Sample records for targeting peripheral proteins

  1. Peripheral Protein Unfolding Drives Membrane Bending.

    PubMed

    Siaw, Hew Ming Helen; Raghunath, Gokul; Dyer, R Brian

    2018-06-20

    Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hy-drophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding-unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA-DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding is directly responsible for membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.

  2. BDNF gene delivery mediated by neuron-targeted nanoparticles is neuroprotective in peripheral nerve injury.

    PubMed

    Lopes, Cátia D F; Gonçalves, Nádia P; Gomes, Carla P; Saraiva, Maria J; Pêgo, Ana P

    2017-03-01

    Neuron-targeted gene delivery is a promising strategy to treat peripheral neuropathies. Here we propose the use of polymeric nanoparticles based on thiolated trimethyl chitosan (TMCSH) to mediate targeted gene delivery to peripheral neurons upon a peripheral and minimally invasive intramuscular administration. Nanoparticles were grafted with the non-toxic carboxylic fragment of the tetanus neurotoxin (HC) to allow neuron targeting and were explored to deliver a plasmid DNA encoding for the brain-derived neurotrophic factor (BDNF) in a peripheral nerve injury model. The TMCSH-HC/BDNF nanoparticle treatment promoted the release and significant expression of BDNF in neural tissues, which resulted in an enhanced functional recovery after injury as compared to control treatments (vehicle and non-targeted nanoparticles), associated with an improvement in key pro-regenerative events, namely, the increased expression of neurofilament and growth-associated protein GAP-43 in the injured nerves. Moreover, the targeted nanoparticle treatment was correlated with a significantly higher density of myelinated axons in the distal stump of injured nerves, as well as with preservation of unmyelinated axon density as compared with controls and a protective role in injury-denervated muscles, preventing them from denervation. These results highlight the potential of TMCSH-HC nanoparticles as non-viral gene carriers to deliver therapeutic genes into the peripheral neurons and thus, pave the way for their use as an effective therapeutic intervention for peripheral neuropathies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. In vivo targeted peripheral nerve imaging with a nerve-specific nanoscale magnetic resonance probe.

    PubMed

    Zheng, Linfeng; Li, Kangan; Han, Yuedong; Wei, Wei; Zheng, Sujuan; Zhang, Guixiang

    2014-11-01

    Neuroimaging plays a pivotal role in clinical practice. Currently, computed tomography (CT), magnetic resonance imaging (MRI), ultrasonography, and positron emission tomography (PET) are applied in the clinical setting as neuroimaging modalities. There is no optimal imaging modality for clinical peripheral nerve imaging even though fluorescence/bioluminescence imaging has been used for preclinical studies on the nervous system. Some studies have shown that molecular and cellular MRI (MCMRI) can be used to visualize and image the cellular and molecular level of the nervous system. Other studies revealed that there are different pathological/molecular changes in the proximal and distal sites after peripheral nerve injury (PNI). Therefore, we hypothesized that in vivo peripheral nerve targets can be imaged using MCMRI with specific MRI probes. Specific probes should have higher penetrability for the blood-nerve barrier (BNB) in vivo. Here, a functional nanometre MRI probe that is based on nerve-specific proteins as targets, specifically, using a molecular antibody (mAb) fragment conjugated to iron nanoparticles as an MRI probe, was constructed for further study. The MRI probe allows for imaging the peripheral nerve targets in vivo. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Modeling peripheral vision for moving target search and detection.

    PubMed

    Yang, Ji Hyun; Huston, Jesse; Day, Michael; Balogh, Imre

    2012-06-01

    Most target search and detection models focus on foveal vision. In reality, peripheral vision plays a significant role, especially in detecting moving objects. There were 23 subjects who participated in experiments simulating target detection tasks in urban and rural environments while their gaze parameters were tracked. Button responses associated with foveal object and peripheral object (PO) detection and recognition were recorded. In an urban scenario, pedestrians appearing in the periphery holding guns were threats and pedestrians with empty hands were non-threats. In a rural scenario, non-U.S. unmanned aerial vehicles (UAVs) were considered threats and U.S. UAVs non-threats. On average, subjects missed detecting 2.48 POs among 50 POs in the urban scenario and 5.39 POs in the rural scenario. Both saccade reaction time and button reaction time can be predicted by peripheral angle and entrance speed of POs. Fast moving objects were detected faster than slower objects and POs appearing at wider angles took longer to detect than those closer to the gaze center. A second-order mixed-effect model was applied to provide each subject's prediction model for peripheral target detection performance as a function of eccentricity angle and speed. About half the subjects used active search patterns while the other half used passive search patterns. An interactive 3-D visualization tool was developed to provide a representation of macro-scale head and gaze movement in the search and target detection task. An experimentally validated stochastic model of peripheral vision in realistic target detection scenarios was developed.

  5. The interactions of peripheral membrane proteins with biological membranes

    DOE PAGES

    Johs, Alexander; Whited, A. M.

    2015-07-29

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

  6. Activation of the unfolded protein response promotes axonal regeneration after peripheral nerve injury.

    PubMed

    Oñate, Maritza; Catenaccio, Alejandra; Martínez, Gabriela; Armentano, Donna; Parsons, Geoffrey; Kerr, Bredford; Hetz, Claudio; Court, Felipe A

    2016-02-24

    Although protein-folding stress at the endoplasmic reticulum (ER) is emerging as a driver of neuronal dysfunction in models of spinal cord injury and neurodegeneration, the contribution of this pathway to peripheral nerve damage remains poorly explored. Here we targeted the unfolded protein response (UPR), an adaptive reaction against ER stress, in mouse models of sciatic nerve injury and found that ablation of the transcription factor XBP1, but not ATF4, significantly delay locomotor recovery. XBP1 deficiency led to decreased macrophage recruitment, a reduction in myelin removal and axonal regeneration. Conversely, overexpression of XBP1s in the nervous system in transgenic mice enhanced locomotor recovery after sciatic nerve crush, associated to an improvement in key pro-regenerative events. To assess the therapeutic potential of UPR manipulation to axonal regeneration, we locally delivered XBP1s or an shRNA targeting this transcription factor to sensory neurons of the dorsal root ganglia using a gene therapy approach and found an enhancement or reduction of axonal regeneration in vivo, respectively. Our results demonstrate a functional role of specific components of the ER proteostasis network in the cellular changes associated to regeneration and functional recovery after peripheral nerve injury.

  7. Activation of the unfolded protein response promotes axonal regeneration after peripheral nerve injury

    PubMed Central

    Oñate, Maritza; Catenaccio, Alejandra; Martínez, Gabriela; Armentano, Donna; Parsons, Geoffrey; Kerr, Bredford; Hetz, Claudio; Court, Felipe A.

    2016-01-01

    Although protein-folding stress at the endoplasmic reticulum (ER) is emerging as a driver of neuronal dysfunction in models of spinal cord injury and neurodegeneration, the contribution of this pathway to peripheral nerve damage remains poorly explored. Here we targeted the unfolded protein response (UPR), an adaptive reaction against ER stress, in mouse models of sciatic nerve injury and found that ablation of the transcription factor XBP1, but not ATF4, significantly delay locomotor recovery. XBP1 deficiency led to decreased macrophage recruitment, a reduction in myelin removal and axonal regeneration. Conversely, overexpression of XBP1s in the nervous system in transgenic mice enhanced locomotor recovery after sciatic nerve crush, associated to an improvement in key pro-regenerative events. To assess the therapeutic potential of UPR manipulation to axonal regeneration, we locally delivered XBP1s or an shRNA targeting this transcription factor to sensory neurons of the dorsal root ganglia using a gene therapy approach and found an enhancement or reduction of axonal regeneration in vivo, respectively. Our results demonstrate a functional role of specific components of the ER proteostasis network in the cellular changes associated to regeneration and functional recovery after peripheral nerve injury. PMID:26906090

  8. A Novel Missense Mutation in Peripheral Myelin Protein-22 Causes Charcot-Marie-Tooth Disease.

    PubMed

    Li, Li-Xi; Dong, Hai-Lin; Xiao, Bao-Guo; Wu, Zhi-Ying

    2017-08-05

    Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. A great number of causative genes have been described in CMT, and among them, the heterozygous duplication of peripheral myelin protein-22 (PMP22) is the major cause. Although the missense mutation in PMP22 is rarely reported, it has been demonstrated to be associated with CMT. This study described a novel missense mutation of PMP22 in a Chinese family with CMT phenotype. Targeted next-generation sequencing (NGS) was used to screen the causative genes in a family featured with an autosomal dominant demyelinating form of CMT. The potential variants identified by targeted NGS were verified by Sanger sequencing and classified according to the American College of Medical Genetics and Genomics standards and guidelines. Further cell transfection studies were performed to characterize the function of the novel variant. Using targeted NGS, a novel heterozygous missense variant in PMP22 (c.320G>A, p.G107D) was identified. In vitro cell functional studies revealed that mutant PMP22 protein carrying p.G107D mutation lost the ability to reach the plasma membrane, was mainly retained in the endoplasmic reticulum, and induced cell apoptosis. This study supported the notion that missense mutations in PMP22 give rise to a CMT phenotype, possibly through a toxic gain-of-function mechanism.

  9. Small cationic antimicrobial peptides delocalize peripheral membrane proteins

    PubMed Central

    Wenzel, Michaela; Chiriac, Alina Iulia; Otto, Andreas; Zweytick, Dagmar; May, Caroline; Schumacher, Catherine; Gust, Ronald; Albada, H. Bauke; Penkova, Maya; Krämer, Ute; Erdmann, Ralf; Metzler-Nolte, Nils; Straus, Suzana K.; Bremer, Erhard; Becher, Dörte; Brötz-Oesterhelt, Heike; Sahl, Hans-Georg; Bandow, Julia Elisabeth

    2014-01-01

    Short antimicrobial peptides rich in arginine (R) and tryptophan (W) interact with membranes. To learn how this interaction leads to bacterial death, we characterized the effects of the minimal pharmacophore RWRWRW-NH2. A ruthenium-substituted derivative of this peptide localized to the membrane in vivo, and the peptide also integrated readily into mixed phospholipid bilayers that resemble Gram-positive membranes. Proteome and Western blot analyses showed that integration of the peptide caused delocalization of peripheral membrane proteins essential for respiration and cell-wall biosynthesis, limiting cellular energy and undermining cell-wall integrity. This delocalization phenomenon also was observed with the cyclic peptide gramicidin S, indicating the generality of the mechanism. Exogenous glutamate increases tolerance to the peptide, indicating that osmotic destabilization also contributes to antibacterial efficacy. Bacillus subtilis responds to peptide stress by releasing osmoprotective amino acids, in part via mechanosensitive channels. This response is triggered by membrane-targeting bacteriolytic peptides of different structural classes as well as by hypoosmotic conditions. PMID:24706874

  10. The Escherichia coli Peripheral Inner Membrane Proteome*

    PubMed Central

    Papanastasiou, Malvina; Orfanoudaki, Georgia; Koukaki, Marina; Kountourakis, Nikos; Sardis, Marios Frantzeskos; Aivaliotis, Michalis; Karamanou, Spyridoula; Economou, Anastassios

    2013-01-01

    Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with

  11. The p38α mitogen-activated protein kinase as a central nervous system drug discovery target

    PubMed Central

    Borders, Aaron S; de Almeida, Lucia; Van Eldik, Linda J; Watterson, D Martin

    2008-01-01

    Protein kinases are critical modulators of a variety of cellular signal transduction pathways, and abnormal phosphorylation events can be a cause or contributor to disease progression in a variety of disorders. This has led to the emergence of protein kinases as an important new class of drug targets for small molecule therapeutics. A serine/threonine protein kinase, p38α mitogen-activated protein kinase (MAPK), is an established therapeutic target for peripheral inflammatory disorders because of its critical role in regulation of proinflammatory cytokine production. There is increasing evidence that p38α MAPK is also an important regulator of proinflammatory cytokine levels in the central nervous system, raising the possibility that the kinase may be a drug discovery target for central nervous system disorders where cytokine overproduction contributes to disease progression. Development of bioavailable, central nervous system-penetrant p38α MAPK inhibitors provides the required foundation for drug discovery campaigns targeting p38α MAPK in neurodegenerative disorders. PMID:19090985

  12. The p38alpha mitogen-activated protein kinase as a central nervous system drug discovery target.

    PubMed

    Borders, Aaron S; de Almeida, Lucia; Van Eldik, Linda J; Watterson, D Martin

    2008-12-03

    Protein kinases are critical modulators of a variety of cellular signal transduction pathways, and abnormal phosphorylation events can be a cause or contributor to disease progression in a variety of disorders. This has led to the emergence of protein kinases as an important new class of drug targets for small molecule therapeutics. A serine/threonine protein kinase, p38alpha mitogen-activated protein kinase (MAPK), is an established therapeutic target for peripheral inflammatory disorders because of its critical role in regulation of proinflammatory cytokine production. There is increasing evidence that p38alpha MAPK is also an important regulator of proinflammatory cytokine levels in the central nervous system, raising the possibility that the kinase may be a drug discovery target for central nervous system disorders where cytokine overproduction contributes to disease progression. Development of bioavailable, central nervous system-penetrant p38alpha MAPK inhibitors provides the required foundation for drug discovery campaigns targeting p38alpha MAPK in neurodegenerative disorders.

  13. Drug Target Protein-Protein Interaction Networks: A Systematic Perspective

    PubMed Central

    2017-01-01

    The identification and validation of drug targets are crucial in biomedical research and many studies have been conducted on analyzing drug target features for getting a better understanding on principles of their mechanisms. But most of them are based on either strong biological hypotheses or the chemical and physical properties of those targets separately. In this paper, we investigated three main ways to understand the functional biomolecules based on the topological features of drug targets. There are no significant differences between targets and common proteins in the protein-protein interactions network, indicating the drug targets are neither hub proteins which are dominant nor the bridge proteins. According to some special topological structures of the drug targets, there are significant differences between known targets and other proteins. Furthermore, the drug targets mainly belong to three typical communities based on their modularity. These topological features are helpful to understand how the drug targets work in the PPI network. Particularly, it is an alternative way to predict potential targets or extract nontargets to test a new drug target efficiently and economically. By this way, a drug target's homologue set containing 102 potential target proteins is predicted in the paper. PMID:28691014

  14. Targeting Peripheral-Derived Regulatory T Cells as a Means of Enhancing Immune Responses Directed against Prostate Cancer

    DTIC Science & Technology

    2017-08-01

    Award Number: W81XWH-15-1-0328 TITLE: Targeting Peripheral-Derived Regulatory T Cells as a Means of Enhancing Immune Responses Directed against...1 August 2016 - 31 July 2017 4. TITLE AND SUBTITLE Targeting Peripheral-Derived Regulatory T Cells as a Means of Enhancing Immune Responses Directed...discovered that a subset of regulatory T cells (Tregs), termed peripheral-derived Tregs (pTregs), impair immune responses directed against tumor

  15. Systematically Ranking the Tightness of Membrane Association for Peripheral Membrane Proteins (PMPs)*

    PubMed Central

    Gao, Liyan; Ge, Haitao; Huang, Xiahe; Liu, Kehui; Zhang, Yuanya; Xu, Wu; Wang, Yingchun

    2015-01-01

    Large-scale quantitative evaluation of the tightness of membrane association for nontransmembrane proteins is important for identifying true peripheral membrane proteins with functional significance. Herein, we simultaneously ranked more than 1000 proteins of the photosynthetic model organism Synechocystis sp. PCC 6803 for their relative tightness of membrane association using a proteomic approach. Using multiple precisely ranked and experimentally verified peripheral subunits of photosynthetic protein complexes as the landmarks, we found that proteins involved in two-component signal transduction systems and transporters are overall tightly associated with the membranes, whereas the associations of ribosomal proteins are much weaker. Moreover, we found that hypothetical proteins containing the same domains generally have similar tightness. This work provided a global view of the structural organization of the membrane proteome with respect to divergent functions, and built the foundation for future investigation of the dynamic membrane proteome reorganization in response to different environmental or internal stimuli. PMID:25505158

  16. Ultrastructural identification of peripheral myelin proteins by a pre-embedding immunogold labeling method.

    PubMed

    Canron, Marie-Hélène; Bouillot, Sandrine; Favereaux, Alexandre; Petry, Klaus G; Vital, Anne

    2003-03-01

    Ultrastructural immunolabeling of peripheral nervous system components is an important tool to study the relation between structure and function. Owing to the scarcity of certain antigens and the dense structure of the peripheral nerve, a pre-embedding technique is likely appropriate. After several investigations on procedures for pre-embedding immunolabeling, we propose a method that offers a good compromise between detection of antigenic sites and preservation of morphology at the ultrastructural level, and that is easy to use and suitable for investigations on peripheral nerve biopsies from humans. Pre-fixation by immersion in paraformaldehyde/glutaraldehyde is necessary to stabilize the ultrastructure. Then, ultrasmall gold particles with silver enhancement are advised. Antibodies against myelin protein zero and myelin basic protein were chosen for demonstration. The same technique was applied to localize a 35 kDa myelin protein.

  17. In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles

    PubMed Central

    Lopes, Cátia DF; Oliveira, Hugo; Estevão, Inês; Pires, Liliana Raquel; Pêgo, Ana Paula

    2016-01-01

    A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine)-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC), which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral neuropathies. PMID:27354797

  18. Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Smith, Elizabeth Myhra

    The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

  19. Lateral Diffusion of Peripheral Membrane Proteins on Supported Lipid Bilayers Is Controlled by the Additive Frictional Drags of 1) Bound Lipids and 2) Protein Domains Penetrating into the Bilayer Hydrocarbon Core

    PubMed Central

    Ziemba, Brian P.; Falke, Joseph J.

    2013-01-01

    Peripheral membrane proteins bound to lipids on bilayer surfaces play central roles in a wide array of cellular processes, including many signaling pathways. These proteins diffuse in the plane of the bilayer and often undergo complex reactions involving the binding of regulatory and substrate lipids and proteins they encounter during their 2-D diffusion. Some peripheral proteins, for example pleckstrin homology (PH) domains, dock to the bilayer in a relatively shallow position with little penetration into the bilayer. Other peripheral proteins exhibit more complex bilayer contacts, for example classical protein kinase C isoforms (PKCs) bind as many as six lipids in stepwise fashion, resulting in the penetration of three PKC domains (C1A, C1B, C2) into the bilayer headgroup and hydrocarbon regions. A molecular understanding of the molecular features that control the diffusion speeds of proteins bound to supported bilayers would enable key molecular information to be extracted from experimental diffusion constants, revealing protein-lipid and protein-bilayer interactions difficult to study by other methods. The present study investigates a range of 11 different peripheral protein constructs comprised by 1 to 3 distinct domains (PH, C1A, C1B, C2, anti-lipid antibody). By combining these constructs with various combinations of target lipids, the study measures 2-D diffusion constants on supported bilayers for 17 different protein-lipid complexes. The resulting experimental diffusion constants, together with the known membrane interaction parameters of each complex, are used to analyze the molecular features correlated with diffusional slowing and bilayer friction. The findings show that both 1) individual bound lipids and 2) individual protein domains that penetrate into the hydrocarbon core make additive contributions to the friction against the bilayer, thereby defining the 2-D diffusion constant. An empirical formula is developed that accurately estimates the

  20. Lateral diffusion of peripheral membrane proteins on supported lipid bilayers is controlled by the additive frictional drags of (1) bound lipids and (2) protein domains penetrating into the bilayer hydrocarbon core.

    PubMed

    Ziemba, Brian P; Falke, Joseph J

    2013-01-01

    Peripheral membrane proteins bound to lipids on bilayer surfaces play central roles in a wide array of cellular processes, including many signaling pathways. These proteins diffuse in the plane of the bilayer and often undergo complex reactions involving the binding of regulatory and substrate lipids and proteins they encounter during their 2D diffusion. Some peripheral proteins, for example pleckstrin homology (PH) domains, dock to the bilayer in a relatively shallow position with little penetration into the bilayer. Other peripheral proteins exhibit more complex bilayer contacts, for example classical protein kinase C isoforms (PKCs) bind as many as six lipids in stepwise fashion, resulting in the penetration of three PKC domains (C1A, C1B, C2) into the bilayer headgroup and hydrocarbon regions. A molecular understanding of the molecular features that control the diffusion speeds of proteins bound to supported bilayers would enable key molecular information to be extracted from experimental diffusion constants, revealing protein-lipid and protein-bilayer interactions difficult to study by other methods. The present study investigates a range of 11 different peripheral protein constructs comprised by 1-3 distinct domains (PH, C1A, C1B, C2, anti-lipid antibody). By combining these constructs with various combinations of target lipids, the study measures 2D diffusion constants on supported bilayers for 17 different protein-lipid complexes. The resulting experimental diffusion constants, together with the known membrane interaction parameters of each complex, are used to analyze the molecular features correlated with diffusional slowing and bilayer friction. The findings show that both (1) individual bound lipids and (2) individual protein domains that penetrate into the hydrocarbon core make additive contributions to the friction against the bilayer, thereby defining the 2D diffusion constant. An empirical formula is developed that accurately estimates the diffusion

  1. α/β-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells

    PubMed Central

    Checco, James W.; Lee, Erinna F.; Evangelista, Marco; Sleebs, Nerida J.; Rogers, Kelly; Pettikiriarachchi, Anne; Kershaw, Nadia J.; Eddinger, Geoffrey A.; Belair, David G.; Wilson, Julia L.; Eller, Chelcie H.; Raines, Ronald T.; Murphy, William L.; Smith, Brian J.; Gellman, Samuel H.; Fairlie, W. Douglas

    2015-01-01

    Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of L-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues (“α/β-peptides”) manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous “α-peptides”. This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a “stapled” Bim BH3 α-peptide, which contains a hydrocarbon crosslink to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain crosslinking to produce synergistic benefits. PMID:26317395

  2. α/β-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells.

    PubMed

    Checco, James W; Lee, Erinna F; Evangelista, Marco; Sleebs, Nerida J; Rogers, Kelly; Pettikiriarachchi, Anne; Kershaw, Nadia J; Eddinger, Geoffrey A; Belair, David G; Wilson, Julia L; Eller, Chelcie H; Raines, Ronald T; Murphy, William L; Smith, Brian J; Gellman, Samuel H; Fairlie, W Douglas

    2015-09-09

    Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues ("α/β-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "α-peptides". This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a "stapled" Bim BH3 α-peptide, which contains a hydrocarbon cross-link to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.

  3. Properties of Protein Drug Target Classes

    PubMed Central

    Bull, Simon C.; Doig, Andrew J.

    2015-01-01

    Accurate identification of drug targets is a crucial part of any drug development program. We mined the human proteome to discover properties of proteins that may be important in determining their suitability for pharmaceutical modulation. Data was gathered concerning each protein’s sequence, post-translational modifications, secondary structure, germline variants, expression profile and drug target status. The data was then analysed to determine features for which the target and non-target proteins had significantly different values. This analysis was repeated for subsets of the proteome consisting of all G-protein coupled receptors, ion channels, kinases and proteases, as well as proteins that are implicated in cancer. Machine learning was used to quantify the proteins in each dataset in terms of their potential to serve as a drug target. This was accomplished by first inducing a random forest that could distinguish between its targets and non-targets, and then using the random forest to quantify the drug target likeness of the non-targets. The properties that can best differentiate targets from non-targets were primarily those that are directly related to a protein’s sequence (e.g. secondary structure). Germline variants, expression levels and interactions between proteins had minimal discriminative power. Overall, the best indicators of drug target likeness were found to be the proteins’ hydrophobicities, in vivo half-lives, propensity for being membrane bound and the fraction of non-polar amino acids in their sequences. In terms of predicting potential targets, datasets of proteases, ion channels and cancer proteins were able to induce random forests that were highly capable of distinguishing between targets and non-targets. The non-target proteins predicted to be targets by these random forests comprise the set of the most suitable potential future drug targets, and should therefore be prioritised when building a drug development programme. PMID

  4. Generation of wavy structure on lipid membrane by peripheral proteins: a linear elastic analysis.

    PubMed

    Mahata, Paritosh; Das, Sovan Lal

    2017-05-01

    We carry out a linear elastic analysis to study wavy structure generation on lipid membrane by peripheral membrane proteins. We model the lipid membrane as linearly elastic and anisotropic material. The hydrophobic insertion by proteins into the lipid membrane has been idealized as penetration of rigid rod-like inclusions into the membrane and the electrostatic interaction between protein and membrane has been modeled by a distributed surface traction acting on the membrane surface. With the proposed model we study curvature generation by several binding domains of peripheral membrane proteins containing BAR domains and amphipathic alpha-helices. It is observed that electrostatic interaction is essential for curvature generation by the BAR domains. © 2017 Federation of European Biochemical Societies.

  5. Central and Peripheral Molecular Targets for Anti-Obesity Pharmacotherapy

    PubMed Central

    Valentino, Michael A.; Lin, Jieru E.; Waldman, Scott A.

    2011-01-01

    Obesity has emerged as one of the principle worldwide health concerns of the modern era, and there exists a tremendous unmet clinical need for safe and effective therapies to combat this global pandemic. The prevalence of obesity and its associated co-morbidities, including cardiovascular and metabolic diseases, has focused drug discovery and development on generating effective modalities for the treatment and prevention of obesity. Early efforts in the field of obesity pharmacotherapy centered on agents with indeterminate mechanisms of action producing treatment paradigms characterized by significant off-target effects. During the past two decades, new insights have been made into the physiologic regulation of energy balance and the subordinate central and peripheral circuits coordinating appetite, metabolism, and lipogenesis. These studies have revealed previously unrecognized molecular targets for controlling appetite and managing weight from which has emerged a new wave of targeted pharmacotherapies to prevent and control obesity. PMID:20445536

  6. Targeted protein degradation by PROTACs.

    PubMed

    Neklesa, Taavi K; Winkler, James D; Crews, Craig M

    2017-06-01

    Targeted protein degradation using the PROTAC technology is emerging as a novel therapeutic method to address diseases driven by the aberrant expression of a disease-causing protein. PROTAC molecules are bifunctional small molecules that simultaneously bind a target protein and an E3-ubiquitin ligase, thus causing ubiquitination and degradation of the target protein by the proteasome. Like small molecules, PROTAC molecules possess good tissue distribution and the ability to target intracellular proteins. Herein, we highlight the advantages of protein degradation using PROTACs, and provide specific examples where degradation offers therapeutic benefit over classical enzyme inhibition. Foremost, PROTACs can degrade proteins regardless of their function. This includes the currently "undruggable" proteome, which comprises approximately 85% of all human proteins. Other beneficial aspects of protein degradation include the ability to target overexpressed and mutated proteins, as well as the potential to demonstrate prolonged pharmacodynamics effect beyond drug exposure. Lastly, due to their catalytic nature and the pre-requisite ubiquitination step, an exquisitely potent molecules with a high degree of degradation selectivity can be designed. Impressive preclinical in vitro and in vivo PROTAC data have been published, and these data have propelled the development of clinically viable PROTACs. With the molecular weight falling in the 700-1000Da range, the delivery and bioavailability of PROTACs remain the largest hurdles on the way to the clinic. Solving these issues and demonstrating proof of concept clinical data will be the focus of many labs over the next few years. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Hot-spot analysis for drug discovery targeting protein-protein interactions.

