets the changes in cancer cells that help them grow, divide, and spread. Learn how targeted therapy works against cancer and about side effects that may occur.

  11. Compressed domain ECG biometric with two-lead features

    NASA Astrophysics Data System (ADS)

    Lee, Wan-Jou; Chang, Wen-Whei

    2016-07-01

    This study presents a new method to combine ECG biometrics with data compression within a common JPEG2000 framework. We target the two-lead ECG configuration that is routinely used in long-term heart monitoring. Incorporation of compressed-domain biometric techniques enables faster person identification as it by-passes the full decompression. Experiments on public ECG databases demonstrate the validity of the proposed method for biometric identification with high accuracies on both healthy and diseased subjects.

  12. Development and Testing of Recombinant Single Domain Antibodies

    DTIC Science & Technology

    2010-12-31

    critical insights to the basis of the sdAb-target interaction and sdAb properties. (a) Papers published in peer-reviewed journals (N/A for none) Structures...Goldman E.R. 2010 Binding Kinetics of anti-Ricin Single Domain Antibodies and Improved Detection Using a B-chain Specific Binder. Analytical...Library. Anal. Chem. 78:8245-55 List of papers submitted or published that acknowledge ARO support during this reporting period. List the papers

  13. Trimerization of the HIV Transmembrane Domain in Lipid Bilayers Modulates Broadly Neutralizing Antibody Binding.

    PubMed

    Reichart, Timothy M; Baksh, Michael M; Rhee, Jin-Kyu; Fiedler, Jason D; Sligar, Stephen G; Finn, M G; Zwick, Michael B; Dawson, Philip E

    2016-02-18

    The membrane-proximal external region (MPER) of HIV gp41 is an established target of antibodies that neutralize a broad range of HIV isolates. To evaluate the role of the transmembrane (TM) domain, synthetic MPER-derived peptides were incorporated into lipid nanoparticles using natural and designed TM domains, and antibody affinity was measured using immobilized and solution-based techniques. Peptides incorporating the native HIV TM domain exhibit significantly stronger interactions with neutralizing antibodies than peptides with a monomeric TM domain. Furthermore, a peptide with a trimeric, three-helix bundle TM domain recapitulates the binding profile of the native sequence. These studies suggest that neutralizing antibodies can bind the MPER when the TM domain is a three-helix bundle and this presentation could influence the binding of neutralizing antibodies to the virus. Lipid-bilayer presentation of viral antigens in Nanodiscs is a new platform for evaluating neutralizing antibodies.

  14. A Method to Examine Content Domain Structures

    ERIC Educational Resources Information Center

    D'Agostino, Jerome; Karpinski, Aryn; Welsh, Megan

    2011-01-01

    After a test is developed, most content validation analyses shift from ascertaining domain definition to studying domain representation and relevance because the domain is assumed to be set once a test exists. We present an approach that allows for the examination of alternative domain structures based on extant test items. In our example based on…

  15. Cellulose binding domain fusion proteins

    SciTech Connect

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  16. Development in the Food Domain.

    ERIC Educational Resources Information Center

    Rozin, Paul

    1990-01-01

    Discusses problems of general interest in developmental psychology that can be successfully studied in the domain of food; these include (1) development of food likes and dislikes; (2) establishment of the edible/inedible distinction; (3) disgust and contagion; (4) transgenerational communication of preferences; and (5) transition to food…

  17. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, O.; Yosef, K.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1998-02-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  18. Partwise cross-parameterization via nonregular convex hull domains.

    PubMed

    Wu, Huai-Yu; Pan, Chunhong; Zha, Hongbin; Yang, Qing; Ma, Songde

    2011-10-01

    In this paper, we propose a novel partwise framework for cross-parameterization between 3D mesh models. Unlike most existing methods that use regular parameterization domains, our framework uses nonregular approximation domains to build the cross-parameterization. Once the nonregular approximation domains are constructed for 3D models, different (and complex) input shapes are transformed into similar (and simple) shapes, thus facilitating the cross-parameterization process. Specifically, a novel nonregular domain, the convex hull, is adopted to build shape correspondence. We first construct convex hulls for each part of the segmented model, and then adopt our convex-hull cross-parameterization method to generate compatible meshes. Our method exploits properties of the convex hull, e.g., good approximation ability and linear convex representation for interior vertices. After building an initial cross-parameterization via convex-hull domains, we use compatible remeshing algorithms to achieve an accurate approximation of the target geometry and to ensure a complete surface matching. Experimental results show that the compatible meshes constructed are well suited for shape blending and other geometric applications.

  19. Intracellular targeting with engineered proteins

    PubMed Central

    Miersch, Shane; Sidhu, Sachdev S.