    PubMed

    Rosell, Mireia; Fernández-Recio, Juan

    2018-04-01

    Protein-protein interactions are important for biological processes and pathological situations, and are attractive targets for drug discovery. However, rational drug design targeting protein-protein interactions is still highly challenging. Hot-spot residues are seen as the best option to target such interactions, but their identification requires detailed structural and energetic characterization, which is only available for a tiny fraction of protein interactions. Areas covered: In this review, the authors cover a variety of computational methods that have been reported for the energetic analysis of protein-protein interfaces in search of hot-spots, and the structural modeling of protein-protein complexes by docking. This can help to rationalize the discovery of small-molecule inhibitors of protein-protein interfaces of therapeutic interest. Computational analysis and docking can help to locate the interface, molecular dynamics can be used to find suitable cavities, and hot-spot predictions can focus the search for inhibitors of protein-protein interactions. Expert opinion: A major difficulty for applying rational drug design methods to protein-protein interactions is that in the majority of cases the complex structure is not available. Fortunately, computational docking can complement experimental data. An interesting aspect to explore in the future is the integration of these strategies for targeting PPIs with large-scale mutational analysis.

  8. Brain amyloidosis ascertainment from cognitive, imaging, and peripheral blood protein measures

    PubMed Central

    Hwang, Kristy S.; Avila, David; Elashoff, David; Kohannim, Omid; Teng, Edmond; Sokolow, Sophie; Jack, Clifford R.; Jagust, William J.; Shaw, Leslie; Trojanowski, John Q.; Weiner, Michael W.; Thompson, Paul M.

    2015-01-01

    Background: The goal of this study was to identify a clinical biomarker signature of brain amyloidosis in the Alzheimer's Disease Neuroimaging Initiative 1 (ADNI1) mild cognitive impairment (MCI) cohort. Methods: We developed a multimodal biomarker classifier for predicting brain amyloidosis using cognitive, imaging, and peripheral blood protein ADNI1 MCI data. We used CSF β-amyloid 1–42 (Aβ42) ≤192 pg/mL as proxy measure for Pittsburgh compound B (PiB)-PET standard uptake value ratio ≥1.5. We trained our classifier in the subcohort with CSF Aβ42 but no PiB-PET data and tested its performance in the subcohort with PiB-PET but no CSF Aβ42 data. We also examined the utility of our biomarker signature for predicting disease progression from MCI to Alzheimer dementia. Results: The CSF training classifier selected Mini-Mental State Examination, Trails B, Auditory Verbal Learning Test delayed recall, education, APOE genotype, interleukin 6 receptor, clusterin, and ApoE protein, and achieved leave-one-out accuracy of 85% (area under the curve [AUC] = 0.8). The PiB testing classifier achieved an AUC of 0.72, and when classifier self-tuning was allowed, AUC = 0.74. The 36-month disease-progression classifier achieved AUC = 0.75 and accuracy = 71%. Conclusions: Automated classifiers based on cognitive and peripheral blood protein variables can identify the presence of brain amyloidosis with a modest level of accuracy. Such methods could have implications for clinical trial design and enrollment in the near future. Classification of evidence: This study provides Class II evidence that a classification algorithm based on cognitive, imaging, and peripheral blood protein measures identifies patients with brain amyloid on PiB-PET with moderate accuracy (sensitivity 68%, specificity 78%). PMID:25609767

  9. Brain amyloidosis ascertainment from cognitive, imaging, and peripheral blood protein measures.

    PubMed

    Apostolova, Liana G; Hwang, Kristy S; Avila, David; Elashoff, David; Kohannim, Omid; Teng, Edmond; Sokolow, Sophie; Jack, Clifford R; Jagust, William J; Shaw, Leslie; Trojanowski, John Q; Weiner, Michael W; Thompson, Paul M

    2015-02-17

    The goal of this study was to identify a clinical biomarker signature of brain amyloidosis in the Alzheimer's Disease Neuroimaging Initiative 1 (ADNI1) mild cognitive impairment (MCI) cohort. We developed a multimodal biomarker classifier for predicting brain amyloidosis using cognitive, imaging, and peripheral blood protein ADNI1 MCI data. We used CSF β-amyloid 1-42 (Aβ42) ≤ 192 pg/mL as proxy measure for Pittsburgh compound B (PiB)-PET standard uptake value ratio ≥ 1.5. We trained our classifier in the subcohort with CSF Aβ42 but no PiB-PET data and tested its performance in the subcohort with PiB-PET but no CSF Aβ42 data. We also examined the utility of our biomarker signature for predicting disease progression from MCI to Alzheimer dementia. The CSF training classifier selected Mini-Mental State Examination, Trails B, Auditory Verbal Learning Test delayed recall, education, APOE genotype, interleukin 6 receptor, clusterin, and ApoE protein, and achieved leave-one-out accuracy of 85% (area under the curve [AUC] = 0.8). The PiB testing classifier achieved an AUC of 0.72, and when classifier self-tuning was allowed, AUC = 0.74. The 36-month disease-progression classifier achieved AUC = 0.75 and accuracy = 71%. Automated classifiers based on cognitive and peripheral blood protein variables can identify the presence of brain amyloidosis with a modest level of accuracy. Such methods could have implications for clinical trial design and enrollment in the near future. This study provides Class II evidence that a classification algorithm based on cognitive, imaging, and peripheral blood protein measures identifies patients with brain amyloid on PiB-PET with moderate accuracy (sensitivity 68%, specificity 78%). © 2015 American Academy of Neurology.

  10. Inhibitory G proteins and their receptors: emerging therapeutic targets for obesity and diabetes

    PubMed Central

    Kimple, Michelle E; Neuman, Joshua C; Linnemann, Amelia K; Casey, Patrick J

    2014-01-01

    The worldwide prevalence of obesity is steadily increasing, nearly doubling between 1980 and 2008. Obesity is often associated with insulin resistance, a major risk factor for type 2 diabetes mellitus (T2DM): a costly chronic disease and serious public health problem. The underlying cause of T2DM is a failure of the beta cells of the pancreas to continue to produce enough insulin to counteract insulin resistance. Most current T2DM therapeutics do not prevent continued loss of insulin secretion capacity, and those that do have the potential to preserve beta cell mass and function are not effective in all patients. Therefore, developing new methods for preventing and treating obesity and T2DM is very timely and of great significance. There is now considerable literature demonstrating a link between inhibitory guanine nucleotide-binding protein (G protein) and G protein-coupled receptor (GPCR) signaling in insulin-responsive tissues and the pathogenesis of obesity and T2DM. These studies are suggesting new and emerging therapeutic targets for these conditions. In this review, we will discuss inhibitory G proteins and GPCRs that have primary actions in the beta cell and other peripheral sites as therapeutic targets for obesity and T2DM, improving satiety, insulin resistance and/or beta cell biology. PMID:24946790

  11. Protein malnutrition after weaning disrupts peripheral clock and daily insulin secretion in mice.

    PubMed

    Borck, Patricia Cristine; Batista, Thiago Martins; Vettorazzi, Jean Franciesco; Camargo, Rafael Ludemann; Boschero, Antonio Carlos; Vieira, Elaine; Carneiro, Everardo Magalhães

    2017-12-01

    Changes in nutritional state may alter circadian rhythms through alterations in expression of clock genes. Protein deficiency has a profound effect on body metabolism, but the effect of this nutrient restriction after weaning on biological clock has not been explored. Thus, this study aims to investigate whether the protein restriction affects the daily oscillation in the behavior and metabolic rhythms, as well as expression of clock genes in peripheral tissues. Male C57BL/6 J mice, after weaning, were fed a normal-protein (NP) diet or a low-protein (LP) diet for 8 weeks. Mice fed an LP diet did not show difference in locomotor activity and energy expenditure, but the food intake was increased, with parallel increased expression of the orexigenic neuropeptide Npy and disruption of the anorexigenic Pomc oscillatory pattern in the hypothalamus. LP mice showed disruption in the daily rhythmic patterns of plasma glucose, triglycerides and insulin. Also, the rhythmic expression of clock genes in peripheral tissues and pancreatic islets was altered in LP mice. In pancreatic islets, the disruption of clock genes was followed by impairment of daily glucose-stimulated insulin secretion and the expression of genes involved in exocytosis. Pharmacological activation of REV-ERBα could not restore the insulin secretion in LP mice. The present study demonstrates that protein restriction, leading to development of malnutrition, alters the peripheral clock and metabolic outputs, suggesting that this nutrient provides important entraining cues to regulate the daily fluctuation of biological clock. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Neutron scattering studies on protein dynamics using the human myelin peripheral membrane protein P2

    NASA Astrophysics Data System (ADS)

    Laulumaa, Saara; Kursula, Petri; Natali, Francesca

    2015-01-01

    Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

  13. Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

    PubMed

    Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

    2014-10-01

    Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets. © 2014 Wiley Periodicals, Inc.

  14. Monocyte Chemoattractant Protein-1 in the choroid plexus: a potential link between vascular pro-inflammatory mediators and the CNS during peripheral tissue inflammation

    PubMed Central

    Mitchell, K.; Yang, H.-Y. T.; Berk, J. D.; Tran, J. H.; Iadarola, M. J.

    2009-01-01

    During peripheral tissue inflammation, inflammatory processes in the CNS can be initiated by blood-borne pro-inflammatory mediators. The choroid plexus, the site of CSF production, is a highly specialized interface between the vascular system and CNS, and thus, this structure may be an important element in communication between the vascular compartment and the CNS during peripheral tissue inflammation. We investigated the potential participation of the choroid plexus in this process during peripheral tissue inflammation by examining expression of the SCYA2 gene which codes for monocyte chemoattractant protein-1 (MCP-1). MCP-1 protein was previously reported to be induced in a variety of cells during peripheral tissue inflammation. In the basal state, SCYA2 is highly expressed in the choroid plexus as compared to other CNS tissues. During hind paw inflammation, SCYA2 expression was significantly elevated in choroid plexus, whereas it remained unchanged in a variety of brain regions. The SCYA2-expressing cells were strongly associated with the choroid plexus as vascular depletion of blood cells by whole-body saline flush did not significantly alter SCYA2 expression in the choroid plexus. In situ hybridization suggested that the SCYA2-expressing cells were localized to the choroid plexus stroma. To elucidate potential molecular mechanisms of SCYA2 increase, we examined genes in the NF-κβ signaling cascade including TNF-α, IL-1β and IκBα in choroid tissue. Given that we also detected increased levels of MCP-1 protein by ELISA, we sought to identify potential downstream targets of MCP-1 and observed altered expression levels of mRNAs encoding tight junction proteins TJP2 and claudin 5. Finally, we detected a substantial up-regulation of the transcript encoding E-selectin, a molecule which could participate in leukocyte recruitment to the choroid plexus along with MCP-1. Together, these results suggest that profound changes occur in the choroid plexus during

  15. Induced oligomerization targets Golgi proteins for degradation in lysosomes.

    PubMed

    Tewari, Ritika; Bachert, Collin; Linstedt, Adam D

    2015-12-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130's cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. © 2015 Tewari et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  16. Targeting endogenous proteins for degradation through the affinity-directed protein missile system.

    PubMed

    Fulcher, Luke J; Hutchinson, Luke D; Macartney, Thomas J; Turnbull, Craig; Sapkota, Gopal P

    2017-05-01

    Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel-Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins. © 2017 The Authors.

  17. Targeting endogenous proteins for degradation through the affinity-directed protein missile system

    PubMed Central

    Fulcher, Luke J.; Hutchinson, Luke D.; Macartney, Thomas J.; Turnbull, Craig

    2017-01-01

    Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel–Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins. PMID:28490657

  18. Protective Effect of a Mitochondria-Targeted Peptide against the Development of Chemotherapy-Induced Peripheral Neuropathy in Mice.

    PubMed

    Toyama, Satoshi; Shimoyama, Naohito; Szeto, Hazel H; Schiller, Peter W; Shimoyama, Megumi

    2018-04-18

    Several chemotherapeutic agents used for cancer treatment induce dose-limiting peripheral neuropathy that compromises patients' quality of life and limits cancer treatment. Recently, mitochondrial dysfunction has been shown to be involved in the mechanism of chemotherapy-induced peripheral neuropathy. SS-20 is a mitochondria-targeted peptide that promotes mitochondrial respiration and restores mitochondrial bioenergetics. In the present study, we examined the protective effect of SS-20 against the development of chemotherapy-induced peripheral neuropathy utilizing a murine model of peripheral neuropathy induced by oxaliplatin, a first-line chemotherapy agent for colon cancer. Weekly administrations of oxaliplatin induced peripheral neuropathy as demonstrated by the development of neuropathic pain and loss of intraepidermal nerve fibers in the hind paw. Continuous administration of SS-20 protected against the development of oxaliplatin-induced neuropathic pain and mitigated the loss of intraepidermal nerve fibers to normal levels. Our findings suggest that SS-20 may be a drug candidate for the prevention of chemotherapy-induced peripheral neuropathy.

  19. Small molecules targeting heterotrimeric G proteins.

    PubMed

    Ayoub, Mohammed Akli

    2018-05-05

    G protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors regulating many human and animal physiological functions. Their implication in human pathophysiology is obvious with almost 30-40% medical drugs commercialized today directly targeting GPCRs as molecular entities. However, upon ligand binding GPCRs signal inside the cell through many key signaling, adaptor and regulatory proteins, including various classes of heterotrimeric G proteins. Therefore, G proteins are considered interesting targets for the development of pharmacological tools that are able to modulate their interaction with the receptors, as well as their activation/deactivation processes. In this review, old attempts and recent advances in the development of small molecules that directly target G proteins will be described with an emphasis on their utilization as pharmacological tools to dissect the mechanisms of activation of GPCR-G protein complexes. These molecules constitute a further asset for research in the "hot" areas of GPCR biology, areas such as multiple G protein coupling/signaling, GPCR-G protein preassembly, and GPCR functional selectivity or bias. Moreover, this review gives a particular focus on studies in vitro and in vivo supporting the potential applications of such small molecules in various GPCR/G protein-related diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Myelin protein zero and its antibody in serum as biomarkers of n-hexane-induced peripheral neuropathy and neurotoxicity effects.

    PubMed

    Jia, Xiaowei; Liu, Qingjun; Zhang, Yanshu; Dai, Yufei; Duan, Huawei; Bin, Ping; Niu, Yong; Liu, Jie; Zhong, Liuzhen; Guo, Jisheng; Liu, Xiaofeng; Zheng, Yuxin

    2014-01-01

    Chronic exposure to n-hexane can lead to peripheral neuropathy that no effective treatment regimen could be applied presently. This study investigated whether myelin protein zero (P0) protein and its antibody could be used to distinguish n-hexane intoxication and protect workers from peripheral neuropathy. We compared P0 protein and its antibody among three levels of n-hexane-exposed groups, which included 18 patients with n-hexane-induced peripheral neuropathy as case group, 120 n-hexane-exposed workers as n-hexaneexposed control group, and 147 non-hexane-exposed participants used as control group. ELISA method was applied to detect P0 protein and its antibody. P0 protein in serum was significantly higher in the case group and n-hexane-exposed control group in comparison with the control group (P < 0.01). Compared with the n-hexane-exposed control group, the case group also had significant increase of P0 protein (P < 0.01). After 6 months therapy, P0 protein was observed to decrease significantly in the case group (P < 0.01). The P0 antibody in serum was significantly higher in the n-hexane-exposed control group than in the control group (P < 0.01), but not significantly different between cases and controls. P0 antibodies in serum may be a short-term effect biomarker for n-hexane exposure. P0 protein in serum may be an early effective biomarker for peripheral nerve neuropathy and its biological limit value needs investigation in the future study.

  1. Detecting target changes in multiple object tracking with peripheral vision: More pronounced eccentricity effects for changes in form than in motion.

    PubMed

    Vater, Christian; Kredel, Ralf; Hossner, Ernst-Joachim

    2017-05-01

    In the current study, dual-task performance is examined with multiple-object tracking as a primary task and target-change detection as a secondary task. The to-be-detected target changes in conditions of either change type (form vs. motion; Experiment 1) or change salience (stop vs. slowdown; Experiment 2), with changes occurring at either near (5°-10°) or far (15°-20°) eccentricities (Experiments 1 and 2). The aim of the study was to test whether changes can be detected solely with peripheral vision. By controlling for saccades and computing gaze distances, we could show that participants used peripheral vision to monitor the targets and, additionally, to perceive changes at both near and far eccentricities. Noticeably, gaze behavior was not affected by the actual target change. Detection rates as well as response times generally varied as a function of change condition and eccentricity, with faster detections for motion changes and near changes. However, in contrast to the effects found for motion changes, sharp declines in detection rates and increased response times were observed for form changes as a function of the eccentricities. This result can be ascribed to properties of the visual system, namely to the limited spatial acuity in the periphery and the comparably receptive motion sensitivity of peripheral vision. These findings show that peripheral vision is functional for simultaneous target monitoring and target-change detection as saccadic information suppression can be avoided and covert attention can be optimally distributed to all targets. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  2. Targeting G protein coupled receptor-related pathways as emerging molecular therapies

    PubMed Central

    Ghanemi, Abdelaziz

    2013-01-01

    G protein coupled receptors (GPCRs) represent the most important targets in modern pharmacology because of the different functions they mediate, especially within brain and peripheral nervous system, and also because of their functional and stereochemical properties. In this paper, we illustrate, via a variety of examples, novel advances about the GPCR-related molecules that have been shown to play diverse roles in GPCR pathways and in pathophysiological phenomena. We have exemplified how those GPCRs’ pathways are, or might constitute, potential targets for different drugs either to stimulate, modify, regulate or inhibit the cellular mechanisms that are hypothesized to govern some pathologic, physiologic, biologic and cellular or molecular aspects both in vivo and in vitro. Therefore, influencing such pathways will, undoubtedly, lead to different therapeutical applications based on the related pharmacological implications. Furthermore, such new properties can be applied in different fields. In addition to offering fruitful directions for future researches, we hope the reviewed data, together with the elements found within the cited references, will inspire clinicians and researchers devoted to the studies on GPCR’s properties. PMID:25972730

  3. Targeting malaria parasite proteins to the erythrocyte.

    PubMed

    Templeton, Thomas J; Deitsch, Kirk W

    2005-09-01

    The intraerythrocytic stages of the protozoan parasite Plasmodium falciparum reside within a parasitophorous vacuole (PV) and set up unique "extraparasite, intraerythrocyte" protein-trafficking pathways that target parasite-encoded proteins to the erythrocyte cytoplasm and cell surface. Two recent articles report the identification of trafficking motifs that regulate the transport of parasite-encoded proteins across the PV. These articles greatly aid the annotation of the parasite "secretome" catalog of proteins that are targeted to the erythrocyte cytoplasm or cell membrane.

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

    PubMed

    Garner, Amanda L; Janda, Kim D

    2011-01-01

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

  5. Protein tyrosine phosphatases as potential therapeutic targets

    PubMed Central

    He, Rong-jun; Yu, Zhi-hong; Zhang, Ruo-yu; Zhang, Zhong-yin

    2014-01-01

    Protein tyrosine phosphorylation is a key regulatory process in virtually all aspects of cellular functions. Dysregulation of protein tyrosine phosphorylation is a major cause of human diseases, such as cancers, diabetes, autoimmune disorders, and neurological diseases. Indeed, protein tyrosine phosphorylation-mediated signaling events offer ample therapeutic targets, and drug discovery efforts to date have brought over two dozen kinase inhibitors to the clinic. Accordingly, protein tyrosine phosphatases (PTPs) are considered next-generation drug targets. For instance, PTP1B is a well-known targets of type 2 diabetes and obesity, and recent studies indicate that it is also a promising target for breast cancer. SHP2 is a bona-fide oncoprotein, mutations of which cause juvenile myelomonocytic leukemia, acute myeloid leukemia, and solid tumors. In addition, LYP is strongly associated with type 1 diabetes and many other autoimmune diseases. This review summarizes recent findings on several highly recognized PTP family drug targets, including PTP1B, Src homology phosphotyrosyl phosphatase 2(SHP2), lymphoid-specific tyrosine phosphatase (LYP), CD45, Fas associated phosphatase-1 (FAP-1), striatal enriched tyrosine phosphatases (STEP), mitogen-activated protein kinase/dual-specificity phosphatase 1 (MKP-1), phosphatases of regenerating liver-1 (PRL), low molecular weight PTPs (LMWPTP), and CDC25. Given that there are over 100 family members, we hope this review will serve as a road map for innovative drug discovery targeting PTPs. PMID:25220640

  6. DNA-protein crosslinks in peripheral lymphocytes of individuals exposed to hexavalent chromium compounds.

    PubMed

    Zhitkovich, A; Lukanova, A; Popov, T; Taioli, E; Cohen, H; Costa, M; Toniolo, P

    1996-01-01

    Abstract DNA-protein crosslinks were measured in peripheral blood lymphocytes of chrome-platers and controls from Bulgaria in order to evaluate a genotoxic effect of human exposure to carcinogenic Cr(VI) compounds. Chrome-platers and most of the unexposed controls were from the industrial city of Jambol; some additional controls were recruited from the seaside town of Burgas. The chrome-platers had significantly elevated levels of chromium in pre- and post-shift urine, erythrocytes and lymphocytes compared with the control subjects. The largest differences between the two groups were found in erythrocyte chromium concentrations which are considered to be indicative of Cr(VI) exposure. Despite the significant differences in internal chromium doses, levels of DNA-protein crosslinks were not significantly different between the combined controls and exposed workers. Individual DNA-protein crosslinks, however, correlated strongly with chromium in erythrocytes at low and moderate doses but at high exposures, such as among the majority of chrome-platers, these DNA adducts were saturated at maximum levels. The saturation of DNA-protein crosslinks seems to occur at 7-8 μg I-(1) chromium in erythrocytes whereas a mean erythrocyte chromium among the chrome platers was as high as 22.8 μg l(-1). Occupationally unexposed subjects exhibited a significant variability with respect to the erythrocyte chromium concentration, however erythrocyte chromium levels correlated closely with DNA-protein crosslinks in lymphocytes. The controls from Jambol had higher chromium concentrations in erythrocytes and elevated levels of DNA-protein crosslinks compared with Burgas controls. Occupational exposure to formaldehyde among furniture factory workers did not change levels of DNA-protein crosslinks in peripheral lymphocytes. DNA-protein crosslink measurements showed a low intraindividual variability and their levels among both controls and exposed indivduals were not affected by smoking, age

  7. Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections.

    PubMed

    Adzemovic, Milena Z; Zeitelhofer, Manuel; Leisser, Marianne; Köck, Ulricke; Kury, Angela; Olsson, Tomas

    2016-11-14

    Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS). Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

  8. MicroRNA-31 negatively regulates peripherally derived regulatory T-cell generation by repressing retinoic acid-inducible protein 3

    PubMed Central

    Zhang, Lingyun; Ke, Fang; Liu, Zhaoyuan; Bai, Jing; Liu, Jinlin; Yan, Sha; Xu, Zhenyao; Lou, Fangzhou; Wang, Hong; Zhu, Huiyuan; Sun, Yang; Cai, Wei; Gao, Yuanyuan; Li, Qun; Yu, Xue-Zhong; Qian, Youcun; Hua, Zichun; Deng, Jiong; Li, Qi-Jing; Wang, Honglin

    2015-01-01

    Peripherally derived regulatory T (pTreg) cell generation requires T-cell receptor (TCR) signalling and the cytokines TGF-β1 and IL-2. Here we show that TCR signalling induces the microRNA miR-31, which negatively regulates pTreg-cell generation. miR-31 conditional deletion results in enhanced induction of pTreg cells, and decreased severity of experimental autoimmune encephalomyelitis (EAE). Unexpectedly, we identify Gprc5a as a direct target of miR-31. Gprc5a is known as retinoic acid-inducible protein 3, and its deficiency leads to impaired pTreg-cell induction and increased EAE severity. By generating miR-31 and Gprc5a double knockout mice, we show that miR-31 promotes the development of EAE through inhibiting Gprc5a. Thus, our data identify miR-31 and its target Gprc5a as critical regulators for pTreg-cell generation, suggesting a previously unrecognized epigenetic mechanism for dysfunctional Treg cells in autoimmune diseases. PMID:26165721

  9. In silico re-identification of properties of drug target proteins.

    PubMed

    Kim, Baeksoo; Jo, Jihoon; Han, Jonghyun; Park, Chungoo; Lee, Hyunju

    2017-05-31

    Computational approaches in the identification of drug targets are expected to reduce time and effort in drug development. Advances in genomics and proteomics provide the opportunity to uncover properties of druggable genomes. Although several studies have been conducted for distinguishing drug targets from non-drug targets, they mainly focus on the sequences and functional roles of proteins. Many other properties of proteins have not been fully investigated. Using the DrugBank (version 3.0) database containing nearly 6,816 drug entries including 760 FDA-approved drugs and 1822 of their targets and human UniProt/Swiss-Prot databases, we defined 1578 non-redundant drug target and 17,575 non-drug target proteins. To select these non-redundant protein datasets, we built four datasets (A, B, C, and D) by considering clustering of paralogous proteins. We first reassessed the widely used properties of drug target proteins. We confirmed and extended that drug target proteins (1) are likely to have more hydrophobic, less polar, less PEST sequences, and more signal peptide sequences higher and (2) are more involved in enzyme catalysis, oxidation and reduction in cellular respiration, and operational genes. In this study, we proposed new properties (essentiality, expression pattern, PTMs, and solvent accessibility) for effectively identifying drug target proteins. We found that (1) drug targetability and protein essentiality are decoupled, (2) druggability of proteins has high expression level and tissue specificity, and (3) functional post-translational modification residues are enriched in drug target proteins. In addition, to predict the drug targetability of proteins, we exploited two machine learning methods (Support Vector Machine and Random Forest). When we predicted drug targets by combining previously known protein properties and proposed new properties, an F-score of 0.8307 was obtained. When the newly proposed properties are integrated, the prediction performance

  10. C-Myc Protein-Protein and Protein-DNA Interactions: Targets for Therapeutic Intervention.

    DTIC Science & Technology

    1997-09-01

    including those of the Myc family. In fact, members of different bHLH protein subgroups, including the Myc proteins, are characterized by conserved BR...important functional consequences, and they provide insights into how different bHLH proteins can act on different targets. The zinc finger protein...roles for a number of BR residues which do not contact bases, yet are conserved within different bHLH protein sub- families (Benezra et al. 1990), and

  11. Essential Function of Protein 4.1G in Targeting of Membrane Protein Palmitoylated 6 into Schmidt-Lanterman Incisures in Myelinated Nerves

    PubMed Central

    Saitoh, Yurika; Ohno, Nobuhiko; Komada, Masayuki; Saitoh, Sei; Peles, Elior; Ohno, Shinichi

    2012-01-01

    Protein 4.1G is a membrane skeletal protein found in specific subcellular structures in myelinated Schwann cells and seminiferous tubules. Here, we show that in the mouse sciatic nerve, protein 4.1G colocalized at Schmidt-Lanterman incisures (SLI) and the paranodes with a member of the membrane-associated guanylate kinase (MAGUK) family, membrane protein palmitoylated 6 (MPP6). Coimmunoprecipitation experiments revealed that MPP6 was interacting with protein 4.1G. In contrast to wild-type nerves, in 4.1G knockout mice, MPP6 was found largely in the cytoplasm near Schwann cell nuclei, indicating an abnormal protein transport. Although the SLI remained in the 4.1G knockout sciatic nerves, as confirmed by E-cadherin immunostaining, their shape was altered in aged 4.1G knockout nerves compared to their shape in wild-type nerves. In the seminiferous tubules, MPP6 was localized similarly to protein 4.1G along cell membranes of the spermatogonium and early spermatocytes. However, in contrast to myelinated peripheral nerves, the specific localization of MPP6 in the seminiferous tubules was unaltered in the absence of protein 4.1G. These results indicate that 4.1G has a specific role in the targeting of MPP6 to the SLI and the assembly of these subcellular structures. PMID:22025680

  12. Earlier saccades to task-relevant targets irrespective of relative gain between peripheral and foveal information.

    PubMed

    Wolf, Christian; Schütz, Alexander C

    2017-06-01

    Saccades bring objects of interest onto the fovea for high-acuity processing. Saccades to rewarded targets show shorter latencies that correlate negatively with expected motivational value. Shorter latencies are also observed when the saccade target is relevant for a perceptual discrimination task. Here we tested whether saccade preparation is equally influenced by informational value as it is by motivational value. We defined informational value as the probability that information is task-relevant times the ratio between postsaccadic foveal and presaccadic peripheral discriminability. Using a gaze-contingent display, we independently manipulated peripheral and foveal discriminability of the saccade target. Latencies of saccades with perceptual task were reduced by 36 ms in general, but they were not modulated by the information saccades provide (Experiments 1 and 2). However, latencies showed a clear negative linear correlation with the probability that the target is task-relevant (Experiment 3). We replicated that the facilitation by a perceptual task is spatially specific and not due to generally heightened arousal (Experiment 4). Finally, the facilitation only emerged when the perceptual task is in the visual but not in the auditory modality (Experiment 5). Taken together, these results suggest that saccade latencies are not equally modulated by informational value as by motivational value. The facilitation by a perceptual task only arises when task-relevant visual information is foveated, irrespective of whether the foveation is useful or not.

  13. Comprehensive peptidomimetic libraries targeting protein-protein interactions.

    PubMed

    Whitby, Landon R; Boger, Dale L

    2012-10-16

    Transient protein-protein interactions (PPIs) are essential components in cellular signaling pathways as well as in important processes such as viral infection, replication, and immune suppression. The unknown or uncharacterized PPIs involved in such interaction networks often represent compelling therapeutic targets for drug discovery. To date, however, the main strategies for discovery of small molecule modulators of PPIs are typically limited to structurally characterized targets. Recent developments in molecular scaffolds that mimic the side chain display of peptide secondary structures have yielded effective designs, but few screening libraries of such mimetics are available to interrogate PPI targets. We initiated a program to prepare a comprehensive small molecule library designed to mimic the three major recognition motifs that mediate PPIs (α-helix, β-turn, and β-strand). Three libraries would be built around templates designed to mimic each such secondary structure and substituted with all triplet combinations of groups representing the 20 natural amino acid side chains. When combined, the three libraries would contain a member capable of mimicking the key interaction and recognition residues of most targetable PPIs. In this Account, we summarize the results of the design, synthesis, and validation of an 8000 member α-helix mimetic library and a 4200 member β-turn mimetic library. We expect that the screening of these libraries will not only provide lead structures against α-helix- or β-turn-mediated protein-protein or peptide-receptor interactions, even if the nature of the interaction is unknown, but also yield key insights into the recognition motif (α-helix or β-turn) and identify the key residues mediating the interaction. Consistent with this expectation, the screening of the libraries against p53/MDM2 and HIV-1 gp41 (α-helix mimetic library) or the opioid receptors (β-turn mimetic library) led to the discovery of library members expected

  14. The Protein Micro-Crystallography Beamlines for Targeted Protein Research Program

    NASA Astrophysics Data System (ADS)

    Hirata, Kunio; Yamamoto, Masaki; Matsugaki, Naohiro; Wakatsuki, Soichi

    In order to collect proper diffraction data from outstanding micro-crystals, a brand-new data collection system should be designed to provide high signal-to noise ratio in diffraction images. SPring-8 and KEK-PF are currently developing two micro-beam beamlines for Targeted Proteins Research Program by MEXT of Japan. The program aims to reveal the structure and function of proteins that are difficult to solve but have great importance in both academic research and industrial application. At SPring-8, a new 1-micron beam beamline for protein micro-crystallography, RIKEN Targeted Proteins Beamline (BL32XU), is developed. At KEK-PF a new low energy micro-beam beamline, BL-1A, is dedicated for SAD micro-crystallography. The two beamlines will start operation in the end of 2010. The present status of the research and development for protein micro-crystallography will be presented.

  15. Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

    PubMed

    Cha, Jaehyun; Kwon, Inchan

    2018-02-27

    Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Design and Preliminary Testing of the International Docking Adapter's Peripheral Docking Target

    NASA Technical Reports Server (NTRS)

    Foster, Christopher W.; Blaschak, Johnathan; Eldridge, Erin A.; Brazzel, Jack P.; Spehar, Peter T.

    2015-01-01

    The International Docking Adapter's Peripheral Docking Target (PDT) was designed to allow a docking spacecraft to judge its alignment relative to the docking system. The PDT was designed to be compatible with relative sensors using visible cameras, thermal imagers, or Light Detection and Ranging (LIDAR) technologies. The conceptual design team tested prototype designs and materials to determine the contrast requirements for the features. This paper will discuss the design of the PDT, the methodology and results of the tests, and the conclusions pertaining to PDT design that were drawn from testing.

  17. Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

    PubMed

    Nissen, Klaus B; Haugaard-Kedström, Linda M; Wilbek, Theis S; Nielsen, Line S; Åberg, Emma; Kristensen, Anders S; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

  18. Japanese neuropathy patients with peripheral myelin protein-22 gene aneuploidy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lebo, R.V.; Li, L.Y.; Flandermeyer, R.R.

    1994-09-01

    Peripheral myelin protein (PMP-22) gene aneuploidy results in Charcot-Marie-Tooth disease Type 1A (CMT1A) and the Hereditary Neuropathy with Liability to Pressure Palsy (HNPP) in Japanese patients as well as Caucasian Americans. Charcot-Marie-Tooth disease (CMT), the most common genetic neuropathy, results when expression of one of at least seven genes is defective. CMT1A, about half of all CMT mutations, is usually associated with a duplication spanning the peripheral myelin protein-22 gene on distal chromosome band 17p11.2. Autosomal dominant HNPP (hereditary pressure and sensory neuropathy, HPSN) results from a deletion of the CMT1A gene region. Multicolor in situ hybridization with PMP-22 genemore » region probe characterized HNPP deletion reliably and detected all different size duplications reported previously. In summary, 72% of 28 Japanese CMT1 (HMSNI) patients tested had the CMT1A duplication, while none of the CMT2 (HMSNII) or CMT3 (HMSNIII) patients had a duplication. Three cases of HNPP were identified by deletion of the CMT1A gene region on chromosome 17p. HNPP and CMT1A have been reported to result simultaneously from the same unequal recombination event. The lower frequency of HNPP compared to CMT1A suggests that HNPP patients have a lower reproductive fitness than CMT1A patients. This result, along with a CMT1A duplication found in an Asian Indian family, demonstrates the broad geographic distribution and high frequency of PMP-22 gene aneuploidy.« less

  19. Druggable orthosteric and allosteric hot spots to target protein-protein interactions.

    PubMed

    Ma, Buyong; Nussinov, Ruth

    2014-01-01

    Drug designing targeting protein-protein interactions is challenging. Because structural elucidation and computational analysis have revealed the importance of hot spot residues in stabilizing these interactions, there have been on-going efforts to develop drugs which bind the hot spots and out-compete the native protein partners. The question arises as to what are the key 'druggable' properties of hot spots in protein-protein interactions and whether these mimic the general hot spot definition. Identification of orthosteric (at the protein- protein interaction site) and allosteric (elsewhere) druggable hot spots is expected to help in discovering compounds that can more effectively modulate protein-protein interactions. For example, are there any other significant features beyond their location in pockets in the interface? The interactions of protein-protein hot spots are coupled with conformational dynamics of protein complexes. Currently increasing efforts focus on the allosteric drug discovery. Allosteric drugs bind away from the native binding site and can modulate the native interactions. We propose that identification of allosteric hot spots could similarly help in more effective allosteric drug discovery. While detection of allosteric hot spots is challenging, targeting drugs to these residues has the potential of greatly increasing the hot spot and protein druggability.

  20. Local GABAergic signaling within sensory ganglia controls peripheral nociceptive transmission

    PubMed Central

    Du, Xiaona; Hao, Han; Yang, Yuehui; Huang, Sha; Wang, Caixue; Gigout, Sylvain; Ramli, Rosmaliza; Li, Xinmeng; Jaworska, Ewa; Edwards, Ian; Yanagawa, Yuchio; Qi, Jinlong; Guan, Bingcai; Jaffe, David B.; Zhang, Hailin

    2017-01-01

    The integration of somatosensory information is generally assumed to be a function of the central nervous system (CNS). Here we describe fully functional GABAergic communication within rodent peripheral sensory ganglia and show that it can modulate transmission of pain-related signals from the peripheral sensory nerves to the CNS. We found that sensory neurons express major proteins necessary for GABA synthesis and release and that sensory neurons released GABA in response to depolarization. In vivo focal infusion of GABA or GABA reuptake inhibitor to sensory ganglia dramatically reduced acute peripherally induced nociception and alleviated neuropathic and inflammatory pain. In addition, focal application of GABA receptor antagonists to sensory ganglia triggered or exacerbated peripherally induced nociception. We also demonstrated that chemogenetic or optogenetic depolarization of GABAergic dorsal root ganglion neurons in vivo reduced acute and chronic peripherally induced nociception. Mechanistically, GABA depolarized the majority of sensory neuron somata, yet produced a net inhibitory effect on the nociceptive transmission due to the filtering effect at nociceptive fiber T-junctions. Our findings indicate that peripheral somatosensory ganglia represent a hitherto underappreciated site of somatosensory signal integration and offer a potential target for therapeutic intervention. PMID:28375159

  1. Proteolysis targeting peptide (PROTAP) strategy for protein ubiquitination and degradation.

    PubMed

    Zheng, Jing; Tan, Chunyan; Xue, Pengcheng; Cao, Jiakun; Liu, Feng; Tan, Ying; Jiang, Yuyang

    2016-02-19

    Ubiquitination proteasome pathway (UPP) is the most important and selective way to degrade proteins in vivo. Here, a novel proteolysis targeting peptide (PROTAP) strategy, composed of a target protein binding peptide, a linker and a ubiquitin E3 ligase recognition peptide, was designed to recruit both target protein and E3 ligase and then induce polyubiquitination and degradation of the target protein through UPP. In our study, the PROTAP strategy was proved to be a general method with high specificity using Bcl-xL protein as model target in vitro and in cells, which indicates that the strategy has great potential for in vivo application. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Spiral Ganglion Neuron Projection Development to the Hindbrain in Mice Lacking Peripheral and/or Central Target Differentiation

    PubMed Central

    Elliott, Karen L.; Kersigo, Jennifer; Pan, Ning; Jahan, Israt; Fritzsch, Bernd

    2017-01-01

    We investigate the importance of the degree of peripheral or central target differentiation for mouse auditory afferent navigation to the organ of Corti and auditory nuclei in three different mouse models: first, a mouse in which the differentiation of hair cells, but not central auditory nuclei neurons is compromised (Atoh1-cre; Atoh1f/f); second, a mouse in which hair cell defects are combined with a delayed defect in central auditory nuclei neurons (Pax2-cre; Atoh1f/f), and third, a mouse in which both hair cells and central auditory nuclei are absent (Atoh1−/−). Our results show that neither differentiated peripheral nor the central target cells of inner ear afferents are needed (hair cells, cochlear nucleus neurons) for segregation of vestibular and cochlear afferents within the hindbrain and some degree of base to apex segregation of cochlear afferents. These data suggest that inner ear spiral ganglion neuron processes may predominantly rely on temporally and spatially distinct molecular cues in the region of the targets rather than interaction with differentiated target cells for a crude topological organization. These developmental data imply that auditory neuron navigation properties may have evolved before auditory nuclei. PMID:28450830

  3. Characterization of the targeting signal in mitochondrial β-barrel proteins

    PubMed Central

    Jores, Tobias; Klinger, Anna; Groß, Lucia E.; Kawano, Shin; Flinner, Nadine; Duchardt-Ferner, Elke; Wöhnert, Jens; Kalbacher, Hubert; Endo, Toshiya; Schleiff, Enrico; Rapaport, Doron

    2016-01-01

    Mitochondrial β-barrel proteins are synthesized on cytosolic ribosomes and must be specifically targeted to the organelle before their integration into the mitochondrial outer membrane. The signal that assures such precise targeting and its recognition by the organelle remained obscure. In the present study we show that a specialized β-hairpin motif is this long searched for signal. We demonstrate that a synthetic β-hairpin peptide competes with the import of mitochondrial β-barrel proteins and that proteins harbouring a β-hairpin peptide fused to passenger domains are targeted to mitochondria. Furthermore, a β-hairpin motif from mitochondrial proteins targets chloroplast β-barrel proteins to mitochondria. The mitochondrial targeting depends on the hydrophobicity of the β-hairpin motif. Finally, this motif interacts with the mitochondrial import receptor Tom20. Collectively, we reveal that β-barrel proteins are targeted to mitochondria by a dedicated β-hairpin element, and this motif is recognized at the organelle surface by the outer membrane translocase. PMID:27345737

  4. Mechanisms of peripheral immune-cell-mediated analgesia in inflammation: clinical and therapeutic implications.

    PubMed

    Hua, Susan; Cabot, Peter J

    2010-09-01

    Peripheral mechanisms of endogenous pain control are significant. In peripheral inflamed tissue, an interaction between immune-cell-derived opioids and opioid receptors localized on sensory nerve terminals results in potent, clinically measurable analgesia. Opioid peptides and the mRNA encoding their precursor proteins are present in immune cells. These cells 'home' preferentially to injured tissue, where they secrete opioids to reduce pain. Investigation of the mechanisms underlying the migration of opioid-containing immune cells to inflamed tissue is an active area of research, with recent data demonstrating the importance of cell adhesion molecules in leukocyte adhesion to both the endothelium in vascular transmigration and to neurons within peripheral inflamed tissue. This review summarizes the physiological mechanisms and clinical significance of this unique endogenous peripheral analgesic pathway and discusses therapeutic implications for the development of novel targeted peripheral analgesics. Copyright 2010 Elsevier Ltd. All rights reserved.

  5. Reversible Folding of Human Peripheral Myelin Protein 22, a Tetraspan Membrane Protein†

    PubMed Central

    Schlebach, Jonathan P.; Peng, Dungeng; Kroncke, Brett M.; Mittendorf, Kathleen F.; Narayan, Malathi; Carter, Bruce D.; Sanders, Charles R.

    2013-01-01

    Misfolding of the α-helical membrane protein peripheral myelin protein 22 (PMP22) has been implicated in the pathogenesis of the common neurodegenerative disease known as Charcot-Marie-Tooth disease (CMTD) and also several other related peripheral neuropathies. Emerging evidence suggests that the propensity of PMP22 to misfold in the cell may be due to an intrinsic lack of conformational stability. Therefore, quantitative studies of the conformational equilibrium of PMP22 are needed to gain insight into the molecular basis of CMTD. In this work, we have investigated the folding and unfolding of wild type (WT) human PMP22 in mixed micelles. Both kinetic and thermodynamic measurements demonstrate that the denaturation of PMP22 by n-lauroyl sarcosine (LS) in dodecylphosphocholine (DPC) micelles is reversible. Assessment of the conformational equilibrium indicates that a significant fraction of unfolded PMP22 persists even in the absence of the denaturing detergent. However, we find the stability of PMP22 is increased by glycerol, which facilitates quantitation of thermodynamic parameters. To our knowledge, this work represents the first report of reversible unfolding of a eukaryotic multispan membrane protein. The results indicate that WT PMP22 possesses minimal conformational stability in micelles, which parallels its poor folding efficiency in the endoplasmic reticulum. Folding equilibrium measurements for PMP22 in mixed micelles may provide an approach to assess the effects of cellular metabolites or potential therapeutic agents on its stability. Furthermore, these results pave the way for future investigation of the effects of pathogenic mutations on the conformational equilibrium of PMP22. PMID:23639031

  6. Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

    PubMed Central

    Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.

    2007-01-01

    Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592

  7. Comprehensive predictions of target proteins based on protein-chemical interaction using virtual screening and experimental verifications.

    PubMed

    Kobayashi, Hiroki; Harada, Hiroko; Nakamura, Masaomi; Futamura, Yushi; Ito, Akihiro; Yoshida, Minoru; Iemura, Shun-Ichiro; Shin-Ya, Kazuo; Doi, Takayuki; Takahashi, Takashi; Natsume, Tohru; Imoto, Masaya; Sakakibara, Yasubumi

    2012-04-05

    Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; however, target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis. We applied our protocol of predicting target proteins combining in silico screening and experimental verification for incednine, which inhibits the anti-apoptotic function of Bcl-xL by an unknown mechanism. One hundred eighty-two target protein candidates were computationally predicted to bind to incednine by the statistical prediction method, and the predictions were verified by in vitro binding of incednine to seven proteins, whose expression can be confirmed in our cell system.As a result, 40% accuracy of the computational predictions was achieved successfully, and we newly found 3 incednine-binding proteins. This study revealed that our proposed protocol of predicting target protein combining in silico screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules.

  8. Molecular Characterization of Monoclonal Antibodies that Inhibit Acetylcholinesterase by Targeting the Peripheral Site and Backdoor Region

    PubMed Central

    Essono, Sosthène; Mondielli, Grégoire; Lamourette, Patricia; Boquet, Didier; Grassi, Jacques; Marchot, Pascale

    2013-01-01

    The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis. PMID:24146971

  9. Hsp70 Protein Complexes as Drug Targets

    PubMed Central

    Assimon, Victoria A.; Gillies, Anne T.; Rauch, Jennifer N.; Gestwicki, Jason E.

    2013-01-01

    Heat shock protein 70 (Hsp70) plays critical roles in proteostasis and is an emerging target for multiple diseases. However, competitive inhibition of the enzymatic activity of Hsp70 has proven challenging and, in some cases, may not be the most productive way to redirect Hsp70 function. Another approach is to inhibit Hsp70’s interactions with important co-chaperones, such as J proteins, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain-containing proteins. These co-chaperones normally bind Hsp70 and guide its many diverse cellular activities. Complexes between Hsp70 and co-chaperones have been shown to have specific functions, such as pro-folding, pro-degradation and pro-trafficking. Thus, a promising strategy may be to block protein-protein interactions between Hsp70 and its co-chaperones or to target allosteric sites that disrupt these contacts. Such an approach might shift the balance of Hsp70 complexes and re-shape the proteome and it has the potential to restore healthy proteostasis. In this review, we discuss specific challenges and opportunities related to those goals. By pursuing Hsp70 complexes as drug targets, we might not only develop new leads for therapeutic development, but also discover new chemical probes for use in understanding Hsp70 biology. PMID:22920901

  10. Complex network theory for the identification and assessment of candidate protein targets.

    PubMed

    McGarry, Ken; McDonald, Sharon

    2018-06-01

    In this work we use complex network theory to provide a statistical model of the connectivity patterns of human proteins and their interaction partners. Our intention is to identify important proteins that may be predisposed to be potential candidates as drug targets for therapeutic interventions. Target proteins usually have more interaction partners than non-target proteins, but there are no hard-and-fast rules for defining the actual number of interactions. We devise a statistical measure for identifying hub proteins, we score our target proteins with gene ontology annotations. The important druggable protein targets are likely to have similar biological functions that can be assessed for their potential therapeutic value. Our system provides a statistical analysis of the local and distant neighborhood protein interactions of the potential targets using complex network measures. This approach builds a more accurate model of drug-to-target activity and therefore the likely impact on treating diseases. We integrate high quality protein interaction data from the HINT database and disease associated proteins from the DrugTarget database. Other sources include biological knowledge from Gene Ontology and drug information from DrugBank. The problem is a very challenging one since the data is highly imbalanced between target proteins and the more numerous nontargets. We use undersampling on the training data and build Random Forest classifier models which are used to identify previously unclassified target proteins. We validate and corroborate these findings from the available literature. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Identification and Herc5-mediated ISGylation of novel target proteins.

    PubMed

    Takeuchi, Tomoharu; Inoue, Satoshi; Yokosawa, Hideyoshi

    2006-09-22

    ISG15, a protein containing two ubiquitin-like domains, is an interferon-stimulated gene product that functions in antiviral response and is conjugated to various cellular proteins (ISGylation) upon interferon stimulation. ISGylation occurs via a pathway similar to the pathway for ubiquitination that requires the sequential action of E1/E2/E3: the E1 (UBE1L), E2 (UbcH8), and E3 (Efp/Herc5) enzymes for ISGylation have been hitherto identified. In this study, we identified six novel candidate target proteins for ISGylation by a proteomic approach. Four candidate target proteins were demonstrated to be ISGylated in UBE1L- and UbcH8-dependent manners, and ISGylation of the respective target proteins was stimulated by Herc5. In addition, Herc5 was capable of binding with the respective target proteins. Thus, these results suggest that Herc5 functions as a general E3 ligase for protein ISGylation.

  12. Exosomes derived from high-glucose-stimulated Schwann cells promote development of diabetic peripheral neuropathy.

    PubMed

    Jia, Longfei; Chopp, Michael; Wang, Lei; Lu, Xuerong; Szalad, Alexandra; Zhang, Zheng Gang

    2018-06-22

    Schwann cells actively interact with axons of dorsal root ganglia (DRG) neurons. Exosomes mediate intercellular communication by transferring their biomaterials, including microRNAs (miRs) into recipient cells. We hypothesized that exosomes derived from Schwann cells stimulated by high glucose (HG) exosomes accelerate development of diabetic peripheral neuropathy and that exosomal cargo miRs contribute to this process. We found that HG exosomes contained high levels of miR-28, -31a, and -130a compared to exosomes derived from non-HG-stimulated Schwann cells. In vitro, treatment of distal axons with HG exosomes resulted in reduction of axonal growth, which was associated with elevation of miR-28, -31a, and -130a and reduction of their target proteins of DNA methyltransferase-3α, NUMB (an endocytic adaptor protein), synaptosome associated protein 25, and growth-associated protein-43 in axons. In vivo, administration of HG exosomes to sciatic nerves of diabetic db/db mice at 7 wk of age promoted occurrence of peripheral neuropathy characterized by impairment of nerve conduction velocity and induction of mechanic and thermal hypoesthesia, which was associated with substantial decreases in intraepidermal nerve fibers. Our findings demonstrate a functional role of exosomes derived from HG-stimulated Schwann cells in mediating development of diabetic peripheral neuropathy.-Jia, L., Chopp, M., Wang, L., Lu, X., Szalad, A., Zhang, Z. G. Exosomes derived from high-glucose-stimulated Schwann cells promote development of diabetic peripheral neuropathy.

  13. [Transcription of protein arginine N-methyltransferase genes in mouse dorsal root ganglia following peripheral nerve injury].