    2016-01-01

    If the isolation, production, and clinical use of insulin marked the inception of the age of biologics as therapeutics, the convergence of molecular biology and combinatorial engineering techniques marked its coming of age. The first wave of recombinant protein-based drugs in the 1980s demonstrated emphatically that proteins could be engineered, formulated, and employed for clinical advantage. Yet despite the successes of protein-based drugs such as antibodies, enzymes, and cytokines, the druggable target space for biologics is currently restricted to targets outside the cell. Insofar as estimates place the number of proteins either secreted or with extracellular domains in the range of 8000 to 9000, this represents only one-third of the proteome and circumscribes the pathways that can be targeted for therapeutic intervention. Clearly, a major objective for this field to reach maturity is to access, interrogate, and modulate the majority of proteins found inside the cell. However, owing to the large size, complex architecture, and general cellular impermeability of existing protein-based drugs, this poses a daunting challenge. In recent years, though, advances on the two related fronts of protein engineering and drug delivery are beginning to bring this goal within reach. First, prompted by the restrictions that limit the applicability of antibodies, intense efforts have been applied to identifying and engineering smaller alternative protein scaffolds for the modulation of intracellular targets. In parallel, innovative solutions for delivering proteins to the intracellular space while maintaining their stability and functional activity have begun to yield successes. This review provides an overview of bioactive intrabodies and alternative protein scaffolds amenable to engineering for intracellular targeting and also outlines advances in protein engineering and formulation for delivery of functional proteins to the interior of the cell to achieve therapeutic action

  20. Impact of Domain Analysis on Reuse Methods

    DTIC Science & Technology

    1989-11-06

    libraries with very different domain models . The semantic network knowledge representation system (differing in this respect from object-oriented approaches...2.1.4 Organizational Strategies ...... ............. 10 2.1.5 Role of Existing Systems ..... .............. ..11 2.2 Process Models for Domain Analysis...Acquire Domain Analysis Resources. ... 15 2.2.4 Develop the Domain Model ..... .............. .16 2.2.4.1 Identification of Domain Objects

  1. Small Molecule-Induced Domain Swapping as a Mechanism for Controlling Protein Function and Assembly

    PubMed Central

    Karchin, Joshua M.; Ha, Jeung-Hoi; Namitz, Kevin E.; Cosgrove, Michael S.; Loh, Stewart N.

    2017-01-01

    Domain swapping is the process by which identical proteins exchange reciprocal segments to generate dimers. Here we introduce induced domain swapping (INDOS) as a mechanism for regulating protein function. INDOS employs a modular design consisting of the fusion of two proteins: a recognition protein that binds a triggering molecule, and a target protein that undergoes a domain swap in response to binding of the triggering ligand. The recognition protein (FK506 binding protein) is inserted into functionally-inactivated point mutants of two target proteins (staphylococcal nuclease and ribose binding protein). Binding of FK506 to the FKBP domain causes the target domain to first unfold, then refold via domain swap. The inactivating mutations become ‘swapped out’ in the dimer, increasing nuclease and ribose binding activities by 100-fold and 15-fold, respectively, restoring them to near wild-type values. INDOS is intended to convert an arbitrary protein into a functional switch, and is the first example of rational design in which a small molecule is used to trigger protein domain swapping and subsequent activation of biological function. PMID:28287617

  2. Hybrid H-2 histocompatibility gene products assign domains recognized by alloreactive T cells.

    PubMed Central

    Ozato, K; Evans, G A; Shykind, B; Margulies, D H; Seidman, J G

    1983-01-01

    In vitro recombination of cloned H-2 genes resulted in two allogeneically novel mouse transplantation antigens in which the C2 domains of H-2Ld and H-2Dd were exchanged. These genes were introduced into mouse L cells by DNA-mediated gene transfer. We have used these transformed fibroblast lines, expressing recombinant H-2 antigens at normal levels, as targets for alloreactive cytotoxic T lymphocytes (CTL). Transformed cell lines expressing native or hybrid H-2 molecules that share the N and C1 domains but not the C2 domain were lysed to an equivalent degree by primary and secondary anti-H-2Dd CTL. Hybrid and native H-2 antigens that have the same N and C1 domains were capable of reciprocally blocking specific lysis by the CTL. Thus, polymorphic determinants recognized by alloreactive T cells are located primarily in the N domain, the C1 domain, or both domains of the H-2 antigen. In contrast, determinants recognized by monoclonal antibodies are present throughout the H-2 protein, including the C2 domain. Antibodies that bind specifically to the C2 domain effectively inhibited CTL activity, possibly due to steric effects. The same antibodies do not competitively inhibit the binding of N/C1-specific antibodies. The predominant reactivity of CTL to the N and C1 domains suggests that humoral and cellular responses "see" a different spectrum of alloantigenic determinants. PMID:6188160

  3. The physical size of transcription factors is key to transcriptional regulation in chromatin domains

    NASA Astrophysics Data System (ADS)