    PubMed

    Xu, Hua-Li; Xu, Shi-Yuan; Mo, Kai

    2017-12-20

    To investigate the changes in the transcription of protein arginine methylation enzyme family genes in the dorsal root ganglia (DRG) following peripheral nerve injury in mice. C57BL6 mouse models of neuropathic pain induced by peripheral nerve injury were established by bilateral L4 spinal nerve ligation (SNL). At 7 days after SNL or sham operation, the DRG tissue was collected for transcriptional analysis of 9 protein arginine methylation enzyme genes (Prmt1?3, Carm1, and Prmt5?9) using RNA?Seq to identify the differentially expressed genes in the injured DRGs. We also established mouse models of lateral L4 SNL and models of chronic constriction injury (CCI) of the sciatic nerve and tested the paw withdrawal frequency (PWF) in response to mechanical stimulation and paw withdrawal latency (PWL) in response to thermal stimulation on 0, 3, 7 and 14 days after SNL or CCI; the expressions of the differentially expressed genes in the injured DRGs were verified in the two models using RT?qPCR. Among the 9 protein arginine methylation enzyme family genes that were tissue?specifically expressed in the DRG, Prmt2 and Prmt3 showed the highest and Prmt6 showed the lowest basal expression. Compared with the sham?operated mice group, the mice receiving SNL exhibited upregulated Carm1 gene transcription (by 1.7 folds) but downregulated Prmt5, Prmt8 and Prmt9 transcription in the injured DRG (Prmt8 gene showed the most significant down?regulation by 16.3 folds). In mouse models of SNL and CCI, Carm1 gene expression increased progressively with time while Prmt8 transcription was obviously lowered on days 3, 7 and 14 after the injury; the transcription levels of Prmt1, Prmt5 and Prmt9 presented with no significant changes following the injuries. Both SNL and CCI induced mechanical allodynia and thermal hypersensitivities in the mice shown by increased PWF and decreased PWL on days 3, 7 and 14 after the injuries. Periphery nerve injury induces Carm1 upregulation and Prmt8

  14. Specifically targeted delivery of protein to phagocytic macrophages

    PubMed Central

    Yu, Min; Chen, Zeming; Guo, Wenjun; Wang, Jin; Feng, Yupeng; Kong, Xiuqi; Hong, Zhangyong

    2015-01-01

    Macrophages play important roles in the pathogenesis of various diseases, and are important potential therapeutic targets. Furthermore, macrophages are key antigen-presenting cells and important in vaccine design. In this study, we report on the novel formulation (bovine serum albumin [BSA]-loaded glucan particles [GMP-BSA]) based on β-glucan particles from cell walls of baker’s yeast for the targeted delivery of protein to macrophages. Using this formulation, chitosan, tripolyphosphate, and alginate were used to fabricate colloidal particles with the model protein BSA via electrostatic interactions, which were caged and incorporated BSA very tightly within the β-glucan particle shells. The prepared GMP-BSA exhibited good protein-release behavior and avoided protein leakage. The particles were also highly specific to phagocytic macrophages, such as Raw 264.7 cells, primary bone marrow-derived macrophages, and peritoneal exudate macrophages, whereas the particles were not taken up by nonphagocytic cells, including NIH3T3, AD293, HeLa, and Caco-2. We hypothesize that these tightly encapsulated protein-loaded glucan particles deliver various types of proteins to macrophages with notably high selectivity, and may have broad applications in targeted drug delivery or vaccine design against macrophages. PMID:25784802

  15. Targeting a KH-domain protein with RNA decoys.

    PubMed

    Makeyev, Aleksandr V; Eastmond, Dawn L; Liebhaber, Stephen A

    2002-09-01

    RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression. Targeted interference with RNA-protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the alphaCP subgroup of KH-domain RNA-binding proteins. These poly(C)-binding proteins have been implicated in a wide spectrum of posttranscriptional controls. Three categories of RNA decoys to alphaCPs were studied: poly(C) homopolymers, native mRNA-binding sites, and a high-affinity structure selected from a combinatorial library. Native chemistry was found to be essential for alphaCP decoy action. Because alphaCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear (U1 snRNA) and cytoplasmic (VA1 RNA) RNA frameworks. Several sequences demonstrated optimal decoy properties when assayed for protein-binding and decoy bioactivity in vitro. A subset of these transcripts was shown to mediate targeted inhibition of alphaCP-dependent translation when expressed in either the nucleus or cytoplasm of transfected cells. Significantly, these studies establish the feasibility of developing RNA decoys that can selectively target biologic functions of abundant and widely expressed RNA binding proteins.

  16. Targeting a KH-domain protein with RNA decoys.

    PubMed Central

    Makeyev, Aleksandr V; Eastmond, Dawn L; Liebhaber, Stephen A

    2002-01-01

    RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression. Targeted interference with RNA-protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the alphaCP subgroup of KH-domain RNA-binding proteins. These poly(C)-binding proteins have been implicated in a wide spectrum of posttranscriptional controls. Three categories of RNA decoys to alphaCPs were studied: poly(C) homopolymers, native mRNA-binding sites, and a high-affinity structure selected from a combinatorial library. Native chemistry was found to be essential for alphaCP decoy action. Because alphaCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear (U1 snRNA) and cytoplasmic (VA1 RNA) RNA frameworks. Several sequences demonstrated optimal decoy properties when assayed for protein-binding and decoy bioactivity in vitro. A subset of these transcripts was shown to mediate targeted inhibition of alphaCP-dependent translation when expressed in either the nucleus or cytoplasm of transfected cells. Significantly, these studies establish the feasibility of developing RNA decoys that can selectively target biologic functions of abundant and widely expressed RNA binding proteins. PMID:12358435

  17. Protein-anchoring therapy to target extracellular matrix proteins to their physiological destinations.

    PubMed

    Ito, Mikako; Ohno, Kinji

    2018-02-20

    Endplate acetylcholinesterase (AChE) deficiency is a form of congenital myasthenic syndrome (CMS) caused by mutations in COLQ, which encodes collagen Q (ColQ). ColQ is an extracellular matrix (ECM) protein that anchors AChE to the synaptic basal lamina. Biglycan, encoded by BGN, is another ECM protein that binds to the dystrophin-associated protein complex (DAPC) on skeletal muscle, which links the actin cytoskeleton and ECM proteins to stabilize the sarcolemma during repeated muscle contractions. Upregulation of biglycan stabilizes the DPAC. Gene therapy can potentially ameliorate any disease that can be recapitulated in cultured cells. However, the difficulty of tissue-specific and developmental stage-specific regulated expression of transgenes, as well as the difficulty of introducing a transgene into all cells in a specific tissue, prevents us from successfully applying gene therapy to many human diseases. In contrast to intracellular proteins, an ECM protein is anchored to the target tissue via its specific binding affinity for protein(s) expressed on the cell surface within the target tissue. Exploiting this unique feature of ECM proteins, we developed protein-anchoring therapy in which a transgene product expressed even in remote tissues can be delivered and anchored to a target tissue using specific binding signals. We demonstrate the application of protein-anchoring therapy to two disease models. First, intravenous administration of adeno-associated virus (AAV) serotype 8-COLQ to Colq-deficient mice, resulting in specific anchoring of ectopically expressed ColQ-AChE at the NMJ, markedly improved motor functions, synaptic transmission, and the ultrastructure of the neuromuscular junction (NMJ). In the second example, Mdx mice, a model for Duchenne muscular dystrophy, were intravenously injected with AAV8-BGN. The treatment ameliorated motor deficits, mitigated muscle histopathologies, decreased plasma creatine kinase activities, and upregulated expression

  18. Targeting protein-trafficking pathways alters melanoma treatment sensitivity

    PubMed Central

    Huang, Zhi-ming; Chinen, Milka; Chang, Philip J.; Xie, Tong; Zhong, Lily; Demetriou, Stephanie; Patel, Mira P.; Scherzer, Rebecca; Sviderskaya, Elena V.; Bennett, Dorothy C.; Millhauser, Glenn L.; Oh, Dennis H.; Cleaver, James E.; Wei, Maria L.

    2012-01-01

    Protein-trafficking pathways are targeted here in human melanoma cells using methods independent of oncogene mutational status, and the ability to up-regulate and down-regulate tumor treatment sensitivity is demonstrated. Sensitivity of melanoma cells to cis-diaminedichloroplatinum II (cDDP, cis-platin), carboplatin, dacarbazine, or temozolomide together with velaparib, an inhibitor of poly (ADP ribose) polymerase 1, is increased by up to 10-fold by targeting genes that regulate both protein trafficking and the formation of melanosomes, intracellular organelles unique to melanocytes and melanoma cells. Melanoma cells depleted of either of the protein-trafficking regulators vacuolar protein sorting 33A protein (VPS33A) or cappuccino protein (CNO) have increased nuclear localization of cDDP, increased nuclear DNA damage by platination, and increased apoptosis, resulting in increased treatment sensitivity. Depleted cells also exhibit a decreased proportion of intracellular, mature melanosomes compared with undepleted cells. Modulation of protein trafficking via cell-surface signaling by binding the melanocortin 1 receptor with the antagonist agouti-signaling protein decreased the proportion of mature melanosomes formed and increased cDDP sensitivity, whereas receptor binding with the agonist melanocyte-stimulating hormone resulted in an increased proportion of mature melanosomes formed and in decreased sensitivity (i.e., increased resistance) to cDDP. Mutation of the protein-trafficking gene Hps6, known to impair the formation of mature melanosomes, also increased cDDP sensitivity. Together, these results indicate that targeting protein-trafficking molecules markedly increases melanoma treatment sensitivity and influences the degree of melanosomes available for sequestration of therapeutic agents. PMID:22203954

  19. Selective targeting of a TNFR decoy receptor pharmaceutical to the primate brain as a receptor-specific IgG fusion protein.

    PubMed

    Boado, Ruben J; Hui, Eric Ka-Wai; Lu, Jeff Zhiqiang; Zhou, Qing-Hui; Pardridge, William M

    2010-03-01

    Decoy receptors, such as the human tumor necrosis factor receptor (TNFR), are potential new therapies for brain disorders. However, decoy receptors are large molecule drugs that are not transported across the blood-brain barrier (BBB). To enable BBB transport of a TNFR decoy receptor, the human TNFR-II extracellular domain was re-engineered as a fusion protein with a chimeric monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb acts as a molecular Trojan horse to ferry the TNFR therapeutic decoy receptor across the BBB. The HIRMAb-TNFR fusion protein was expressed in stably transfected CHO cells, and was analyzed with electrophoresis, Western blotting, size exclusion chromatography, and binding assays for the HIR and TNFalpha. The HIRMAb-TNFR fusion protein was radio-labeled by trititation, in parallel with the radio-iodination of recombinant TNFR:Fc fusion protein, and the proteins were co-injected in the adult Rhesus monkey. The TNFR:Fc fusion protein did not cross the primate BBB in vivo, but the uptake of the HIRMAb-TNFR fusion protein was high and 3% of the injected dose was taken up by the primate brain. The TNFR was selectively targeted to brain, relative to peripheral organs, following fusion to the HIRMAb. This study demonstrates that decoy receptors may be re-engineered as IgG fusion proteins with a BBB molecular Trojan horse that selectively targets the brain, and enables penetration of the BBB in vivo. IgG-decoy receptor fusion proteins represent a new class of human neurotherapeutics. Copyright 2010 Elsevier B.V. All rights reserved.

  20. Targeting BET bromodomain proteins in solid tumors

    PubMed Central

    Sahai, Vaibhav; Redig, Amanda J.; Collier, Katharine A.; Eckerdt, Frank D.; Munshi, Hidayatullah G.

    2016-01-01

    There is increasing interest in inhibitors targeting BET (bromodomain and extra-terminal) proteins because of the association between this family of proteins and cancer progression. BET inhibitors were initially shown to have efficacy in hematologic malignancies; however, a number of studies have now shown that BET inhibitors can also block progression of non-hematologic malignancies. In this Review, we summarize the efficacy of BET inhibitors in select solid tumors; evaluate the role of BET proteins in mediating resistance to current targeted therapies; and consider potential toxicities of BET inhibitors. We also evaluate recently characterized mechanisms of resistance to BET inhibitors; summarize ongoing clinical trials with these inhibitors; and discuss potential future roles of BET inhibitors in patients with solid tumors. PMID:27283767

  1. POEMS Syndrome Diagnosed 10 Years after Disabling Peripheral Neuropathy.

    PubMed

    Nguyen, Viet H

    2011-01-01

    Peripheral neuropathy is characterized as a generalized, relatively homogeneous process affecting many peripheral nerves and predominantly affecting distal nerves. The epidemiology of peripheral neuropathy is limited since the disease presents with varying etiology, pathology, and severity. Toxic, inflammatory, hereditary, and infectious factors can cause damage to the peripheral nerves resulting in peripheral neuropathy. Peripheral neuropathy is most commonly caused by diabetes, alcohol, HIV infection, and malignancy. We report a case of a 42-year-old female with 10-year history of progressively worsening peripheral neuropathy, hypothyroidism, and skin changes who presents with dyspnea secondary to recurrent pleural and pericardial effusions. Prior to her arrival, her peripheral neuropathy was believed to be secondary to chronic demyelinating inflammatory polyneuropathy (CDIP) given elevated protein in the cerebral spinal fluid (CSF) which was treated with intravenous immunoglobulin (IVIG) and corticosteroids. Unfortunately, her peripheral neuropathy did not have any improvement. Incidentally, patient was found to have splenomegaly and papilledema on physical exam. Serum protein electrophoresis showed a monoclonal pattern of IgA lambda. Patient met the diagnostic criteria for POEMS (polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes) syndrome. An underlying diagnosis of POEMS syndrome should be considered in patients with chronic debilitating neuropathy and an elevated protein in the CSF.

  2. POEMS Syndrome Diagnosed 10 Years after Disabling Peripheral Neuropathy

    PubMed Central

    Nguyen, Viet H.

    2011-01-01

    Peripheral neuropathy is characterized as a generalized, relatively homogeneous process affecting many peripheral nerves and predominantly affecting distal nerves. The epidemiology of peripheral neuropathy is limited since the disease presents with varying etiology, pathology, and severity. Toxic, inflammatory, hereditary, and infectious factors can cause damage to the peripheral nerves resulting in peripheral neuropathy. Peripheral neuropathy is most commonly caused by diabetes, alcohol, HIV infection, and malignancy. We report a case of a 42-year-old female with 10-year history of progressively worsening peripheral neuropathy, hypothyroidism, and skin changes who presents with dyspnea secondary to recurrent pleural and pericardial effusions. Prior to her arrival, her peripheral neuropathy was believed to be secondary to chronic demyelinating inflammatory polyneuropathy (CDIP) given elevated protein in the cerebral spinal fluid (CSF) which was treated with intravenous immunoglobulin (IVIG) and corticosteroids. Unfortunately, her peripheral neuropathy did not have any improvement. Incidentally, patient was found to have splenomegaly and papilledema on physical exam. Serum protein electrophoresis showed a monoclonal pattern of IgA lambda. Patient met the diagnostic criteria for POEMS (polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes) syndrome. An underlying diagnosis of POEMS syndrome should be considered in patients with chronic debilitating neuropathy and an elevated protein in the CSF. PMID:22013451

  3. Aptamers as inhibitors of target proteins.

    PubMed

    Missailidis, S; Hardy, A

    2009-08-01

    Aptamers as inhibitors of proteins in therapeutic applications offer great advantages over their antibody counterparts and the promise to be developed into the next generation therapeutic agents. However, the control of aptamer intellectual property (IP) by two major players has made aptamers an area difficult to operate and often off-putting for academic and commercial organisations. Yet, their great potential is keeping aptamers at the research forefront, with one aptamer in the clinic and various at different stages of clinical trials. To provide a comprehensive review of the aptamer IP landscape and the issues associated with aptamer therapeutics against protein targets. Extensive review of the scientific and patent literature. Following our experience in developing, patenting and commercialising our aptamers against MUC1 and an extensive review of the literature, we have identified a variety of issues pertaining to the development of aptamers against protein targets for therapeutic applications, their patenting and granting of patents, the original IP holders and their policy, as well as the current market and traits. Despite a slow start, aptamers have been developed against various therapeutic proteins and offer the promise of providing a novel generation of therapeutic entities with a variety of applications.

  4. Protein phosphorylation in human peripheral blood lymphocytes. Phosphorylation of endogenous plasma membrane and cytoplasmic proteins

    PubMed Central

    Chaplin, David D.; Wedner, H. James; Parker, Charles W.

    1979-01-01

    Phosphorylation of endogenous proteins in subcellular fractions of human peripheral-blood lymphocytes was studied by one- and two-dimensional polyacrylamide-gel electrophoresis. Studies using extensively purified subcellular fractions indicated that the endogenous phosphorylating activity in the particulate fractions was derived primarily from the plasma membrane. Electrophoresis of 32P-labelled subcellular fractions in two dimensions [O'Farrell (1975) J. Biol. Chem. 250, 4007–4021] provided much greater resolution of the endogenous phosphoproteins than electrophoresis in one dimension, facilitating their excision from gels for quantification of 32P content. More than 100 cytoplasmic and 20 plasma-membrane phosphorylated species were observed. Phosphorylation of more than 10 cytoplasmic proteins was absolutely dependent on cyclic AMP. In the plasma membrane, cyclic AMP-dependent phosphoproteins were observed with mol.wts. of 42000, 42000, 80000 and 90000 and pI values of 6.1, 6.3, 6.25 and 6.5 respectively. Phosphorylation of endogenous cytoplasmic and plasma-membrane proteins was rapid with t½=5–12s at 25°C. Between 40 and 70% of the 32P was recovered as phosphoserine and phosphothreonine when acid hydrolysates of isolated plasma-membrane phosphoproteins were analysed by high-voltage paper electrophoresis. The presence of cyclic AMP-dependent protein kinase and endogenous phosphate-acceptor proteins in the plasma membranes of lymphocytes provides a mechanism by which these cells might respond to plasma-membrane pools of cyclic AMP generated in response to stimulation by mitogens or physiological modulators of lymphocyte function. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4. PMID:228657

  5. Wiskott-Aldrich syndrome protein deficiency in B cells results in impaired peripheral homeostasis

    PubMed Central

    Meyer-Bahlburg, Almut; Becker-Herman, Shirly; Humblet-Baron, Stephanie; Khim, Socheath; Weber, Michele; Bouma, Gerben; Thrasher, Adrian J.; Batista, Facundo D.

    2008-01-01

    To more precisely identify the B-cell phenotype in Wiskott-Aldrich syndrome (WAS), we used 3 distinct murine in vivo models to define the cell intrinsic requirements for WAS protein (WASp) in central versus peripheral B-cell development. Whereas WASp is dispensable for early bone marrow B-cell development, WASp deficiency results in a marked reduction in each of the major mature peripheral B-cell subsets, exerting the greatest impact on marginal zone and B1a B cells. Using in vivo bromodeoxyuridine labeling and in vitro functional assays, we show that these deficits reflect altered peripheral homeostasis, partially resulting from an impairment in integrin function, rather than a developmental defect. Consistent with these observations, we also show that: (1) WASp expression levels increase with cell maturity, peaking in those subsets exhibiting the greatest sensitivity to WASp deficiency; (2) WASp+ murine B cells exhibit a marked selective advantage beginning at the late transitional B-cell stage; and (3) a similar in vivo selective advantage is manifest by mature WASp+ human B cells. Together, our data provide a better understanding of the clinical phenotype of WAS and suggest that gene therapy might be a useful approach to rescue altered B-cell homeostasis in this disease. PMID:18687984

  6. Receptor Tyrosine Kinases as Targets for Treatment of Peripheral Nerve Sheath Tumors in NF 1 Patients

    DTIC Science & Technology

    2010-03-01

    targeted therapy of MPNST. Acknowledgements We thank Kathrein Stichling and Petra Matylewski for their technical assist- ance and Prof. Karl Riabowol...neurofibromatosis. Oncogene, 17, 795–800. 13.Leroy,K., Dumas,V., Martin -Garcia,N. et al. (2001) Malignant peripheral nerve sheath tumors associated with...specific inhibitors. Finally, a combination of drugs is likely to be most effec- tive in combating MPNSTs. Acknowledgments We thank Petra Matylewski

  7. TARGETED DELIVERY OF INHALED PROTEINS

    EPA Science Inventory

    ETD-02-047 (Martonen) GPRA # 10108

    TARGETED DELIVERY OF INHALED PROTEINS
    T. B. Martonen1, J. Schroeter2, Z. Zhang3, D. Hwang4, and J. S. Fleming5
    1Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Research Triangle Park...

  8. Nonhistone protein acetylation as cancer therapy targets

    PubMed Central

    Singh, Brahma N; Zhang, Guanghua; Hwa, Yi L; Li, Jinping; Dowdy, Sean C; Jiang, Shi-Wen

    2012-01-01

    Acetylation and deacetylation are counteracting, post-translational modifications that affect a large number of histone and nonhistone proteins. The significance of histone acetylation in the modification of chromatin structure and dynamics, and thereby gene transcription regulation, has been well recognized. A steadily growing number of nonhistone proteins have been identified as acetylation targets and reversible lysine acetylation in these proteins plays an important role(s) in the regulation of mRNA stability, protein localization and degradation, and protein–protein and protein–DNA interactions. The recruitment of histone acetyltransferases (HATs) and histone deacetylases (HDACs) to the transcriptional machinery is a key element in the dynamic regulation of genes controlling cellular proliferation, differentiation and apoptosis. Many nonhistone proteins targeted by acetylation are the products of oncogenes or tumor-suppressor genes and are directly involved in tumorigenesis, tumor progression and metastasis. Aberrant activity of HDACs has been documented in several types of cancers and HDAC inhibitors (HDACi) have been employed for therapeutic purposes. Here we review the published literature in this field and provide updated information on the regulation and function of nonhistone protein acetylation. While concentrating on the molecular mechanism and pathways involved in the addition and removal of the acetyl moiety, therapeutic modalities of HDACi are also discussed. PMID:20553216

  9. Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling.

    PubMed

    Arriaga, Gustavo; Macopson, Joshua J; Jarvis, Erich D

    2015-09-14

    Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)(1). PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site(2,3). Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)(4,5). This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3'-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites(6-11).

  10. Targeted Quantitation of Proteins by Mass Spectrometry

    PubMed Central

    2013-01-01

    Quantitative measurement of proteins is one of the most fundamental analytical tasks in a biochemistry laboratory, but widely used immunochemical methods often have limited specificity and high measurement variation. In this review, we discuss applications of multiple-reaction monitoring (MRM) mass spectrometry, which allows sensitive, precise quantitative analyses of peptides and the proteins from which they are derived. Systematic development of MRM assays is permitted by databases of peptide mass spectra and sequences, software tools for analysis design and data analysis, and rapid evolution of tandem mass spectrometer technology. Key advantages of MRM assays are the ability to target specific peptide sequences, including variants and modified forms, and the capacity for multiplexing that allows analysis of dozens to hundreds of peptides. Different quantitative standardization methods provide options that balance precision, sensitivity, and assay cost. Targeted protein quantitation by MRM and related mass spectrometry methods can advance biochemistry by transforming approaches to protein measurement. PMID:23517332

  11. Targeted quantitation of proteins by mass spectrometry.

    PubMed

    Liebler, Daniel C; Zimmerman, Lisa J

    2013-06-04

    Quantitative measurement of proteins is one of the most fundamental analytical tasks in a biochemistry laboratory, but widely used immunochemical methods often have limited specificity and high measurement variation. In this review, we discuss applications of multiple-reaction monitoring (MRM) mass spectrometry, which allows sensitive, precise quantitative analyses of peptides and the proteins from which they are derived. Systematic development of MRM assays is permitted by databases of peptide mass spectra and sequences, software tools for analysis design and data analysis, and rapid evolution of tandem mass spectrometer technology. Key advantages of MRM assays are the ability to target specific peptide sequences, including variants and modified forms, and the capacity for multiplexing that allows analysis of dozens to hundreds of peptides. Different quantitative standardization methods provide options that balance precision, sensitivity, and assay cost. Targeted protein quantitation by MRM and related mass spectrometry methods can advance biochemistry by transforming approaches to protein measurement.

  12. The search of the target of promotion: Phenylbenzoate esterase activities in hen peripheral nerve

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Moretto, A.; Nicolli, A.; Lotti, M.

    2007-03-15

    Certain esterase inhibitors, such as carbamates, phosphinates and sulfonyl halides, do not cause neuropathy as some organophosphates, but they may exacerbate chemical or traumatic insults to axons. This phenomenon is called promotion of axonopathies. Given the biochemical and toxicological characteristics of these compounds, the hypothesis was made that the target of promotion is a phenyl valerate (PV) esterase similar to neuropathy target esterase (NTE), the target of organophosphate induced delayed polyneuropathy. However, attempts to identify a PV esterase in hen peripheral nerve have been, so far, unsuccessful. We tested several esters, other than PV, as substrates of esterases from crudemore » homogenate of the hen peripheral nerve. The ideal substrate should be poorly hydrolysed by NTE but extensively by enzyme(s) that are insensitive to non-promoters, such as mipafox, and sensitive to promoters, such as phenyl methane sulfonyl fluoride (PMSF). When phenyl benzoate (PB) was used as substrate, about 65% of total activity was resistant to the non-promoter mipafox (up to 0.5 mM, 20 min, pH 8.0), that inhibits NTE and other esterases. More than 90% of this resistant activity was sensitive to the classical promoter PMSF (1 mM, 20 min, pH 8.0) with an IC{sub 50} of about 0.08 mM (20 min, pH 8.0). On the contrary, the non-promoter p-toluene sulfonyl fluoride caused only about 10% inhibition at 0.5 mM. Several esterase inhibitors including, paraoxon, phenyl benzyl carbamate, di-n-butyl dichlorovinyl phosphate and di-isopropyl fluorophosphate, were tested both in vitro and in vivo for inhibition of this PB activity. Mipafox-resistant PMSF-sensitive PB esterase activity(ies) was inhibited by promoters but not by non promoters and neuropathic compounds.« less

  13. Cell cycle proteins as promising targets in cancer therapy.

    PubMed

    Otto, Tobias; Sicinski, Piotr

    2017-01-27

    Cancer is characterized by uncontrolled tumour cell proliferation resulting from aberrant activity of various cell cycle proteins. Therefore, cell cycle regulators are considered attractive targets in cancer therapy. Intriguingly, animal models demonstrate that some of these proteins are not essential for proliferation of non-transformed cells and development of most tissues. By contrast, many cancers are uniquely dependent on these proteins and hence are selectively sensitive to their inhibition. After decades of research on the physiological functions of cell cycle proteins and their relevance for cancer, this knowledge recently translated into the first approved cancer therapeutic targeting of a direct regulator of the cell cycle. In this Review, we focus on proteins that directly regulate cell cycle progression (such as cyclin-dependent kinases (CDKs)), as well as checkpoint kinases, Aurora kinases and Polo-like kinases (PLKs). We discuss the role of cell cycle proteins in cancer, the rationale for targeting them in cancer treatment and results of clinical trials, as well as the future therapeutic potential of various cell cycle inhibitors.

  14. A Network-Biology Informed Computational Drug Repositioning Strategy to Target Disease Risk Trajectories and Comorbidities of Peripheral Artery Disease.

    PubMed

    Shameer, Khader; Dow, Garrett; Glicksberg, Benjamin S; Johnson, Kipp W; Ze, Yi; Tomlinson, Max S; Readhead, Ben; Dudley, Joel T; Kullo, Iftikhar J

    2018-01-01

    Currently, drug discovery approaches focus on the design of therapies that alleviate an index symptom by reengineering the underlying biological mechanism in agonistic or antagonistic fashion. For example, medicines are routinely developed to target an essential gene that drives the disease mechanism. Therapeutic overloading where patients get multiple medications to reduce the primary and secondary side effect burden is standard practice. This single-symptom based approach may not be scalable, as we understand that diseases are more connected than random and molecular interactions drive disease comorbidities. In this work, we present a proof-of-concept drug discovery strategy by combining network biology, disease comorbidity estimates, and computational drug repositioning, by targeting the risk factors and comorbidities of peripheral artery disease, a vascular disease associated with high morbidity and mortality. Individualized risk estimation and recommending disease sequelae based therapies may help to lower the mortality and morbidity of peripheral artery disease.

  15. Parkinson's disease proteins: Novel mitochondrial targets for cardioprotection

    PubMed Central

    Mukherjee, Uma A.; Ong, Sang-Bing; Ong, Sang-Ging; Hausenloy, Derek J.

    2015-01-01

    Ischemic heart disease (IHD) is the leading cause of death and disability worldwide. Therefore, novel therapeutic targets for protecting the heart against acute ischemia/reperfusion injury (IRI) are required to attenuate cardiomyocyte death, preserve myocardial function, and prevent the onset of heart failure. In this regard, a specific group of mitochondrial proteins, which have been linked to familial forms of Parkinson's disease (PD), may provide novel therapeutic targets for cardioprotection. In dopaminergic neurons of the substantia nigra, these PD proteins, which include Parkin, PINK1, DJ-1, LRRK2, and α-synuclein, play essential roles in preventing cell death—through maintaining normal mitochondrial function, protecting against oxidative stress, mediating mitophagy, and preventing apoptosis. These rare familial forms of PD may therefore provide important insights into the pathophysiology underlying mitochondrial dysfunction and the development of PD. Interestingly, these PD proteins are also present in the heart, but their role in myocardial health and disease is not clear. In this article, we review the role of these PD proteins in the heart and explore their potential as novel mitochondrial targets for cardioprotection. PMID:26481155

  16. Protein-Protein Interactions of Viroporins in Coronaviruses and Paramyxoviruses: New Targets for Antivirals?

    PubMed Central

    Torres, Jaume; Surya, Wahyu; Li, Yan; Liu, Ding Xiang

    2015-01-01

    Viroporins are members of a rapidly growing family of channel-forming small polypeptides found in viruses. The present review will be focused on recent structural and protein-protein interaction information involving two viroporins found in enveloped viruses that target the respiratory tract; (i) the envelope protein in coronaviruses and (ii) the small hydrophobic protein in paramyxoviruses. Deletion of these two viroporins leads to viral attenuation in vivo, whereas data from cell culture shows involvement in the regulation of stress and inflammation. The channel activity and structure of some representative members of these viroporins have been recently characterized in some detail. In addition, searches for protein-protein interactions using yeast-two hybrid techniques have shed light on possible functional roles for their exposed cytoplasmic domains. A deeper analysis of these interactions should not only provide a more complete overview of the multiple functions of these viroporins, but also suggest novel strategies that target protein-protein interactions as much needed antivirals. These should complement current efforts to block viroporin channel activity. PMID:26053927

  17. Protein targeting and integration signal for the chloroplastic outer envelope membrane.

    PubMed Central

    Li, H M; Chen, L J

    1996-01-01

    Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol. With the exception of most quter envelope membrane proteins, nuclear-encoded chloroplastic proteins are synthesized with N-terminal extensions that contain the chloroplast targeting information of these proteins. Most outer membrane proteins, however, are synthesized without extensions in the cytosol. Therefore, it is not clear where the chloroplastic outer membrane targeting information resides within these polypeptides. We have analyzed a chloroplastic outer membrane protein, OEP14 (outer envelope membrane protein of 14 kD, previously named OM14), and localized its outer membrane targeting and integration signal to the first 30 amino acids of the protein. This signal consists of a positively charged N-terminal portion followed by a hydrophobic core, bearing resemblance to the signal peptides of proteins targeted to the endoplasmic reticulum. However, a chimeric protein containing this signal fused to a passenger protein did not integrate into the endoplasmic reticulum membrane. Furthermore, membrane topology analysis indicated that the signal inserts into the chloroplastic outer membrane in an orientation opposite to that predicted by the "positive inside" rule. PMID:8953775

  18. Non-Invasive Targeted Peripheral Nerve Ablation Using 3D MR Neurography and MRI-Guided High-Intensity Focused Ultrasound (MR-HIFU): Pilot Study in a Swine Model.

    PubMed

    Huisman, Merel; Staruch, Robert M; Ladouceur-Wodzak, Michelle; van den Bosch, Maurice A; Burns, Dennis K; Chhabra, Avneesh; Chopra, Rajiv

    2015-01-01

    Ultrasound (US)-guided high intensity focused ultrasound (HIFU) has been proposed for noninvasive treatment of neuropathic pain and has been investigated in in-vivo studies. However, ultrasound has important limitations regarding treatment guidance and temperature monitoring. Magnetic resonance (MR)-imaging guidance may overcome these limitations and MR-guided HIFU (MR-HIFU) has been used successfully for other clinical indications. The primary purpose of this study was to evaluate the feasibility of utilizing 3D MR neurography to identify and guide ablation of peripheral nerves using a clinical MR-HIFU system. Volumetric MR-HIFU was used to induce lesions in the peripheral nerves of the lower limbs in three pigs. Diffusion-prep MR neurography and T1-weighted images were utilized to identify the target, plan treatment and immediate post-treatment evaluation. For each treatment, one 8 or 12 mm diameter treatment cell was used (sonication duration 20 s and 36 s, power 160-300 W). Peripheral nerves were extracted < 3 hours after treatment. Ablation dimensions were calculated from thermal maps, post-contrast MRI and macroscopy. Histological analysis included standard H&E staining, Masson's trichrome and toluidine blue staining. All targeted peripheral nerves were identifiable on MR neurography and T1-weighted images and could be accurately ablated with a single exposure of focused ultrasound, with peak temperatures of 60.3 to 85.7°C. The lesion dimensions as measured on MR neurography were similar to the lesion dimensions as measured on CE-T1, thermal dose maps, and macroscopy. Histology indicated major hyperacute peripheral nerve damage, mostly confined to the location targeted for ablation. Our preliminary results indicate that targeted peripheral nerve ablation is feasible with MR-HIFU. Diffusion-prep 3D MR neurography has potential for guiding therapy procedures where either nerve targeting or avoidance is desired, and may also have potential for post

  19. Non-Invasive Targeted Peripheral Nerve Ablation Using 3D MR Neurography and MRI-Guided High-Intensity Focused Ultrasound (MR-HIFU): Pilot Study in a Swine Model

    PubMed Central

    Huisman, Merel; Staruch, Robert M.; Ladouceur-Wodzak, Michelle; van den Bosch, Maurice A.; Burns, Dennis K.; Chhabra, Avneesh; Chopra, Rajiv

    2015-01-01

    Purpose Ultrasound (US)-guided high intensity focused ultrasound (HIFU) has been proposed for noninvasive treatment of neuropathic pain and has been investigated in in-vivo studies. However, ultrasound has important limitations regarding treatment guidance and temperature monitoring. Magnetic resonance (MR)-imaging guidance may overcome these limitations and MR-guided HIFU (MR-HIFU) has been used successfully for other clinical indications. The primary purpose of this study was to evaluate the feasibility of utilizing 3D MR neurography to identify and guide ablation of peripheral nerves using a clinical MR-HIFU system. Methods Volumetric MR-HIFU was used to induce lesions in the peripheral nerves of the lower limbs in three pigs. Diffusion-prep MR neurography and T1-weighted images were utilized to identify the target, plan treatment and immediate post-treatment evaluation. For each treatment, one 8 or 12 mm diameter treatment cell was used (sonication duration 20 s and 36 s, power 160–300 W). Peripheral nerves were extracted < 3 hours after treatment. Ablation dimensions were calculated from thermal maps, post-contrast MRI and macroscopy. Histological analysis included standard H&E staining, Masson’s trichrome and toluidine blue staining. Results All targeted peripheral nerves were identifiable on MR neurography and T1-weighted images and could be accurately ablated with a single exposure of focused ultrasound, with peak temperatures of 60.3 to 85.7°C. The lesion dimensions as measured on MR neurography were similar to the lesion dimensions as measured on CE-T1, thermal dose maps, and macroscopy. Histology indicated major hyperacute peripheral nerve damage, mostly confined to the location targeted for ablation. Conclusion Our preliminary results indicate that targeted peripheral nerve ablation is feasible with MR-HIFU. Diffusion-prep 3D MR neurography has potential for guiding therapy procedures where either nerve targeting or avoidance is desired, and may

  20. Deployment of spatial attention to words in central and peripheral vision.

    PubMed

    Ducrot, Stéphanie; Grainger, Jonathan

    2007-05-01

    Four perceptual identification experiments examined the influence of spatial cues on the recognition of words presented in central vision (with fixation on either the first or last letter of the target word) and in peripheral vision (displaced left or right of a central fixation point). Stimulus location had a strong effect on word identification accuracy in both central and peripheral vision, showing a strong right visual field superiority that did not depend on eccentricity. Valid spatial cues improved word identification for peripherally presented targets but were largely ineffective for centrally presented targets. Effects of spatial cuing interacted with visual field effects in Experiment 1, with valid cues reducing the right visual field superiority for peripherally located targets, but this interaction was shown to depend on the type of neutral cue. These results provide further support for the role of attentional factors in visual field asymmetries obtained with targets in peripheral vision but not with centrally presented targets.

  1. Identifying protein kinase target preferences using mass spectrometry

    PubMed Central

    Douglass, Jacqueline; Gunaratne, Ruwan; Bradford, Davis; Saeed, Fahad; Hoffert, Jason D.; Steinbach, Peter J.; Pisitkun, Trairak

    2012-01-01

    A general question in molecular physiology is how to identify candidate protein kinases corresponding to a known or hypothetical phosphorylation site in a protein of interest. It is generally recognized that the amino acid sequence surrounding the phosphorylation site provides information that is relevant to identification of the cognate protein kinase. Here, we present a mass spectrometry-based method for profiling the target specificity of a given protein kinase as well as a computational tool for the calculation and visualization of the target preferences. The mass spectrometry-based method identifies sites phosphorylated in response to in vitro incubation of protein mixtures with active recombinant protein kinases followed by standard phosphoproteomic methodologies. The computational tool, called “PhosphoLogo,” uses an information-theoretic algorithm to calculate position-specific amino acid preferences and anti-preferences from the mass-spectrometry data (http://helixweb.nih.gov/PhosphoLogo/). The method was tested using protein kinase A (catalytic subunit α), revealing the well-known preference for basic amino acids in positions −2 and −3 relative to the phosphorylated amino acid. It also provides evidence for a preference for amino acids with a branched aliphatic side chain in position +1, a finding compatible with known crystal structures of protein kinase A. The method was also employed to profile target preferences and anti-preferences for 15 additional protein kinases with potential roles in regulation of epithelial transport: CK2, p38, AKT1, SGK1, PKCδ, CaMK2δ, DAPK1, MAPKAPK2, PKD3, PIM1, OSR1, STK39/SPAK, GSK3β, Wnk1, and Wnk4. PMID:22723110

  2. Microtubule-Targeting Agents Eribulin and Paclitaxel Differentially Affect Neuronal Cell Bodies in Chemotherapy-Induced Peripheral Neuropathy.

    PubMed

    Benbow, Sarah J; Wozniak, Krystyna M; Kulesh, Bridget; Savage, April; Slusher, Barbara S; Littlefield, Bruce A; Jordan, Mary Ann; Wilson, Leslie; Feinstein, Stuart C

    2017-07-01

    Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of anticancer treatment with microtubule-targeted agents (MTAs). The frequency of severe CIPN, which can be dose limiting and even life threatening, varies widely among different MTAs. For example, paclitaxel induces a higher frequency of severe CIPN than does eribulin. Different MTAs also possess distinct mechanisms of microtubule-targeted action. Recently, we demonstrated that paclitaxel and eribulin differentially affect sciatic nerve axons, with paclitaxel inducing more pronounced neurodegenerative effects and eribulin inducing greater microtubule stabilizing biochemical effects. Here, we complement and extend these axonal studies by assessing the effects of paclitaxel and eribulin in the cell bodies of sciatic nerve axons, housed in the dorsal root ganglia (DRG). Importantly, the microtubule network in cell bodies is known to be significantly more dynamic than in axons. Paclitaxel induced activating transcription factor 3 expression, a marker of neuronal stress/injury. Paclitaxel also increased expression levels of acetylated tubulin and end binding protein 1, markers of microtubule stability and growth, respectively. These effects are hypothesized to be detrimental to the dynamic microtubule network within the cell bodies. In contrast, eribulin had no significant effect on any of these parameters in the cell bodies. Taken together, DRG cell bodies and their axons, two distinct neuronal cell compartments, contain functionally distinct microtubule networks that exhibit unique biochemical responses to different MTA treatments. We hypothesize that these distinct mechanistic actions may underlie the variability seen in the initiation, progression, persistence, and recovery from CIPN.

  3. Protein painting reveals solvent-excluded drug targets hidden within native protein–protein interfaces

    PubMed Central

    Luchini, Alessandra; Espina, Virginia; Liotta, Lance A.

    2014-01-01

    Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein–protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets. PMID:25048602

  4. Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks

    DTIC Science & Technology

    2012-09-21

    Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks Paulo Shakarian1*, J. Kenneth Wickiser2 1 Paulo Shakarian...significantly attacked. Citation: Shakarian P, Wickiser JK (2012) Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks...to 00-00-2012 4. TITLE AND SUBTITLE Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks 5a. CONTRACT NUMBER 5b

  5. Genomes2Drugs: Identifies Target Proteins and Lead Drugs from Proteome Data

    PubMed Central

    Toomey, David; Hoppe, Heinrich C.; Brennan, Marian P.; Nolan, Kevin B.; Chubb, Anthony J.

    2009-01-01

    Background Genome sequencing and bioinformatics have provided the full hypothetical proteome of many pathogenic organisms. Advances in microarray and mass spectrometry have also yielded large output datasets of possible target proteins/genes. However, the challenge remains to identify new targets for drug discovery from this wealth of information. Further analysis includes bioinformatics and/or molecular biology tools to validate the findings. This is time consuming and expensive, and could fail to yield novel drugs if protein purification and crystallography is impossible. To pre-empt this, a researcher may want to rapidly filter the output datasets for proteins that show good homology to proteins that have already been structurally characterised or proteins that are already targets for known drugs. Critically, those researchers developing novel antibiotics need to select out the proteins that show close homology to any human proteins, as future inhibitors are likely to cross-react with the host protein, causing off-target toxicity effects later in clinical trials. Methodology/Principal Findings To solve many of these issues, we have developed a free online resource called Genomes2Drugs which ranks sequences to identify proteins that are (i) homologous to previously crystallized proteins or (ii) targets of known drugs, but are (iii) not homologous to human proteins. When tested using the Plasmodium falciparum malarial genome the program correctly enriched the ranked list of proteins with known drug target proteins. Conclusions/Significance Genomes2Drugs rapidly identifies proteins that are likely to succeed in drug discovery pipelines. This free online resource helps in the identification of potential drug targets. Importantly, the program further highlights proteins that are likely to be inhibited by FDA-approved drugs. These drugs can then be rapidly moved into Phase IV clinical studies under ‘change-of-application’ patents. PMID:19593435

  6. Phage protein-targeted cancer nanomedicines

    PubMed Central

    Petrenko, V.A.; Jayanna, P.K.

    2015-01-01

    Nanoencapsulation of anticancer drugs improves their therapeutic indices by virtue of the enhanced permeation and retention effect which achieves passive targeting of nanoparticles in tumors. This effect can be significantly enhanced by active targeting of nanovehicles to tumors. Numerous ligands have been proposed and used in various studies with peptides being considered attractive alternatives to antibodies. This is further reinforced by the availability of peptide phage display libraries which offer an unlimited reservoir of target-specific probes. In particular landscape phages with multivalent display of target-specific peptides which enable the phage particle itself to become a nanoplatform creates a paradigm for high throughput selection of nanoprobes setting the stage for personalized cancer management. Despite its promise, this conjugate of combinatorial chemistry and nanotechnology has not made a significant clinical impact in cancer management due to a lack of using robust processes that facilitate scale-up and manufacturing. To this end we proposed the use of phage fusion protein as the navigating modules of novel targeted nanomedicine platforms which are described in this review. PMID:24269681

  7. Structural Basis for Induction of Peripheral Neuropathy by Microtubule-Targeting Cancer Drugs.

    PubMed

    Smith, Jennifer A; Slusher, Barbara S; Wozniak, Krystyna M; Farah, Mohamed H; Smiyun, Gregoriy; Wilson, Leslie; Feinstein, Stuart; Jordan, Mary Ann

    2016-09-01

    Peripheral neuropathy is a serious, dose-limiting side effect of cancer treatment with microtubule-targeting drugs. Symptoms present in a "stocking-glove" distribution, with longest nerves affected most acutely, suggesting a length-dependent component to the toxicity. Axonal transport of ATP-producing mitochondria along neuronal microtubules from cell body to synapse is crucial to neuronal function. We compared the effects of the drugs paclitaxel and ixabepilone that bind along the lengths of microtubules and the drugs eribulin and vincristine that bind at microtubule ends, on mitochondrial trafficking in cultured human neuronal SK-N-SH cells and on axonal transport in mouse sciatic nerves. Antiproliferative concentrations of paclitaxel and ixabepilone significantly inhibited the anterograde transport velocity of mitochondria in neuronal cells, whereas eribulin and vincristine inhibited transport only at significantly higher concentrations. Confirming these observations, anterogradely transported amyloid precursor protein accumulated in ligated sciatic nerves of control and eribulin-treated mice, but not in paclitaxel-treated mice, indicating that paclitaxel inhibited anterograde axonal transport, whereas eribulin did not. Electron microscopy of sciatic nerves of paclitaxel-treated mice showed reduced organelle accumulation proximal to the ligation consistent with inhibition of anterograde (kinesin based) transport by paclitaxel. In contrast, none of the drugs significantly affected retrograde (dynein based) transport in neuronal cells or mouse nerves. Collectively, these results suggest that paclitaxel and ixabepilone, which bind along the lengths and stabilize microtubules, inhibit kinesin-based axonal transport, but not dynein-based transport, whereas the microtubule-destabilizing drugs, eribulin and vincristine, which bind preferentially to microtubule ends, have significantly less effect on all microtubule-based axonal transport. Cancer Res; 76(17); 5115-23.

  8. Mechanism-based Proteomic Screening Identifies Targets of Thioredoxin-like Proteins*

    PubMed Central

    Nakao, Lia S.; Everley, Robert A.; Marino, Stefano M.; Lo, Sze M.; de Souza, Luiz E.; Gygi, Steven P.; Gladyshev, Vadim N.

    2015-01-01

    Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes. PMID:25561728

  9. A Network-Biology Informed Computational Drug Repositioning Strategy to Target Disease Risk Trajectories and Comorbidities of Peripheral Artery Disease

    PubMed Central

    Shameer, Khader; Dow, Garrett; Glicksberg, Benjamin S.; Johnson, Kipp W.; Ze, Yi; Tomlinson, Max S.; Readhead, Ben; Dudley, Joel T.; Kullo, Iftikhar J.

    2018-01-01

    Currently, drug discovery approaches focus on the design of therapies that alleviate an index symptom by reengineering the underlying biological mechanism in agonistic or antagonistic fashion. For example, medicines are routinely developed to target an essential gene that drives the disease mechanism. Therapeutic overloading where patients get multiple medications to reduce the primary and secondary side effect burden is standard practice. This single-symptom based approach may not be scalable, as we understand that diseases are more connected than random and molecular interactions drive disease comorbidities. In this work, we present a proof-of-concept drug discovery strategy by combining network biology, disease comorbidity estimates, and computational drug repositioning, by targeting the risk factors and comorbidities of peripheral artery disease, a vascular disease associated with high morbidity and mortality. Individualized risk estimation and recommending disease sequelae based therapies may help to lower the mortality and morbidity of peripheral artery disease. PMID:29888052

  10. Acquisition, Conservation, and Loss of Dual-Targeted Proteins in Land Plants1[W][OA

    PubMed Central

    Xu, Lin; Carrie, Chris; Law, Simon R.; Murcha, Monika W.; Whelan, James

    2013-01-01

    The dual-targeting ability of a variety of proteins from Physcomitrella patens, rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) was tested to determine when dual targeting arose and to what extent it was conserved in land plants. Overall, the targeting ability of over 80 different proteins from rice and P. patens, representing 42 dual-targeted proteins in Arabidopsis, was tested. We found that dual targeting arose early in land plant evolution, as it was evident in many cases with P. patens proteins that were conserved in rice and Arabidopsis. Furthermore, we found that the acquisition of dual-targeting ability is still occurring, evident in P. patens as well as rice and Arabidopsis. The loss of dual-targeting ability appears to be rare, but does occur. Ascorbate peroxidase represents such an example. After gene duplication in rice, individual genes encode proteins that are targeted to a single organelle. Although we found that dual targeting was generally conserved, the ability to detect dual-targeted proteins differed depending on the cell types used. Furthermore, it appears that small changes in the targeting signal can result in a loss (or gain) of dual-targeting ability. Overall, examination of the targeting signals within this study did not reveal any clear patterns that would predict dual-targeting ability. The acquisition of dual-targeting ability also appears to be coordinated between proteins. Mitochondrial intermembrane space import and assembly protein40, a protein involved in oxidative folding in mitochondria and peroxisomes, provides an example where acquisition of dual targeting is accompanied by the dual targeting of substrate proteins. PMID:23257241

  11. Multi-Conformer Ensemble Docking to Difficult Protein Targets

    DOE PAGES

    Ellingson, Sally R.; Miao, Yinglong; Baudry, Jerome; ...

    2014-09-08

    We investigate large-scale ensemble docking using five proteins from the Directory of Useful Decoys (DUD, dud.docking.org) for which docking to crystal structures has proven difficult. Molecular dynamics trajectories are produced for each protein and an ensemble of representative conformational structures extracted from the trajectories. Docking calculations are performed on these selected simulation structures and ensemble-based enrichment factors compared with those obtained using docking in crystal structures of the same protein targets or random selection of compounds. We also found simulation-derived snapshots with improved enrichment factors that increased the chemical diversity of docking hits for four of the five selected proteins.more » A combination of all the docking results obtained from molecular dynamics simulation followed by selection of top-ranking compounds appears to be an effective strategy for increasing the number and diversity of hits when using docking to screen large libraries of chemicals against difficult protein targets.« less

  12. Targeting protein-protein interaction between MLL1 and reciprocal proteins for leukemia therapy.

    PubMed

    Wang, Zhi-Hui; Li, Dong-Dong; Chen, Wei-Lin; You, Qi-Dong; Guo, Xiao-Ke

    2018-01-15

    The mixed lineage leukemia protein-1 (MLL1), as a lysine methyltransferase, predominantly regulates the methylation of histone H3 lysine 4 (H3K4) and functions in hematopoietic stem cell (HSC) self-renewal. MLL1 gene fuses with partner genes that results in the generation of MLL1 fusion proteins (MLL1-FPs), which are frequently detected in acute leukemia. In the progress of leukemogenesis, a great deal of proteins cooperate with MLL1 to form multiprotein complexes serving for the dysregulation of H3K4 methylation, the overexpression of homeobox (HOX) cluster genes, and the consequent generation of leukemia. Hence, disrupting the interactions between MLL1 and the reciprocal proteins has been considered to be a new treatment strategy for leukemia. Here, we reviewed potential protein-protein interactions (PPIs) between MLL1 and its reciprocal proteins, and summarized the inhibitors to target MLL1 PPIs. The druggability of MLL1 PPIs for leukemia were also discussed. Copyright © 2017. Published by Elsevier Ltd.

  13. Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system.

    PubMed

    Bortfeld, M; Rius, M; König, J; Herold-Mende, C; Nies, A T; Keppler, D

    2006-01-01

    Dehydroepiandrosterone 3-sulfate and other neurosteroids are synthesized in the CNS and peripheral nervous system where they may modulate neuronal excitability by interacting with ligand-gated ion channels. For this modulatory activity, neurosteroids have to be locally released from either neurons or glial cells. We here identify the integral membrane protein ABCC11 (multidrug resistance protein 8) as an ATP-dependent efflux pump for steroid sulfates, including dehydroepiandrosterone 3-sulfate, and localize it to axons of the human CNS and peripheral nervous system. ABCC11 mRNA was detected in human brain by real-time polymerase chain reaction. Antibodies raised against ABCC11 served to detect the protein in brain by immunoblotting and immunofluorescence microscopy. ABCC11 was preferentially found in the white matter of the brain and co-localized with neurofilaments indicating that it is an axonal protein. Additionally, ABCC11 was localized to axons of the peripheral nervous system. For functional studies, ABCC11 was expressed in polarized Madin-Darby canine kidney cells where it was sorted to the apical membrane. This apical sorting is in accordance with the localization of ABCC11 to the axonal membrane of neurons. Inside-out plasma membrane vesicles containing recombinant ABCC11 mediated ATP-dependent transport of dehydroepiandrosterone 3-sulfate with a Km value of 21 microM. This transport function together with the localization of the ABCC11 protein in vicinity to GABAA receptors is consistent with a role of ABCC11 in dehydroepiandrosterone 3-sulfate release from neurons to sites of dehydroepiandrosterone 3-sulfate-mediated receptor modulation. Our findings may provide a basis for the characterization of mutations in the human ABCC11 gene and their linkage with neurological disorders.