    Maeshima, Kazuhiro; Kaizu, Kazunari; Tamura, Sachiko; Nozaki, Tadasu; Kokubo, Tetsuro; Takahashi, Koichi

    2015-02-01

    Genetic information, which is stored in the long strand of genomic DNA as chromatin, must be scanned and read out by various transcription factors. First, gene-specific transcription factors, which are relatively small (˜50 kDa), scan the genome and bind regulatory elements. Such factors then recruit general transcription factors, Mediators, RNA polymerases, nucleosome remodellers, and histone modifiers, most of which are large protein complexes of 1-3 MDa in size. Here, we propose a new model for the functional significance of the size of transcription factors (or complexes) for gene regulation of chromatin domains. Recent findings suggest that chromatin consists of irregularly folded nucleosome fibres (10 nm fibres) and forms numerous condensed domains (e.g., topologically associating domains). Although the flexibility and dynamics of chromatin allow repositioning of genes within the condensed domains, the size exclusion effect of the domain may limit accessibility of DNA sequences by transcription factors. We used Monte Carlo computer simulations to determine the physical size limit of transcription factors that can enter condensed chromatin domains. Small gene-specific transcription factors can penetrate into the chromatin domains and search their target sequences, whereas large transcription complexes cannot enter the domain. Due to this property, once a large complex binds its target site via gene-specific factors it can act as a ‘buoy’ to keep the target region on the surface of the condensed domain and maintain transcriptional competency. This size-dependent specialization of target-scanning and surface-tethering functions could provide novel insight into the mechanisms of various DNA transactions, such as DNA replication and repair/recombination.

  4. Cross-domain question classification in community question answering via kernel mapping

    NASA Astrophysics Data System (ADS)

    Su, Lei; Hu, Zuoliang; Yang, Bin; Li, Yiyang; Chen, Jun

    2015-10-01

    An increasingly popular method for retrieving information is via the community question answering (CQA) systems such as Yahoo! Answers and Baidu Knows. In CQA, question classification plays an important role to find the answers. However, the labeled training examples for statistical question classifier are fairly expensive to obtain, as they require the experienced human efforts. Meanwhile, unlabeled data are readily available. This paper employs the method of domain adaptation via kernel mapping to solve this problem. In detail, the kernel approach is utilized to map the target-domain data and the source-domain data into a common space, where the question classifiers are trained under the closer conditional probabilities. The kernel mapping function is constructed by domain knowledge. Therefore, domain knowledge could be transferred from the labeled examples in the source domain to the unlabeled ones in the targeted domain. The statistical training model can be improved by using a large number of unlabeled data. Meanwhile, the Hadoop Platform is used to construct the mapping mechanism to reduce the time complexity. Map/Reduce enable kernel mapping for domain adaptation in parallel in the Hadoop Platform. Experimental results show that the accuracy of question classification could be improved by the method of kernel mapping. Furthermore, the parallel method in the Hadoop Platform could effective schedule the computing resources to reduce the running time.

  5. Development and Evaluation of Single Domain Antibodies for Vaccinia and the L1 Antigen

    PubMed Central

    Walper, Scott A.; Liu, Jinny L.; Zabetakis, Daniel; Anderson, George P.; Goldman, Ellen R.

    2014-01-01

    There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10−9 M to 7.0×10−10 M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×105 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies. PMID:25211488

  6. SH2 domain proteins as high-affinity receptor tyrosine kinase substrates.

    PubMed

    Sierke, S L; Koland, J G

    1993-09-28

    Activation of a growth factor receptor tyrosine kinase (RTK) is accompanied by a rapid autophosphorylation of the receptor on tyrosine residues. Receptor activation has been shown to promote the association of signal-transducing proteins containing SH2 domains (second domain of src homology). These receptor-associated proteins can, in turn, be phosphorylated by the RTK, an event which presumably regulates their activities. It has been suggested that SH2 domains in signal-transducing proteins target these proteins as substrates of the activated RTK. To test this hypothesis, recombinant proteins were generated that contained tyrosine phosphorylation sites of the erbB3 receptor and/or the SH2 domain of c-src. Incorporation of the SH2 domain led to a decrease in KM and an increase in Vmax for the substrate. The KM determined for one chimeric SH2/erbB3 substrate was among the lowest reported for epidermal growth factor RTK substrates. Experiments with a truncated kinase lacking C-terminal autophosphorylation sites indicated that the reduction in KM for these substrates was mediated by interactions between the substrate SH2 domain and phosphotyrosine residues of the RTK. These interactions could also inhibit RTK activity. These results demonstrate that the SH2 domain can effectively target substrates to a RTK and that SH2 domain proteins can regulate RTK activity.

  7. Polarized internal target apparatus

    DOEpatents

    Holt, R.J.

    1984-10-10

    A polarized internal target apparatus with a polarized gas target of improved polarization and density (achieved by mixing target gas atoms with a small amount of alkali metal gas atoms, an