  14. Phage display selection of peptides that target calcium-binding proteins.

    PubMed

    Vetter, Stefan W

    2013-01-01

    Phage display allows to rapidly identify peptide sequences with binding affinity towards target proteins, for example, calcium-binding proteins (CBPs). Phage technology allows screening of 10(9) or more independent peptide sequences and can identify CBP binding peptides within 2 weeks. Adjusting of screening conditions allows selecting CBPs binding peptides that are either calcium-dependent or independent. Obtained peptide sequences can be used to identify CBP target proteins based on sequence homology or to quickly obtain peptide-based CBP inhibitors to modulate CBP-target interactions. The protocol described here uses a commercially available phage display library, in which random 12-mer peptides are displayed on filamentous M13 phages. The library was screened against the calcium-binding protein S100B.

  15. A cell death assay for assessing the mitochondrial targeting of proteins.

    PubMed

    Camara Teixeira, Daniel; Cordonier, Elizabeth L; Wijeratne, Subhashinee S K; Huebbe, Patricia; Jamin, Augusta; Jarecke, Sarah; Wiebe, Matthew; Zempleni, Janos

    2018-06-01

    The mitochondrial proteome comprises 1000 to 1500 proteins, in addition to proteins for which the mitochondrial localization is uncertain. About 800 diseases have been linked with mutations in mitochondrial proteins. We devised a cell survival assay for assessing the mitochondrial localization in a high-throughput format. This protocol allows us to assess the mitochondrial localization of proteins and their mutants, and to identify drugs and nutrients that modulate the mitochondrial targeting of proteins. The assay works equally well for proteins directed to the outer mitochondrial membrane, inner mitochondrial membrane mitochondrial and mitochondrial matrix, as demonstrated by assessing the mitochondrial targeting of the following proteins: carnitine palmitoyl transferase 1 (consensus sequence and R123C mutant), acetyl-CoA carboxylase 2, uncoupling protein 1 and holocarboxylase synthetase. Our screen may be useful for linking the mitochondrial proteome with rare diseases and for devising drug- and nutrition-based strategies for altering the mitochondrial targeting of proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Plastoglobules: a new address for targeting recombinant proteins in the chloroplast

    PubMed Central

    Vidi, Pierre-Alexandre; Kessler, Felix; Bréhélin, Claire

    2007-01-01

    Background The potential of transgenic plants for cost-effective production of pharmaceutical molecules is now becoming apparent. Plants have the advantage over established fermentation systems (bacterial, yeast or animal cell cultures) to circumvent the risk of pathogen contamination, to be amenable to large scaling up and to necessitate only established farming procedures. Chloroplasts have proven a useful cellular compartment for protein accumulation owing to their large size and number, as well as the possibility for organellar transformation. They therefore represent the targeting destination of choice for recombinant proteins in leaf crops such as tobacco. Extraction and purification of recombinant proteins from leaf material contribute to a large extent to the production costs. Developing new strategies facilitating these processes is therefore necessary. Results Here, we evaluated plastoglobule lipoprotein particles as a new subchloroplastic destination for recombinant proteins. The yellow fluorescent protein as a trackable cargo was targeted to plastoglobules when fused to plastoglobulin 34 (PGL34) as the carrier. Similar to adipocyte differentiation related protein (ADRP) in animal cells, most of the protein sequence of PGL34 was necessary for targeting to lipid bodies. The recombinant protein was efficiently enriched in plastoglobules isolated by simple flotation centrifugation. The viability of plants overproducing the recombinant protein was not affected, indicating that plastoglobule targeting did not significantly impair photosynthesis or sugar metabolism. Conclusion Our data identify plastoglobules as a new targeting destination for recombinant protein in leaf crops. The wide-spread presence of plastoglobules and plastoglobulins in crop species promises applications comparable to those of transgenic oilbody-oleosin technology in molecular farming. PMID:17214877

  17. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    NASA Astrophysics Data System (ADS)

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-11-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases.

  18. Detection of Sirtuin-1 protein expression in peripheral blood leukocytes in dogs.

    PubMed

    Yoshimura, Kuniko; Matsuu, Aya; Sasaki, Kai; Momoi, Yasuyuki

    2018-05-11

    Sirtuin-1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD + )-dependent histone deacetylase with a large number of protein substrates. It has attracted a lot of attention in association with extending lifespan. The objective of this study was to enable the evaluation of SIRT1 expression in peripheral blood mononuclear cells (PBMCs) from dogs by flow cytometry. Three transcript variants were amplified from PBMCs by reverse transcription PCR and the nucleotide sequences were analyzed. On the basis deduced amino acid sequence, a monoclonal antibody against human SIRT1, 1F3, was selected to detect canine SIRT1. Canine SIRT1 in peripheral blood mononuclear cells was successfully detected by western blotting using this antibody. Intracellular canine SIRT1 was also detected in permeabilized 293T cells transfected with a canine SIRT1 expression plasmid by flow cytometry using this antibody. SIRT1 was detected in all leukocyte subsets including lymphocytes, granulocytes and monocytes. The expression level was markedly different among individual dogs. These results indicated that the method applied in this study is useful for evaluating canine SIRT1 levels in PBMCs from dogs.

  19. Rapid detection of proteins in transgenic crops without protein reference standards by targeted proteomic mass spectrometry.

    PubMed

    Schacherer, Lindsey J; Xie, Weiping; Owens, Michaela A; Alarcon, Clara; Hu, Tiger X

    2016-09-01

    Liquid chromatography coupled with tandem mass spectrometry is increasingly used for protein detection for transgenic crops research. Currently this is achieved with protein reference standards which may take a significant time or efforts to obtain and there is a need for rapid protein detection without protein reference standards. A sensitive and specific method was developed to detect target proteins in transgenic maize leaf crude extract at concentrations as low as ∼30 ng mg(-1) dry leaf without the need of reference standards or any sample enrichment. A hybrid Q-TRAP mass spectrometer was used to monitor all potential tryptic peptides of the target proteins in both transgenic and non-transgenic samples. The multiple reaction monitoring-initiated detection and sequencing (MIDAS) approach was used for initial peptide/protein identification via Mascot database search. Further confirmation was achieved by direct comparison between transgenic and non-transgenic samples. Definitive confirmation was provided by running the same experiments of synthetic peptides or protein standards, if available. A targeted proteomic mass spectrometry method using MIDAS approach is an ideal methodology for detection of new proteins in early stages of transgenic crop research and development when neither protein reference standards nor antibodies are available. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  20. Analysis of protein targets in pathogen-host interaction in infectious diseases: a case study on Plasmodium falciparum and Homo sapiens interaction network.

    PubMed

    Saha, Sovan; Sengupta, Kaustav; Chatterjee, Piyali; Basu, Subhadip; Nasipuri, Mita

    2017-09-23

    Infection and disease progression is the outcome of protein interactions between pathogen and host. Pathogen, the role player of Infection, is becoming a severe threat to life as because of its adaptability toward drugs and evolutionary dynamism in nature. Identifying protein targets by analyzing protein interactions between host and pathogen is the key point. Proteins with higher degree and possessing some topologically significant graph theoretical measures are found to be drug targets. On the other hand, exceptional nodes may be involved in infection mechanism because of some pathway process and biologically unknown factors. In this article, we attempt to investigate characteristics of host-pathogen protein interactions by presenting a comprehensive review of computational approaches applied on different infectious diseases. As an illustration, we have analyzed a case study on infectious disease malaria, with its causative agent Plasmodium falciparum acting as 'Bait' and host, Homo sapiens/human acting as 'Prey'. In this pathogen-host interaction network based on some interconnectivity and centrality properties, proteins are viewed as central, peripheral, hub and non-hub nodes and their significance on infection process. Besides, it is observed that because of sparseness of the pathogen and host interaction network, there may be some topologically unimportant but biologically significant proteins, which can also act as Bait/Prey. So, functional similarity or gene ontology mapping can help us in this case to identify these proteins. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  1. Nuclear AgNOR protein enhancement in nucleoplasms of peripheral blood lymphocytes of babies/children with Down syndrome.

    PubMed

    Imamoglu, Nalan; Eroz, Recep; Canatan, Halit; Demirtas, Halil; Saatci, Çetin

    2016-03-01

    Down syndrome (DS) is one of the most common chromosomal disorders. The factors contributing to the mental retardation together with other defects in this syndrome have not been fully explained. Individuals with DS have extra rRNA gene family since they carry an extra chromosome 21. The few reports available are on the relationship between the nucleolus organizer regions (NORs) and DS phenotype. The in vivo regulation of NORs expression on the extra chromosome 21 is not completely understood. Previous studies have shown that nucleoli of lymphocytes from infants (mostly neonates) with DS contain more in vivo and in vitro nucleolar AgNOR proteins when compared with healthy infants. The objective of this study is to compare the in vivo nuclear AgNOR protein level in nucleoplasms (also called as karyoplasm) of nonstimulated peripheral blood lymphocytes from babies/children with DS and healthy controls. Peripheral blood samples obtained from 20 babies/children with DS and 20 matched healthy controls were smeared on clean glass slides and then AgNOR staining was performed. The AgNOR protein level in nucleoplasms of lymphocytes from both groups was calculated using a computer program. Nearly 100 interphase nuclei per individual were analysed. Average nuclear AgNOR protein levels in nucleoplasms of lymphocytes from babies/children with DS were found to be significantly higher than those of the controls (P < 0.001). On the basis of our present results, we propose that the increase of nuclear AgNOR protein in in vivo conditions may contribute to the formation of DS phenotypes. © 2016 Wiley Periodicals, Inc.

  2. Identification of neuronal target genes for CCAAT/Enhancer Binding Proteins

    PubMed Central

    Kfoury, N.; Kapatos, G.

    2009-01-01

    CCAAT/Enhancer Binding Proteins (C/EBPs) play pivotal roles in development and plasticity of the nervous system. Identification of the physiological targets of C/EBPs (C/EBP target genes) should therefore provide insight into the underlying biology of these processes. We used unbiased genome-wide mapping to identify 115 C/EBPβ target genes in PC12 cells that include transcription factors, neurotransmitter receptors, ion channels, protein kinases and synaptic vesicle proteins. C/EBPβ binding sites were located primarily within introns, suggesting novel regulatory functions, and were associated with binding sites for other developmentally important transcription factors. Experiments using dominant negatives showed C/EBPβ to repress transcription of a subset of target genes. Target genes in rat brain were subsequently found to preferentially bind C/EBPα, β and δ. Analysis of the hippocampal transcriptome of C/EBPβ knockout mice revealed dysregulation of a high percentage of transcripts identified as C/EBP target genes. These results support the hypothesis that C/EBPs play non-redundant roles in the brain. PMID:19103292

  3. Targeted Approach for Proteomic Analysis of a Hidden Membrane Protein.

    PubMed

    Martins-Marques, Tania; Anjo, Sandra I; Ribeiro-Rodrigues, Teresa; Manadas, Bruno; Girao, Henrique

    2017-01-01

    Given the properties of plasma membrane proteins, namely, their hydrophobicity, low solubility, and high resistance to digestion and extraction, their identification by traditional mass spectrometry (MS) has been a challenging task. Hence, proteomic studies involving the transmembrane protein connexin43 (Cx43) are scarce. Additionally, studies demonstrating the presence of proteins embedded in the lipid bilayer of extracellular vesicles (EVs) are difficult to perform and require specific changes and fine adjustments in the experimental and technical procedure to allow their detection by MS. In this review, we provide a detailed description of the protocol we have used to detect Cx43 in EVs of human peripheral blood. This includes some of the modifications that we have introduced in order to improve the detection of Cx43 in EVs, including an optimization of vesicle isolation, Cx43 purification, MS acquisition data, and further analysis.

  4. Platelet amyloid precursor protein isoform expression in Alzheimer's disease: evidence for peripheral marker.

    PubMed

    Vignini, A; Sartini, D; Morganti, S; Nanetti, L; Luzzi, S; Provinciali, L; Mazzanti, L; Emanuelli, M

    2011-01-01

    Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by a progressive cognitive and memory decline. Among peripheral markers of AD, great interest has been focused on the amyloid precursor protein (APP). In this regard, platelets represent an important peripheral source of APP since it has been demonstrated that the three major isoforms, that are constituted of 770, 751 and 695 aa residues, are inserted in the membrane of resting platelets. APP 751 and APP 770 contain a Kunitz-type serine protease inhibitor domain (APP KPI) and APP 695 lacks this domain. To address this issue, we first examined the platelet APP isoform mRNAs prospectively as biomarker for the diagnosis of AD by means of real-time quantitative PCR, and then evaluated the correlation between APP mRNA expression levels and cognitive impairment of enrolled subjects. Differential gene expression measurements in the AD patient group (n=18) revealed a significant up-regulation of APP TOT (1.52-fold), APP KPI (1.32-fold), APP 770 (1.33-fold) and APP 751 (1.26-fold) compared to controls (n=22). Moreover, a statistically significant positive correlation was found between APP mRNA levels (TOT, KPI, 770 and 751) and cognitive impairment. Since AD definitive diagnosis still relies on pathological evaluation at autopsy, the present results are consistent with the hypothesis that platelet APP could be considered a potential reliable peripheral marker for studying AD and could contribute to define a signature for the presence of AD pathology.

  5. Targeting protein-protein interactions in hematologic malignancies: still a challenge or a great opportunity for future therapies?

    PubMed Central

    Cierpicki, Tomasz; Grembecka, Jolanta

    2015-01-01

    Summary Over the past several years, there has been an increasing research effort focused on inhibition of protein-protein interactions (PPIs) to develop novel therapeutic approaches for cancer, including hematologic malignancies. These efforts have led to development of small molecule inhibitors of PPIs, some of which already advanced to the stage of clinical trials while others are at different stages of pre-clinical optimization, emphasizing PPIs as an emerging and attractive class of drug targets. Here, we review several examples of recently developed inhibitors of protein-protein interactions highly relevant to hematologic cancers. We address the existing skepticism about feasibility of targeting PPIs and emphasize potential therapeutic benefit from blocking PPIs in hematologic malignancies. We then use these examples to discuss the approaches for successful identification of PPI inhibitors and provide analysis of the protein-protein interfaces, with the goal to address ‘druggability’ of new PPIs relevant to hematology. We discuss lessons learned to improve the success of targeting new protein-protein interactions and evaluate prospects and limits of the research in this field. We conclude that not all PPIs are equally tractable for blocking by small molecules, and detailed analysis of PPI interfaces is critical for selection of those with the highest chance of success. Together, our analysis uncovers patterns that should help to advance drug discovery in hematologic malignancies by successful targeting of new protein-protein interactions. PMID:25510283

  6. The Advantages of Targeted Protein Degradation Over Inhibition: An RTK Case Study.

    PubMed

    Burslem, George M; Smith, Blake E; Lai, Ashton C; Jaime-Figueroa, Saul; McQuaid, Daniel C; Bondeson, Daniel P; Toure, Momar; Dong, Hanqing; Qian, Yimin; Wang, Jing; Crew, Andrew P; Hines, John; Crews, Craig M

    2018-01-18

    Proteolysis targeting chimera (PROTAC) technology has emerged over the last two decades as a powerful tool for targeted degradation of endogenous proteins. Herein we describe the development of PROTACs for receptor tyrosine kinases, a protein family yet to be targeted for induced protein degradation. The use of VHL-recruiting PROTACs against this protein family reveals several advantages of degradation over inhibition alone: direct comparisons of fully functional, target-degrading PROTACs with target-inhibiting variants that contain an inactivated E3 ligase-recruiting ligand show that degradation leads to more potent inhibition of cell proliferation and a more durable and sustained downstream signaling response, and thus addresses the kinome rewiring challenge seen with many receptor tyrosine kinase inhibitors. Combined, these findings demonstrate the ability to target receptor tyrosine kinases for degradation using the PROTAC technology and outline the advantages of this degradation-based approach. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Thyroxine differentially modulates the peripheral clock: lessons from the human hair follicle.

    PubMed

    Hardman, Jonathan A; Haslam, Iain S; Farjo, Nilofer; Farjo, Bessam; Paus, Ralf

    2015-01-01

    The human hair follicle (HF) exhibits peripheral clock activity, with knock-down of clock genes (BMAL1 and PER1) prolonging active hair growth (anagen) and increasing pigmentation. Similarly, thyroid hormones prolong anagen and stimulate pigmentation in cultured human HFs. In addition they are recognized as key regulators of the central clock that controls circadian rhythmicity. Therefore, we asked whether thyroxine (T4) also influences peripheral clock activity in the human HF. Over 24 hours we found a significant reduction in protein levels of BMAL1 and PER1, with their transcript levels also decreasing significantly. Furthermore, while all clock genes maintained their rhythmicity in both the control and T4 treated HFs, there was a significant reduction in the amplitude of BMAL1 and PER1 in T4 (100 nM) treated HFs. Accompanying this, cell-cycle progression marker Cyclin D1 was also assessed appearing to show an induced circadian rhythmicity by T4 however, this was not significant. Contrary to short term cultures, after 6 days, transcript and/or protein levels of all core clock genes (BMAL1, PER1, clock, CRY1, CRY2) were up-regulated in T4 treated HFs. BMAL1 and PER1 mRNA was also up-regulated in the HF bulge, the location of HF epithelial stem cells. Together this provides the first direct evidence that T4 modulates the expression of the peripheral molecular clock. Thus, patients with thyroid dysfunction may also show a disordered peripheral clock, which raises the possibility that short term, pulsatile treatment with T4 might permit one to modulate circadian activity in peripheral tissues as a target to treat clock-related disease.

  8. Selective Targeting of Proteins within Secretory Pathway for Endoplasmic Reticulum-associated Degradation

    PubMed Central

    Vecchi, Lara; Petris, Gianluca; Bestagno, Marco; Burrone, Oscar R.

    2012-01-01

    The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity. PMID:22523070

  9. Mammalian plasma membrane proteins as potential biomarkers and drug targets.

    PubMed

    Rucevic, Marijana; Hixson, Douglas; Josic, Djuro

    2011-06-01

    Defining the plasma membrane proteome is crucial to understand the role of plasma membrane in fundamental biological processes. Change in membrane proteins is one of the first events that take place under pathological conditions, making plasma membrane proteins a likely source of potential disease biomarkers with prognostic or diagnostic potential. Membrane proteins are also potential targets for monoclonal antibodies and other drugs that block receptors or inhibit enzymes essential to the disease progress. Despite several advanced methods recently developed for the analysis of hydrophobic proteins and proteins with posttranslational modifications, integral membrane proteins are still under-represented in plasma membrane proteome. Recent advances in proteomic investigation of plasma membrane proteins, defining their roles as diagnostic and prognostic disease biomarkers and as target molecules in disease treatment, are presented. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Intraoperative Ultrasound for Peripheral Nerve Applications.

    PubMed

    Willsey, Matthew; Wilson, Thomas J; Henning, Phillip Troy; Yang, Lynda J-S

    2017-10-01

    Offering real-time, high-resolution images via intraoperative ultrasound is advantageous for a variety of peripheral nerve applications. To highlight the advantages of ultrasound, its extraoperative uses are reviewed. The current intraoperative uses, including nerve localization, real-time evaluation of peripheral nerve tumors, and implantation of leads for peripheral nerve stimulation, are reviewed. Although intraoperative peripheral nerve localization has been performed previously using guide wires and surgical dyes, the authors' approach using ultrasound-guided instrument clamps helps guide surgical dissection to the target nerve, which could lead to more timely operations and shorter incisions. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Sympathetic reinnervation of peripheral targets following bilateral axotomy of the adult superior cervical ganglion

    PubMed Central

    Hesp, Zoe C.; Zhu, Zheng; Morris, Teresa A.; Walker, Ryan G.; Isaacson, L.G.

    2012-01-01

    The ability of adult injured postganglionic axons to reinnervate cerebrovascular targets is unknown, yet these axons can influence cerebral blood flow, particularly during REM sleep. The objective of the present study was to assess quantitatively the sympathetic reinnervation of vascular as well as non-vascular targets following bilateral axotomy of the superior cervical ganglion (SCG) at short term (1 day, 7 days) and long term (8 weeks, 12 weeks) survival time points. The sympathetic innervation of representative extracerebral blood vessels [internal carotid artery (ICA), basilar artery (BA), middle cerebral artery (MCA)], the submandibular gland (SMG), and pineal gland was quantified following injury using an antibody to tyrosine hydroxylase (TH). Changes in TH innervation were related to TH protein content in the SCG. At 7 days following bilateral SCG axotomy, all targets were significantly depleted of TH innervation, and the exact site on the BA where SCG input was lost could be discerned. Complete sympathetic reinnervation of the ICA was observed at long term survival times, yet TH innervation of other vascular targets showed significant decreases even at 12 weeks following axotomy. The SMG was fully reinnervated by 12 weeks, yet TH innervation of the pineal gland remained significantly decreased. TH protein in the SCG was significantly decreased at both short term and long term time points and showed little evidence of recovery. Our data demonstrate a slow reinnervation of most vascular targets following axotomy of the SCG with only minimal recovery of TH protein in the SCG at 12 weeks following injury. PMID:22842079

  12. Membrane and inclusion body targeting of lyssavirus matrix proteins.

    PubMed

    Pollin, Reiko; Granzow, Harald; Köllner, Bernd; Conzelmann, Karl-Klaus; Finke, Stefan

    2013-02-01

    Lyssavirus matrix proteins (M) support virus budding and have accessory functions that may contribute to host cell manipulation and adaptation to specific hosts. Here, we show that rabies virus (RABV) and European Bat Lyssavirus Type 1 (EBLV-1) M proteins differ in targeting and accumulation at cellular membranes. In contrast to RABV M, EBLV-1 M expressed from authentic EBLV-1 or chimeric RABV accumulated at the Golgi apparatus. Chimeric M proteins revealed that Golgi association depends on the integrity of the entire EBLV-1 M protein. Since RABV and EBLV-1 M differ in the use of cellular membranes for particle formation, differential membrane targeting and transport of M might determine the site of virus production. Moreover, both RABV and EBLV-1 M were for the first time detected within the nucleus and in Negri body-like inclusions bodies. Whereas nuclear M may imply hitherto unknown functions of lyssavirus M in host cell manipulation, the presence of M in inclusion bodies may correlate with regulatory functions of M in virus RNA synthesis. The data strongly support a model in which targeting of lyssavirus M proteins to distinctintracellular sites is a key determinant of diverse features in lyssavirus replication, host adaptation and pathogenesis. © 2012 Blackwell Publishing Ltd.

  13. Heterologous mitochondrial targeting sequences can deliver functional proteins into mitochondria.

    PubMed

    Marcus, Dana; Lichtenstein, Michal; Cohen, Natali; Hadad, Rita; Erlich-Hadad, Tal; Greif, Hagar; Lorberboum-Galski, Haya

    2016-12-01

    Mitochondrial Targeting Sequences (MTSs) are responsible for trafficking nuclear-encoded proteins into mitochondria. Once entering the mitochondria, the MTS is recognized and cleaved off. Some MTSs are long and undergo two-step processing, as in the case of the human frataxin (FXN) protein (80aa), implicated in Friedreich's ataxia (FA). Therefore, we chose the FXN protein to examine whether nuclear-encoded mitochondrial proteins can efficiently be targeted via a heterologous MTS (hMTS) and deliver a functional protein into mitochondria. We examined three hMTSs; that of citrate synthase (cs), lipoamide deydrogenase (LAD) and C6ORF66 (ORF), as classically MTS sequences, known to be removed by one-step processing, to deliver FXN into mitochondria, in the form of fusion proteins. We demonstrate that using hMTSs for delivering FXN results in the production of 4-5-fold larger amounts of the fusion proteins, and at 4-5-fold higher concentrations. Moreover, hMTSs delivered a functional FXN protein into the mitochondria even more efficiently than the native MTSfxn, as evidenced by the rescue of FA patients' cells from oxidative stress; demonstrating a 18%-54% increase in cell survival; and a 13%-33% increase in ATP levels, as compared to the fusion protein carrying the native MTS. One fusion protein with MTScs increased aconitase activity within patients' cells, by 400-fold. The implications form our studies are of vast importance for both basic and translational research of mitochondrial proteins as any mitochondrial protein can be delivered efficiently by an hMTS. Moreover, effective targeting of functional proteins is important for restoration of mitochondrial function and treatment of related disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. How proteins bind to DNA: target discrimination and dynamic sequence search by the telomeric protein TRF1

    PubMed Central

    2017-01-01

    Abstract Target search as performed by DNA-binding proteins is a complex process, in which multiple factors contribute to both thermodynamic discrimination of the target sequence from overwhelmingly abundant off-target sites and kinetic acceleration of dynamic sequence interrogation. TRF1, the protein that binds to telomeric tandem repeats, faces an intriguing variant of the search problem where target sites are clustered within short fragments of chromosomal DNA. In this study, we use extensive (>0.5 ms in total) MD simulations to study the dynamical aspects of sequence-specific binding of TRF1 at both telomeric and non-cognate DNA. For the first time, we describe the spontaneous formation of a sequence-specific native protein–DNA complex in atomistic detail, and study the mechanism by which proteins avoid off-target binding while retaining high affinity for target sites. Our calculated free energy landscapes reproduce the thermodynamics of sequence-specific binding, while statistical approaches allow for a comprehensive description of intermediate stages of complex formation. PMID:28633355

  15. An affinity-directed protein missile system for targeted proteolysis.

    PubMed

    Fulcher, Luke J; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro; Sapkota, Gopal P

    2016-10-01

    The von Hippel-Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. © 2016 The Authors.

  16. An affinity-directed protein missile system for targeted proteolysis

    PubMed Central

    Fulcher, Luke J.; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro

    2016-01-01

    The von Hippel–Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. PMID:27784791

  17. Orally active-targeted drug delivery systems for proteins and peptides.

    PubMed

    Li, Xiuying; Yu, Miaorong; Fan, Weiwei; Gan, Yong; Hovgaard, Lars; Yang, Mingshi

    2014-09-01

    In the past decade, extensive efforts have been devoted to designing 'active targeted' drug delivery systems (ATDDS) to improve oral absorption of proteins and peptides. Such ATDDS enhance cellular internalization and permeability of proteins and peptides via molecular recognition processes such as ligand-receptor or antigen-antibody interaction, and thus enhance drug absorption. This review focuses on recent advances with orally ATDDS, including ligand-protein conjugates, recombinant ligand-protein fusion proteins and ligand-modified carriers. In addition to traditional intestinal active transport systems of substrates and their corresponding receptors, transporters and carriers, new targets such as intercellular adhesion molecule-1 and β-integrin are also discussed. ATDDS can improve oral absorption of proteins and peptides. However, currently, no clinical studies on ATDDS for proteins and peptides are underway, perhaps due to the complexity and limited knowledge of transport mechanisms. Therefore, more research is warranted to optimize ATDDS efficiency.

  18. A Single Peroxisomal Targeting Signal Mediates Matrix Protein Import in Diatoms

    PubMed Central

    Gonzalez, Nicola H.; Felsner, Gregor; Schramm, Frederic D.; Klingl, Andreas; Maier, Uwe-G.; Bolte, Kathrin

    2011-01-01

    Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1. PMID:21966495

  19. Targeted Degradation of Proteins Localized in Subcellular Compartments by Hybrid Small Molecules.

    PubMed

    Okuhira, Keiichiro; Shoda, Takuji; Omura, Risa; Ohoka, Nobumichi; Hattori, Takayuki; Shibata, Norihito; Demizu, Yosuke; Sugihara, Ryo; Ichino, Asato; Kawahara, Haruka; Itoh, Yukihiro; Ishikawa, Minoru; Hashimoto, Yuichi; Kurihara, Masaaki; Itoh, Susumu; Saito, Hiroyuki; Naito, Mikihiko

    2017-03-01

    Development of novel small molecules that selectively degrade pathogenic proteins would provide an important advance in targeted therapy. Recently, we have devised a series of hybrid small molecules named SNIPER (specific and nongenetic IAP-dependent protein ERaser) that induces the degradation of target proteins via the ubiquitin-proteasome system. To understand the localization of proteins that can be targeted by this protein knockdown technology, we examined whether SNIPER molecules are able to induce degradation of cellular retinoic acid binding protein II (CRABP-II) proteins localized in subcellular compartments of cells. CRABP-II is genetically fused with subcellular localization signals, and they are expressed in the cells. SNIPER(CRABP) with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1 compound, induce the proteasomal degradation of wild-type (WT), cytosolic, nuclear, and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity toward the mitochondrial CRABP-II protein. The small interfering RNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP) against WT and cytosolic CRABP-II proteins, indicating that cIAP1 is the E3 ligase responsible for degradation of these proteins. Against membrane-localized CRABP-II protein, cIAP1 is also a primary E3 ligase in the cells, but another E3 ligase distinct from cIAP2 and X-linked inhibitor of apoptosis protein (XIAP) could also be involved in the SNIPER(CRABP)-11-induced degradation. However, for the degradation of nuclear and mitochondrial CRABP-II proteins, E3 ligases other than cIAP1, cIAP2, and XIAP play a role in the SNIPER-mediated protein knockdown. These results indicate that SNIPER can target cytosolic, nuclear, membrane-localized, and mitochondrial proteins for degradation, but the responsible E3 ligase is different, depending on the localization of the target protein. Copyright © 2017 by

  20. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

    PubMed

    Wilkinson, Trevor C I

    2016-06-15

    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  1. Structure of the Get3 targeting factor in complex with its membrane protein cargo

    DOE PAGES

    Mateja, Agnieszka; Paduch, Marcin; Chang, Hsin-Yang; ...

    2015-03-06

    Tail-anchored (TA) proteins are a physiologically important class of membrane proteins targeted to the endoplasmic reticulum by the conserved guided-entry of TA proteins (GET) pathway. During transit, their hydrophobic transmembrane domains (TMDs) are chaperoned by the cytosolic targeting factor Get3, but the molecular nature of the functional Get3-TA protein targeting complex remains unknown. In this paper, we reconstituted the physiologic assembly pathway for a functional targeting complex and showed that it comprises a TA protein bound to a Get3 homodimer. Crystal structures of Get3 bound to different TA proteins showed an α-helical TMD occupying a hydrophobic groove that spans themore » Get3 homodimer. Finally, our data elucidate the mechanism of TA protein recognition and shielding by Get3 and suggest general principles of hydrophobic domain chaperoning by cellular targeting factors.« less

  2. Atomic resolution view into the structure–function relationships of the human myelin peripheral membrane protein P2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ruskamo, Salla; University of Oulu, Oulu; Yadav, Ravi P.

    2014-01-01

    The structure of the human myelin peripheral membrane protein P2 has been refined at 0.93 Å resolution. In combination with functional experiments in vitro, in vivo and in silico, the fine details of the structure–function relationships in P2 are emerging. P2 is a fatty acid-binding protein expressed in vertebrate peripheral nerve myelin, where it may function in bilayer stacking and lipid transport. P2 binds to phospholipid membranes through its positively charged surface and a hydrophobic tip, and accommodates fatty acids inside its barrel structure. The structure of human P2 refined at the ultrahigh resolution of 0.93 Å allows detailed structuralmore » analyses, including the full organization of an internal hydrogen-bonding network. The orientation of the bound fatty-acid carboxyl group is linked to the protonation states of two coordinating arginine residues. An anion-binding site in the portal region is suggested to be relevant for membrane interactions and conformational changes. When bound to membrane multilayers, P2 has a preferred orientation and is stabilized, and the repeat distance indicates a single layer of P2 between membranes. Simulations show the formation of a double bilayer in the presence of P2, and in cultured cells wild-type P2 induces membrane-domain formation. Here, the most accurate structural and functional view to date on P2, a major component of peripheral nerve myelin, is presented, showing how it can interact with two membranes simultaneously while going through conformational changes at its portal region enabling ligand transfer.« less

  3. The potential of targeting Ras proteins in lung cancer.

    PubMed

    McCormick, Frank

    2015-04-01

    The Ras pathway is a major driver in lung adenocarcinoma: over 75% of all cases harbor mutations that activate this pathway. While spectacular clinical successes have been achieved by targeting activated receptor tyrosine kinases in this pathway, little, if any, significant progress has been achieved targeting Ras proteins themselves or cancers driven by oncogenic Ras mutants. New approaches to drug discovery, new insights into Ras function, new ways of attacking undruggable proteins through RNA interference and new ways of harnessing the immune system could change this landscape in the relatively near future.

  4. Genetically engineered and self-assembled oncolytic protein nanoparticles for targeted cancer therapy.

    PubMed

    Lee, Joong-Jae; Kang, Jung Ae; Ryu, Yiseul; Han, Sang-Soo; Nam, You Ree; Rho, Jong Kook; Choi, Dae Seong; Kang, Sun-Woong; Lee, Dong-Eun; Kim, Hak-Sung

    2017-03-01

    The integration of a targeted delivery with a tumour-selective agent has been considered an ideal platform for achieving high therapeutic efficacy and negligible side effects in cancer therapy. Here, we present engineered protein nanoparticles comprising a tumour-selective oncolytic protein and a targeting moiety as a new format for the targeted cancer therapy. Apoptin from chicken anaemia virus (CAV) was used as a tumour-selective apoptotic protein. An EGFR-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was employed to play a dual role as a tumour-targeting moiety and a fusion partner for producing apoptin nanoparticles in E. coli, respectively. The repebody was genetically fused to apoptin, and the resulting fusion protein was shown to self-assemble into supramolecular repebody-apoptin nanoparticles with high homogeneity and stability as a soluble form when expressed in E. coli. The repebody-apoptin nanoparticles showed a remarkable anti-tumour activity with negligible side effects in xenograft mice through a cooperative action of the two protein components with distinct functional roles. The repebody-apoptin nanoparticles can be developed as a systemic injectable and tumour-selective therapeutic protein for targeted cancer treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. RNA-modifying proteins as anticancer drug targets.

    PubMed

    Boriack-Sjodin, P Ann; Ribich, Scott; Copeland, Robert A

    2018-06-01

    All major biological macromolecules (DNA, RNA, proteins and lipids) undergo enzyme-catalysed covalent modifications that impact their structure, function and stability. A variety of covalent modifications of RNA have been identified and demonstrated to affect RNA stability and translation to proteins; these mechanisms of translational control have been termed epitranscriptomics. Emerging data suggest that some epitranscriptomic mechanisms are altered in human cancers as well as other human diseases. In this Review, we examine the current understanding of RNA modifications with a focus on mRNA methylation, highlight their possible roles in specific cancer indications and discuss the emerging potential of RNA-modifying proteins as therapeutic targets.

  6. 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.

  7. Hsp42 is required for sequestration of protein aggregates into deposition sites in Saccharomyces cerevisiae

    PubMed Central

    Specht, Sebastian; Miller, Stephanie B.M.

    2011-01-01

    The aggregation of proteins inside cells is an organized process with cytoprotective function. In Saccharomyces cerevisiae, aggregating proteins are spatially sequestered to either juxtanuclear or peripheral sites, which target distinct quality control pathways for refolding and degradation. The cellular machinery driving the sequestration of misfolded proteins to these sites is unknown. In this paper, we show that one of the two small heat shock proteins of yeast, Hsp42, is essential for the formation of peripheral aggregates during physiological heat stress. Hsp42 preferentially localizes to peripheral aggregates but is largely absent from juxtanuclear aggregates, which still form in hsp42Δ cells. Transferring the amino-terminal domain of Hsp42 to Hsp26, which does not participate in aggregate sorting, enables Hsp26 to replace Hsp42 function. Our data suggest that Hsp42 acts via its amino-terminal domain to coaggregate with misfolded proteins and perhaps link such complexes to further sorting factors. PMID:22065637

  8. Improved protein model quality assessments by changing the target function.

    PubMed

    Uziela, Karolis; Menéndez Hurtado, David; Shu, Nanjiang; Wallner, Björn; Elofsson, Arne

    2018-06-01

    Protein modeling quality is an important part of protein structure prediction. We have for more than a decade developed a set of methods for this problem. We have used various types of description of the protein and different machine learning methodologies. However, common to all these methods has been the target function used for training. The target function in ProQ describes the local quality of a residue in a protein model. In all versions of ProQ the target function has been the S-score. However, other quality estimation functions also exist, which can be divided into superposition- and contact-based methods. The superposition-based methods, such as S-score, are based on a rigid body superposition of a protein model and the native structure, while the contact-based methods compare the local environment of each residue. Here, we examine the effects of retraining our latest predictor, ProQ3D, using identical inputs but different target functions. We find that the contact-based methods are easier to predict and that predictors trained on these measures provide some advantages when it comes to identifying the best model. One possible reason for this is that contact based methods are better at estimating the quality of multi-domain targets. However, training on the S-score gives the best correlation with the GDT_TS score, which is commonly used in CASP to score the global model quality. To take the advantage of both of these features we provide an updated version of ProQ3D that predicts local and global model quality estimates based on different quality estimates. © 2018 Wiley Periodicals, Inc.

  9. Peripheral nerve proteins as potential autoantigens in acute and chronic inflammatory demyelinating polyneuropathies.

    PubMed

    Lim, Jia Pei; Devaux, Jérôme; Yuki, Nobuhiro

    2014-10-01

    Guillain-Barré syndrome is classified into acute inflammatory demyelinating polyneuropathy and acute motor axonal neuropathy. Whereas autoantibodies to GM1 or GD1a induce the development of acute motor axonal neuropathy, pathogenic autoantibodies have yet to be identified in acute inflammatory demyelinating polyneuropathy and chronic inflammatory demyelinating polyneuropathy. This review highlights the importance of autoantibodies to peripheral nerve proteins in the physiopathology of acute and chronic inflammatory demyelinating polyneuropathies. Moreover, we listed up other potential antigens, which may become helpful biomarkers for acquired, dysimmune demyelinating neuropathies based on their critical functions during myelination and their implications in hereditary demyelinating neuropathies. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Chemical probes targeting epigenetic proteins: Applications beyond oncology

    PubMed Central

    Ackloo, Suzanne; Brown, Peter J.; Müller, Susanne

    2017-01-01

    ABSTRACT Epigenetic chemical probes are potent, cell-active, small molecule inhibitors or antagonists of specific domains in a protein; they have been indispensable for studying bromodomains and protein methyltransferases. The Structural Genomics Consortium (SGC), comprising scientists from academic and pharmaceutical laboratories, has generated most of the current epigenetic chemical probes. Moreover, the SGC has shared about 4 thousand aliquots of these probes, which have been used primarily for phenotypic profiling or to validate targets in cell lines or primary patient samples cultured in vitro. Epigenetic chemical probes have been critical tools in oncology research and have uncovered mechanistic insights into well-established targets, as well as identify new therapeutic starting points. Indeed, the literature primarily links epigenetic proteins to oncology, but applications in inflammation, viral, metabolic and neurodegenerative diseases are now being reported. We summarize the literature of these emerging applications and provide examples where existing probes might be used. PMID:28080202

  11. High frequency of mutations in codon 98 of the peripheral myelin protein Po gene in 20 French CMT1 patients

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rougher, H.; LeGuern, E. Gouider, R.

    1996-03-01

    Charcot-Marie-Tooth disease, characterized by distal muscle weakness and amyotrophy, decreased or absent tendon reflexes, and high arched feet, is the most common inherited peripheral neuropathy, with a prevalence of 1 in 2,500. Two types of CMT have been distinguished on the basis of nerve conduction velocities. CMT type 1 is the most frequent, with markedly slowed velocities ({<=}40 m/s) associated with hypertrophic onion bulb changes on nerve biopsy. Autosomal dominant CMT1 is genetically heterogeneous: CMT1A is caused by a 1.5-Mb duplication in 17p11.2 and, more rarely, by a point mutation in tha PMP22 (peripheral myelin protein, 22 kD) gene locatedmore » in the duplicated region; CMT1B results from mutations in the Po (peripheral myelin protein zero) gene in 1q22-23. Forty-five percent (7/16) of the published mutations associated with CMT1 occur in exon 3 of Po. In order to determine the cause of CMT1 in 20 unrelated patients without 17p11.2 duplications, mutations were sought in exon 3 of Po with three techniques: nonradioactive SSCP, automated sequencing, and PCR enzymatic restriction. 18 refs., 2 figs.« less

  12. Comparative proteomic analysis of differentially expressed proteins between peripheral sensory and motor nerves.

    PubMed

    He, Qianru; Man, Lili; Ji, Yuhua; Zhang, Shuqiang; Jiang, Maorong; Ding, Fei; Gu, Xiaosong

    2012-06-01

    Peripheral sensory and motor nerves have different functions and different approaches to regeneration, especially their distinct ability to accurately reinervate terminal nerve pathways. To understand the molecular aspects underlying these differences, the proteomics technique by coupling isobaric tags for relative and absolute quantitation (iTRAQ) with online two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) was used to investigate the protein profile of sensory and motor nerve samples from rats. A total of 1472 proteins were identified in either sensory or motor nerve. Of them, 100 proteins showed differential expressions between both nerves, and some of them were validated by quantitative real time RT-PCR, Western blot analysis, and immunohistochemistry. In the light of functional categorization, the differentially expressed proteins in sensory and motor nerves, belonging to a broad range of classes, were related to a diverse array of biological functions, which included cell adhesion, cytoskeleton, neuronal plasticity, neurotrophic activity, calcium-binding, signal transduction, transport, enzyme catalysis, lipid metabolism, DNA-binding, synaptosome function, actin-binding, ATP-binding, extracellular matrix, and commitment to other lineages. The relatively higher expressed proteins in either sensory or motor nerve were tentatively discussed in combination with their specific molecular characteristics. It is anticipated that the database generated in this study will provide a solid foundation for further comprehensive investigation of functional differences between sensory and motor nerves, including the specificity of their regeneration.

  13. [Molecular markers of Alzheimer disease early diagnostic: investigation perspectives of peripheral tissues.

    PubMed

    Paltsev, M A; Zuev, V A; Kozhevnikova, E O; Linkova, N S; Kvetnaia, T V; Polyakova, V O; Kvetnoy, I M

    2017-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disorder of elderly and old age people. For intravital diagnosis of the expression of signaling molecules - AD markers, cerebrospinal fluid (CSF) and peripheral tissues are used: lymphocytes and blood platelets, buccal and olfactory epithelium, skin fibroblasts. There are several changes in the production of hyper phosphorylated form of τ-protein, BACE1 and peptide Аβ42 in CSF in case of AD, but CSF taking may have a number of side effects. Less traumatic taking of sampling tissues for the diagnosis of AD is in use of epithelium biopsy and blood portion. An increase in the expression of the hyper phosphorylated form of τ-protein is shown in blood lymphocytes of AD patients. An increase in the content of high molecular weight forms of phosphorylated t-protein and amyloid precursor protein-APP was also revealed in blood platelets of AD patients. Changes in the amount of 2 miRNA families - miR-132 family and miR-134 family were revealed in blood cells 1-5 years before the manifestation of clinical signs of AD. An increase in the concentration of bound calcium, synthesis of peptides Aβ40 and Aβ42, τ protein was observed in AD skin fibroblasts. In the olfactory and buccal epithelium an increase in the expression of hyper phosphorylated form of τ-protein and Aβ peptide was detected in patients with AD. Verification of AD markers in peripheral tissues for biopsy have the important significant for life diagnostics, prevention and and target AD treatment.

  14. Functional evaluation of peripheral nerve regeneration and target reinnervation in animal models: a critical overview.

    PubMed

    Navarro, Xavier

    2016-02-01

    Peripheral nerve injuries usually lead to severe loss of motor, sensory and autonomic functions in the patients. Due to the complex requirements for adequate axonal regeneration, functional recovery is often poorly achieved. Experimental models are useful to investigate the mechanisms related to axonal regeneration and tissue reinnervation, and to test new therapeutic strategies to improve functional recovery. Therefore, objective and reliable evaluation methods should be applied for the assessment of regeneration and function restitution after nerve injury in animal models. This review gives an overview of the most useful methods to assess nerve regeneration, target reinnervation and recovery of complex sensory and motor functions, their values and limitations. The selection of methods has to be adequate to the main objective of the research study, either enhancement of axonal regeneration, improving regeneration and reinnervation of target organs by different types of nerve fibres, or increasing recovery of complex sensory and motor functions. It is generally recommended to use more than one functional method for each purpose, and also to perform morphological studies of the injured nerve and the reinnervated targets. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  15. T Cells that Recognize HPV Protein Can Target Virus-Infected Cells | Center for Cancer Research

    Cancer.gov

    Adoptive T-cell transfer (ACT) is a promising form of cancer immunotherapy. Treating patients with T cells isolated from a tumor and subsequently expanded in the lab can cause the complete regression of some melanomas and cervical cancers, but the treatment is currently restricted to a few cancer types. An approach that may be applied to a wider array of cancers involves modifying peripheral blood T cells with chimeric antigen receptors or T-cell receptors (TCR) that target specific tumor antigens. Unfortunately, epithelial cancers, which are the vast majority of cancers diagnosed, have proven difficult to treat this way because most identified antigens are shared with healthy tissues and targeting them leads to toxic side effects. However, cancers caused by persistent human papillomavirus (HPV) infection, including cervical, head and neck, anal, vaginal, vulvar, and penile cancers, may be particularly amenable to the latter form of ACT since the E6 and E7 viral proteins are essential for cancer formation but are not produced in normal tissues. To test this idea, Christian Hinrichs, M.D., and his colleagues examined tumor infiltrating lymphocytes (TILs) from a patient who experienced a prolonged disease-free period after her second surgical removal of metastatic anal cancer in the hopes of identifying a TCR against one of the HPV oncoproteins.

  16. Massively parallel de novo protein design for targeted therapeutics.

    PubMed

    Chevalier, Aaron; Silva, Daniel-Adriano; Rocklin, Gabriel J; Hicks, Derrick R; Vergara, Renan; Murapa, Patience; Bernard, Steffen M; Zhang, Lu; Lam, Kwok-Ho; Yao, Guorui; Bahl, Christopher D; Miyashita, Shin-Ichiro; Goreshnik, Inna; Fuller, James T; Koday, Merika T; Jenkins, Cody M; Colvin, Tom; Carter, Lauren; Bohn, Alan; Bryan, Cassie M; Fernández-Velasco, D Alejandro; Stewart, Lance; Dong, Min; Huang, Xuhui; Jin, Rongsheng; Wilson, Ian A; Fuller, Deborah H; Baker, David

    2017-10-05

    De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37-43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing.

  17. Massively parallel de novo protein design for targeted therapeutics

    NASA Astrophysics Data System (ADS)

    Chevalier, Aaron; Silva, Daniel-Adriano; Rocklin, Gabriel J.; Hicks, Derrick R.; Vergara, Renan; Murapa, Patience; Bernard, Steffen M.; Zhang, Lu; Lam, Kwok-Ho; Yao, Guorui; Bahl, Christopher D.; Miyashita, Shin-Ichiro; Goreshnik, Inna; Fuller, James T.; Koday, Merika T.; Jenkins, Cody M.; Colvin, Tom; Carter, Lauren; Bohn, Alan; Bryan, Cassie M.; Fernández-Velasco, D. Alejandro; Stewart, Lance; Dong, Min; Huang, Xuhui; Jin, Rongsheng; Wilson, Ian A.; Fuller, Deborah H.; Baker, David

    2017-10-01

    De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37-43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing.

  18. Massively parallel de novo protein design for targeted therapeutics

    PubMed Central

    Chevalier, Aaron; Silva, Daniel-Adriano; Rocklin, Gabriel J.; Hicks, Derrick R.; Vergara, Renan; Murapa, Patience; Bernard, Steffen M.; Zhang, Lu; Lam, Kwok-Ho; Yao, Guorui; Bahl, Christopher D.; Miyashita, Shin-Ichiro; Goreshnik, Inna; Fuller, James T.; Koday, Merika T.; Jenkins, Cody M.; Colvin, Tom; Carter, Lauren; Bohn, Alan; Bryan, Cassie M.; Fernández-Velasco, D. Alejandro; Stewart, Lance; Dong, Min; Huang, Xuhui; Jin, Rongsheng; Wilson, Ian A.; Fuller, Deborah H.; Baker, David

    2018-01-01

    De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37–43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing. PMID:28953867

  19. TALE-PvuII fusion proteins--novel tools for gene targeting.

    PubMed

    Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

    2013-01-01

    Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.

  20. Targeted intracellular delivery of proteins with spatial and temporal control.

    PubMed

    Morales, Demosthenes P; Braun, Gary B; Pallaoro, Alessia; Chen, Renwei; Huang, Xiao; Zasadzinski, Joseph A; Reich, Norbert O

    2015-02-02

    While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery.

  1. Comprehensive adipocytic and neurogenic tissue microarray analysis of NY-ESO-1 expression - a promising immunotherapy target in malignant peripheral nerve sheath tumor and liposarcoma

    PubMed Central

    Shurell, Elizabeth; Vergara-Lluri, Maria E.; Li, Yunfeng; Crompton, Joseph G.; Singh, Arun; Bernthal, Nicholas; Wu, Hong; Eilber, Fritz C.; Dry, Sarah M.

    2016-01-01

    Background Immunotherapy targeting cancer-testis antigen NY-ESO-1 shows promise for tumors with poor response to chemoradiation. Malignant peripheral nerve sheath tumors (MPNSTs) and liposarcomas (LPS) are chemoresistant and have few effective treatment options. Materials Methods Using a comprehensive tissue microarray (TMA) of both benign and malignant tumors in primary, recurrent, and metastatic samples, we examined NY-ESO-1 expression in peripheral nerve sheath tumor (PNST) and adipocytic tumors. The PNST TMA included 42 MPNSTs (spontaneous n = 26, NF1-associated n = 16), 35 neurofibromas (spontaneous n = 22, NF-1 associated n = 13), 11 schwannomas, and 18 normal nerves. The LPS TMA included 48 well-differentiated/dedifferentiated (WD/DD) LPS, 13 myxoid/round cell LPS, 3 pleomorphic LPS, 8 lipomas, 1 myelolipoma, and 3 normal adipocytic tissue samples. Stained in triplicate, NY-ESO-1 intensity and density were scored. Results NY-ESO-1 expression was exclusive to malignant tumors. 100% of myxoid/round cell LPS demonstrated NY-ESO-1 expression, while only 6% of WD/DD LPS showed protein expression, one of which was WD LPS. Of MPNST, 4/26 (15%) spontaneous and 2/16 (12%) NF1-associated MPNSTs demonstrated NY-ESO-1 expression. Strong NY-ESO-1 expression was observed in myxoid/round cell and dedifferentiated LPS, and MPNST in primary, neoadjuvant, and metastatic settings. Conclusions We found higher prevalence of NY-ESO-1 expression in MPNSTs than previously reported, highlighting a subset of MPNST patients who may benefit from immunotherapy. This study expands our understanding of NY-ESO-1 in WD/DD LPS and is the first demonstration of staining in a WD LPS and metastatic/recurrent myxoid/round cell LPS. These results suggest immunotherapy targeting NY-ESO-1 may benefit patients with aggressive tumors resistant to conventional therapy. PMID:27655679

  2. Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae

    PubMed Central

    Tacchi, Jessica L.; Raymond, Benjamin B. A.; Haynes, Paul A.; Berry, Iain J.; Widjaja, Michael; Bogema, Daniel R.; Woolley, Lauren K.; Jenkins, Cheryl; Minion, F. Chris; Padula, Matthew P.; Djordjevic, Steven P.

    2016-01-01

    Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC–MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity. PMID:26865024

  3. Inferring protein domains associated with drug side effects based on drug-target interaction network.

    PubMed

    Iwata, Hiroaki; Mizutani, Sayaka; Tabei, Yasuo; Kotera, Masaaki; Goto, Susumu; Yamanishi, Yoshihiro

    2013-01-01

    Most phenotypic effects of drugs are involved in the interactions between drugs and their target proteins, however, our knowledge about the molecular mechanism of the drug-target interactions is very limited. One of challenging issues in recent pharmaceutical science is to identify the underlying molecular features which govern drug-target interactions. In this paper, we make a systematic analysis of the correlation between drug side effects and protein domains, which we call "pharmacogenomic features," based on the drug-target interaction network. We detect drug side effects and protein domains that appear jointly in known drug-target interactions, which is made possible by using classifiers with sparse models. It is shown that the inferred pharmacogenomic features can be used for predicting potential drug-target interactions. We also discuss advantages and limitations of the pharmacogenomic features, compared with the chemogenomic features that are the associations between drug chemical substructures and protein domains. The inferred side effect-domain association network is expected to be useful for estimating common drug side effects for different protein families and characteristic drug side effects for specific protein domains.

  4. Twenty-four-hour central blood pressure is not better associated with hypertensive target organ damage than 24-h peripheral blood pressure.

    PubMed

    de la Sierra, Alejandro; Pareja, Julia; Fernández-Llama, Patricia; Armario, Pedro; Yun, Sergi; Acosta, Eva; Calero, Francesca; Vázquez, Susana; Blanch, Pedro; Sierra, Cristina; Oliveras, Anna

    2017-10-01

    Central blood pressure (BP) is increasingly considered as a better estimator of hypertension associated risks. We aimed to evaluate the association of 24-h central BP, in comparison with 24-h peripheral BP, with the presence of target organ damage (TOD). Cross-sectional study of 208 hypertensive patients, aged 57 ± 12 years, 34% women. Office (mean of 4 measurements) and 24-h central and peripheral BP were measured by the oscillometric Mobil-O-Graph device. TOD was assessed at cardiac (left ventricular hypertrophy by echocardiography), renal (reduction of glomerular filtration rate and/or microalbuminuria), and arterial (increased aortic pulse wave velocity) levels. A total of 107 patients (51.4%) had TOD (77, 35% patients left ventricular hypertrophy; 54, 25.9% renal abnormalities; and 40, 19.2% arterial stiffness). All SBP and pulse BP estimates (office, 24-h, daytime, and night-time) were associated with the presence of TOD, after adjustment for age, sex, and antihypertensive treatment, with higher odds ratios for ambulatory-derived values. Odds ratios for central and peripheral BP were similar for all office, 24-h, daytime, and night-time BP. After simultaneous adjustment, peripheral, but not central, 24-h and night-time SBP and pulse pressures were associated with the presence of TOD. TOD in hypertension is associated with BP elevation, independently of the type of measurement (office or ambulatory, central or peripheral). Central BP, even monitored during 24 h, is not better associated with TOD than peripheral BP. These results do not support a routine measurement of 24-h central BP.

  5. Axonal ensheathment and septate junction formation in the peripheral nervous system of Drosophila.

    PubMed

    Banerjee, Swati; Pillai, Anilkumar M; Paik, Raehum; Li, Jingjun; Bhat, Manzoor A

    2006-03-22

    Axonal insulation is critical for efficient action potential propagation and normal functioning of the nervous system. In Drosophila, the underlying basis of nerve ensheathment is the axonal insulation by glial cells and the establishment of septate junctions (SJs) between glial cell membranes. However, the details of the cellular and molecular mechanisms underlying axonal insulation and SJ formation are still obscure. Here, we report the characterization of axonal insulation in the Drosophila peripheral nervous system (PNS). Targeted expression of tau-green fluorescent protein in the glial cells and ultrastructural analysis of the peripheral nerves allowed us to visualize the glial ensheathment of axons. We show that individual or a group of axons are ensheathed by inner glial processes, which in turn are ensheathed by the outer perineurial glial cells. SJs are formed between the inner and outer glial membranes. We also show that Neurexin IV, Contactin, and Neuroglian are coexpressed in the peripheral glial membranes and that these proteins exist as a complex in the Drosophila nervous system. Mutations in neurexin IV, contactin, and neuroglian result in the disruption of blood-nerve barrier function in the PNS, and ultrastructural analyses of the mutant embryonic peripheral nerves show loss of glial SJs. Interestingly, the murine homologs of Neurexin IV, Contactin, and Neuroglian are expressed at the paranodal SJs and play a key role in axon-glial interactions of myelinated axons. Together, our data suggest that the molecular machinery underlying axonal insulation and axon-glial interactions may be conserved across species.

  6. Molecular design and nanoparticle-mediated intracellular delivery of functional proteins to target cellular pathways

    NASA Astrophysics Data System (ADS)

    Shah, Dhiral Ashwin

    Intracellular delivery of specific proteins and peptides represents a novel method to influence stem cells for gain-of-function and loss-of-function. Signaling control is vital in stem cells, wherein intricate control of and interplay among critical pathways directs the fate of these cells into either self-renewal or differentiation. The most common route to manipulate cellular function involves the introduction of genetic material such as full-length genes and shRNA into the cell to generate (or prevent formation of) the target protein, and thereby ultimately alter cell function. However, viral-mediated gene delivery may result in relatively slow expression of proteins and prevalence of oncogene insertion into the cell, which can alter cell function in an unpredictable fashion, and non-viral delivery may lead to low efficiency of genetic delivery. For example, the latter case plagues the generation of induced pluripotent stem cells (iPSCs) and hinders their use for in vivo applications. Alternatively, introducing proteins into cells that specifically recognize and influence target proteins, can result in immediate deactivation or activation of key signaling pathways within the cell. In this work, we demonstrate the cellular delivery of functional proteins attached to hydrophobically modified silica (SiNP) nanoparticles to manipulate specifically targeted cell signaling proteins. In the Wnt signaling pathway, we have targeted the phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta) by designing a chimeric protein and delivering it in neural stem cells. Confocal imaging indicates that the SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, indicated by the elevation of beta-catenin levels, and increased transcription of Wnt target genes, such as c-MYC. The results presented here suggest that

  7. Reverse screening methods to search for the protein targets of chemopreventive compounds

    NASA Astrophysics Data System (ADS)

    Huang, Hongbin; Zhang, Guigui; Zhou, Yuquan; Lin, Chenru; Chen, Suling; Lin, Yutong; Mai, Shangkang; Huang, Zunnan

    2018-05-01

    This article is a systematic review of reverse screening methods used to search for the protein targets of chemopreventive compounds or drugs. Typical chemopreventive compounds include components of traditional Chinese medicine, natural compounds and Food and Drug Administration (FDA)-approved drugs. Such compounds are somewhat selective but are predisposed to bind multiple protein targets distributed throughout diverse signaling pathways in human cells. In contrast to conventional virtual screening, which identifies the ligands of a targeted protein from a compound database, reverse screening is used to identify the potential targets or unintended targets of a given compound from a large number of receptors by examining their known ligands or crystal structures. This method, also known as in silico or computational target fishing, is highly valuable for discovering the target receptors of query molecules from terrestrial or marine natural products, exploring the molecular mechanisms of chemopreventive compounds, finding alternative indications of existing drugs by drug repositioning, and detecting adverse drug reactions and drug toxicity. Reverse screening can be divided into three major groups: shape screening, pharmacophore screening and reverse docking. Several large software packages, such as Schrödinger and Discovery Studio; typical software/network services such as ChemMapper, PharmMapper, idTarget and INVDOCK; and practical databases of known target ligands and receptor crystal structures, such as ChEMBL, BindingDB and the Protein Data Bank (PDB), are available for use in these computational methods. Different programs, online services and databases have different applications and constraints. Here, we conducted a systematic analysis and multilevel classification of the computational programs, online services and compound libraries available for shape screening, pharmacophore screening and reverse docking to enable non-specialist users to quickly learn and

  8. Reverse Screening Methods to Search for the Protein Targets of Chemopreventive Compounds.

    PubMed

    Huang, Hongbin; Zhang, Guigui; Zhou, Yuquan; Lin, Chenru; Chen, Suling; Lin, Yutong; Mai, Shangkang; Huang, Zunnan

    2018-01-01

    This article is a systematic review of reverse screening methods used to search for the protein targets of chemopreventive compounds or drugs. Typical chemopreventive compounds include components of traditional Chinese medicine, natural compounds and Food and Drug Administration (FDA)-approved drugs. Such compounds are somewhat selective but are predisposed to bind multiple protein targets distributed throughout diverse signaling pathways in human cells. In contrast to conventional virtual screening, which identifies the ligands of a targeted protein from a compound database, reverse screening is used to identify the potential targets or unintended targets of a given compound from a large number of receptors by examining their known ligands or crystal structures. This method, also known as in silico or computational target fishing, is highly valuable for discovering the target receptors of query molecules from terrestrial or marine natural products, exploring the molecular mechanisms of chemopreventive compounds, finding alternative indications of existing drugs by drug repositioning, and detecting adverse drug reactions and drug toxicity. Reverse screening can be divided into three major groups: shape screening, pharmacophore screening and reverse docking. Several large software packages, such as Schrödinger and Discovery Studio; typical software/network services such as ChemMapper, PharmMapper, idTarget, and INVDOCK; and practical databases of known target ligands and receptor crystal structures, such as ChEMBL, BindingDB, and the Protein Data Bank (PDB), are available for use in these computational methods. Different programs, online services and databases have different applications and constraints. Here, we conducted a systematic analysis and multilevel classification of the computational programs, online services and compound libraries available for shape screening, pharmacophore screening and reverse docking to enable non-specialist users to quickly learn

  9. Reverse Screening Methods to Search for the Protein Targets of Chemopreventive Compounds

    PubMed Central

    Huang, Hongbin; Zhang, Guigui; Zhou, Yuquan; Lin, Chenru; Chen, Suling; Lin, Yutong; Mai, Shangkang; Huang, Zunnan

    2018-01-01

    This article is a systematic review of reverse screening methods used to search for the protein targets of chemopreventive compounds or drugs. Typical chemopreventive compounds include components of traditional Chinese medicine, natural compounds and Food and Drug Administration (FDA)-approved drugs. Such compounds are somewhat selective but are predisposed to bind multiple protein targets distributed throughout diverse signaling pathways in human cells. In contrast to conventional virtual screening, which identifies the ligands of a targeted protein from a compound database, reverse screening is used to identify the potential targets or unintended targets of a given compound from a large number of receptors by examining their known ligands or crystal structures. This method, also known as in silico or computational target fishing, is highly valuable for discovering the target receptors of query molecules from terrestrial or marine natural products, exploring the molecular mechanisms of chemopreventive compounds, finding alternative indications of existing drugs by drug repositioning, and detecting adverse drug reactions and drug toxicity. Reverse screening can be divided into three major groups: shape screening, pharmacophore screening and reverse docking. Several large software packages, such as Schrödinger and Discovery Studio; typical software/network services such as ChemMapper, PharmMapper, idTarget, and INVDOCK; and practical databases of known target ligands and receptor crystal structures, such as ChEMBL, BindingDB, and the Protein Data Bank (PDB), are available for use in these computational methods. Different programs, online services and databases have different applications and constraints. Here, we conducted a systematic analysis and multilevel classification of the computational programs, online services and compound libraries available for shape screening, pharmacophore screening and reverse docking to enable non-specialist users to quickly learn

  10. Lupus autoantibodies target ribosomal P proteins

    PubMed Central

    1985-01-01

    All nine SLE (systemic lupus erythematosus) sera with antiribosomal antibody activity targeted the same three ribosomal protein antigens, of molecular masses 38 and 17/19 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. One serum reacted with an additional protein of approximately kD. Ribosomal subunit fractionation by composite gel electrophoresis and sucrose density ultracentrifugation showed that these proteins were part of the large subunit. Isoelectric focusing in agarose, and two-dimensional polyacrylamide gel electrophoresis revealed that the antigens had pI between 4.5 and 6.5, but that the 17/19 kD antigens were more acidic than the 38 kD antigen. Similarities in the molecular masses, charges, as well as the presence of highly conserved crossreactive epitopes, failure to bind to carboxymethylcellulose at pH 4.2, and extractability of the 17/19 kD proteins by 400 mM NH4Cl-ethanol at 0 degrees C indicated that these antigens were analogous to the proteins P0 (38 kD) and P1/P2 (17/19 kD) described previously (25, 36). Co-identity was confirmed using reference antibodies and antigen. Although antibodies to these proteins were only found in 5-10% of more than 50 sera screened by radioimmunoassay or Western blotting, the selective production of antibodies to epitopes on three (out of a total of more than 80) ribosomal proteins may provide further clues to autoantibody induction of SLE. PMID:2410526

  11. Impact of 4D-(18)FDG-PET/CT imaging on target volume delineation in SBRT patients with central versus peripheral lung tumors. Multi-reader comparative study.

    PubMed

    Chirindel, Alin; Adebahr, Sonja; Schuster, Daniel; Schimek-Jasch, Tanja; Schanne, Daniel H; Nemer, Ursula; Mix, Michael; Meyer, Philipp; Grosu, Anca-Ligia; Brunner, Thomas; Nestle, Ursula

    2015-06-01

    Evaluation of the effect of co-registered 4D-(18)FDG-PET/CT for SBRT target delineation in patients with central versus peripheral lung tumors. Analysis of internal target volume (ITV) delineation of central and peripheral lung lesions in 21 SBRT-patients. Manual delineation was performed by 4 observers in 2 contouring phases: on respiratory gated 4DCT with diagnostic 3DPET available aside (CT-ITV) and on co-registered 4DPET/CT (PET/CT-ITV). Comparative analysis of volumes and inter-reader agreement. 11 cases of peripheral and 10 central lesions were evaluated. In peripheral lesions, average CT-ITV was 6.2 cm(3) and PET/CT-ITV 8.6 cm(3), resembling a mean change in hypothetical radius of 2 mm. For both CT-ITVs and PET/CT-ITVs inter reader agreement was good and unchanged (0.733 and 0.716; p=0.58). All PET/CT-ITVs stayed within the PTVs derived from CT-ITVs. In central lesions, average CT-ITVs were 42.1 cm(3), PET/CT-ITVs 44.2 cm(3), without significant overall volume changes. Inter-reader agreement improved significantly (0.665 and 0.750; p<0.05). 2/10 PET/CT-ITVs exceeded the PTVs derived from CT-ITVs by >1 ml in average for all observers. The addition of co-registered 4DPET data to 4DCT based target volume delineation for SBRT of centrally located lung tumors increases the inter-observer agreement and may help to avoid geographic misses. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  12. Sequence- and Interactome-Based Prediction of Viral Protein Hotspots Targeting Host Proteins: A Case Study for HIV Nef

    PubMed Central

    Sarmady, Mahdi; Dampier, William; Tozeren, Aydin

    2011-01-01

    Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk. PMID:21738584

  13. Ras-Driven Transcriptome Analysis Identifies Aurora Kinase A as a Potential Malignant Peripheral Nerve Sheath Tumor Therapeutic Target

    PubMed Central

    Patel, Ami V.; Eaves, David; Jessen, Walter J.; Rizvi, Tilat A.; Ecsedy, Jeffrey A.; Qian, Mark G.; Aronow, Bruce J.; Perentesis, John P.; Serra, Eduard; Cripe, Timothy P.; Miller, Shyra J.; Ratner, Nancy

    2013-01-01

    Purpose Patients with Neurofibromatosis Type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNST) which are often inoperable and do not respond well to current chemotherapies or radiation. The goal of this study was to utilize comprehensive gene expression analysis to identify novel therapeutic targets. Experimental Design Nerve Schwann cells and/or their precursors are the tumorigenic cell types in MPNST due to the loss of the NF1 gene, which encodes the RasGAP protein neurofibromin. Therefore, we created a transgenic mouse model, CNP-HRas12V, expressing constitutively-active HRas in Schwann cells and defined a Ras-induced gene expression signature to drive a Bayesian factor regression model analysis of differentially expressed genes in mouse and human neurofibromas and MPNSTs. We tested functional significance of Aurora kinase over-expression in MPNST in vitro and in vivo using Aurora kinase shRNAs and compounds that inhibit Aurora kinase. Results We identified 2000 genes with probability of linkage to nerve Ras signaling of which 339 were significantly differentially expressed in mouse and human NF1-related tumor samples relative to normal nerves, including Aurora kinase A (AURKA). AURKA was dramatically over-expressed and genomically amplified in MPNSTs but not neurofibromas. Aurora kinase shRNAs and Aurora kinase inhibitors blocked MPNST cell growth in vitro. Furthermore, an AURKA selective inhibitor, MLN8237, stabilized tumor volume and significantly increased survival of mice with MPNST xenografts. Conclusion Integrative cross-species transcriptome analyses combined with preclinical testing has provided an effective method for identifying candidates for molecular-targeted therapeutics. Blocking Aurora kinases may be a viable treatment platform for MPNST. PMID:22811580

  14. Identification of poly(rC) binding protein 2 (PCBP2) as a target protein of immunosuppressive agent 15-deoxyspergualin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Murahashi, Masataka; Simizu, Siro; Morioka, Masahiko

    15-Deoxyspergualin (DSG) is an immunosuppressive agent being clinically used. Unlike tacrolimus and cyclosporine A, it does not inhibit the calcineurin pathway, and its mechanism of action and target molecule have not been elucidated. Therefore, we previously prepared biotinylated derivative of DSG (BDSG) to fish up the target protein. In the present research, we identified poly(rC) binding protein 2 (PCBP2) as a DSG-binding protein using this probe. DSG was confirmed to bind to PCBP2 by pull-down assay. Intracellular localization of PCBP2 was changed from the nucleus to the cytoplasm by DSG treatment. DSG inhibited the cell growth, and over-expression of PCBP2more » reduced the anti-proliferative activity of DSG. PCBP2 is known to regulate various proteins including STAT1/2. Thus, we found PCBP2 as the first target protein of DSG that can explain the immunosuppressive activity. -- Highlights: •Fifteen-deoxyspergualin (DSG) is an immunosuppressive agent clinically used. •We have identified PCBP2, an RNA-binding protein, as a molecular target of DSG. •Alteration of PCBP2 activity may explain the immunosuppressive activity of DSG.« less

  15. Target identification in Fusobacterium nucleatum by subtractive genomics approach and enrichment analysis of host-pathogen protein-protein interactions.

    PubMed

    Kumar, Amit; Thotakura, Pragna Lakshmi; Tiwary, Basant Kumar; Krishna, Ramadas

    2016-05-12

    Fusobacterium nucleatum, a well studied bacterium in periodontal diseases, appendicitis, gingivitis, osteomyelitis and pregnancy complications has recently gained attention due to its association with colorectal cancer (CRC) progression. Treatment with berberine was shown to reverse F. nucleatum-induced CRC progression in mice by balancing the growth of opportunistic pathogens in tumor microenvironment. Intestinal microbiota imbalance and the infections caused by F. nucleatum might be regulated by therapeutic intervention. Hence, we aimed to predict drug target proteins in F. nucleatum, through subtractive genomics approach and host-pathogen protein-protein interactions (HP-PPIs). We also carried out enrichment analysis of host interacting partners to hypothesize the possible mechanisms involved in CRC progression due to F. nucleatum. In subtractive genomics approach, the essential, virulence and resistance related proteins were retrieved from RefSeq proteome of F. nucleatum by searching against Database of Essential Genes (DEG), Virulence Factor Database (VFDB) and Antibiotic Resistance Gene-ANNOTation (ARG-ANNOT) tool respectively. A subsequent hierarchical screening to identify non-human homologous, metabolic pathway-independent/pathway-specific and druggable proteins resulted in eight pathway-independent and 27 pathway-specific druggable targets. Co-aggregation of F. nucleatum with host induces proinflammatory gene expression thereby potentiates tumorigenesis. Hence, proteins from IBDsite, a database for inflammatory bowel disease (IBD) research and those involved in colorectal adenocarcinoma as interpreted from The Cancer Genome Atlas (TCGA) were retrieved to predict drug targets based on HP-PPIs with F. nucleatum proteome. Prediction of HP-PPIs exhibited 186 interactions contributed by 103 host and 76 bacterial proteins. Bacterial interacting partners were accounted as putative targets. And enrichment analysis of host interacting partners showed statistically

  16. Prm3p is a pheromone-induced peripheral nuclear envelope protein required for yeast nuclear fusion.

    PubMed

    Shen, Shu; Tobery, Cynthia E; Rose, Mark D

    2009-05-01

    Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex.

  17. Acyl-protein thioesterase 2 catalyzes the deacylation of peripheral membrane-associated GAP-43.

    PubMed

    Tomatis, Vanesa M; Trenchi, Alejandra; Gomez, Guillermo A; Daniotti, Jose L

    2010-11-30

    An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.

  18. Target Highlights in CASP9: Experimental Target Structures for the Critical Assessment of Techniques for Protein Structure Prediction

    PubMed Central

    Kryshtafovych, Andriy; Moult, John; Bartual, Sergio G.; Bazan, J. Fernando; Berman, Helen; Casteel, Darren E.; Christodoulou, Evangelos; Everett, John K.; Hausmann, Jens; Heidebrecht, Tatjana; Hills, Tanya; Hui, Raymond; Hunt, John F.; Jayaraman, Seetharaman; Joachimiak, Andrzej; Kennedy, Michael A.; Kim, Choel; Lingel, Andreas; Michalska, Karolina; Montelione, Gaetano T.; Otero, José M.; Perrakis, Anastassis; Pizarro, Juan C.; van Raaij, Mark J.; Ramelot, Theresa A.; Rousseau, Francois; Tong, Liang; Wernimont, Amy K.; Young, Jasmine; Schwede, Torsten

    2011-01-01

    One goal of the CASP Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction is to identify the current state of the art in protein structure prediction and modeling. A fundamental principle of CASP is blind prediction on a set of relevant protein targets, i.e. the participating computational methods are tested on a common set of experimental target proteins, for which the experimental structures are not known at the time of modeling. Therefore, the CASP experiment would not have been possible without broad support of the experimental protein structural biology community. In this manuscript, several experimental groups discuss the structures of the proteins which they provided as prediction targets for CASP9, highlighting structural and functional peculiarities of these structures: the long tail fibre protein gp37 from bacteriophage T4, the cyclic GMP-dependent protein kinase Iβ (PKGIβ) dimerization/docking domain, the ectodomain of the JTB (Jumping Translocation Breakpoint) transmembrane receptor, Autotaxin (ATX) in complex with an inhibitor, the DNA-Binding J-Binding Protein 1 (JBP1) domain essential for biosynthesis and maintenance of DNA base-J (β-D-glucosyl-hydroxymethyluracil) in Trypanosoma and Leishmania, an so far uncharacterized 73 residue domain from Ruminococcus gnavus with a fold typical for PDZ-like domains, a domain from the Phycobilisome (PBS) core-membrane linker (LCM) phycobiliprotein ApcE from Synechocystis, the Heat shock protein 90 (Hsp90) activators PFC0360w and PFC0270w from Plasmodium falciparum, and 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae. PMID:22020785

  19. A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering.

    PubMed

    Belsare, Ketaki D; Andorfer, Mary C; Cardenas, Frida S; Chael, Julia R; Park, Hyun June; Lewis, Jared C

    2017-03-17

    Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450 BM3 , pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.

  20. Identifying relationships between unrelated pharmaceutical target proteins on the basis of shared active compounds.

    PubMed

    Miljković, Filip; Kunimoto, Ryo; Bajorath, Jürgen

    2017-08-01

    Computational exploration of small-molecule-based relationships between target proteins from different families. Target annotations of drugs and other bioactive compounds were systematically analyzed on the basis of high-confidence activity data. A total of 286 novel chemical links were established between distantly related or unrelated target proteins. These relationships involved a total of 1859 bioactive compounds including 147 drugs and 141 targets. Computational analysis of large amounts of compounds and activity data has revealed unexpected relationships between diverse target proteins on the basis of compounds they share. These relationships are relevant for drug discovery efforts. Target pairs that we have identified and associated compound information are made freely available.

  1. Nanoscale strategies: treatment for peripheral vascular disease and critical limb ischemia.

    PubMed

    Tu, Chengyi; Das, Subhamoy; Baker, Aaron B; Zoldan, Janeta; Suggs, Laura J

    2015-01-01

    Peripheral vascular disease (PVD) is one of the most prevalent vascular diseases in the U.S. afflicting an estimated 8 million people. Obstruction of peripheral arteries leads to insufficient nutrients and oxygen supply to extremities, which, if not treated properly, can potentially give rise to a severe condition called critical limb ischemia (CLI). CLI is associated with extremely high morbidities and mortalities. Conventional treatments such as angioplasty, atherectomy, stent implantation and bypass surgery have achieved some success in treating localized macrovascular disease but are limited by their invasiveness. An emerging alternative is the use of growth factor (delivered as genes or proteins) and cell therapy for PVD treatment. By delivering growth factors or cells to the ischemic tissue, one can stimulate the regeneration of functional vasculature network locally, re-perfuse the ischemic tissue, and thus salvage the limb. Here we review recent advance in nanomaterials, and discuss how their application can improve and facilitate growth factor or cell therapies. Specifically, nanoparticles (NPs) can serve as drug carrier and target to ischemic tissues and achieve localized and sustained release of pro-angiogenic proteins. As nonviral vectors, NPs can greatly enhance the transfection of target cells with pro-angiogenic genes with relatively fewer safety concern. Further, NPs may also be used in combination with cell therapy to enhance cell retention, cell survival and secretion of angiogenic factors. Lastly, nano/micro fibrous vascular grafts can be engineered to better mimic the structure and composition of native vessels, and hopefully overcome many complications/limitations associated with conventional synthetic grafts.

  2. Inferring protein domains associated with drug side effects based on drug-target interaction network

    PubMed Central

    2013-01-01

    Background Most phenotypic effects of drugs are involved in the interactions between drugs and their target proteins, however, our knowledge about the molecular mechanism of the drug-target interactions is very limited. One of challenging issues in recent pharmaceutical science is to identify the underlying molecular features which govern drug-target interactions. Results In this paper, we make a systematic analysis of the correlation between drug side effects and protein domains, which we call "pharmacogenomic features," based on the drug-target interaction network. We detect drug side effects and protein domains that appear jointly in known drug-target interactions, which is made possible by using classifiers with sparse models. It is shown that the inferred pharmacogenomic features can be used for predicting potential drug-target interactions. We also discuss advantages and limitations of the pharmacogenomic features, compared with the chemogenomic features that are the associations between drug chemical substructures and protein domains. Conclusion The inferred side effect-domain association network is expected to be useful for estimating common drug side effects for different protein families and characteristic drug side effects for specific protein domains. PMID:24565527

  3. Mover Is a Homomeric Phospho-Protein Present on Synaptic Vesicles

    PubMed Central

    Kremer, Thomas; Hoeber, Jan; Kiran Akula, Asha; Urlaub, Henning; Islinger, Markus; Kirsch, Joachim; Dean, Camin; Dresbach, Thomas

    2013-01-01

    With remarkably few exceptions, the molecules mediating synaptic vesicle exocytosis at active zones are structurally and functionally conserved between vertebrates and invertebrates. Mover was found in a yeast-2-hybrid assay using the vertebrate-specific active zone scaffolding protein bassoon as a bait. Peptides of Mover have been reported in proteomics screens for self-interacting proteins, phosphorylated proteins, and synaptic vesicle proteins, respectively. Here, we tested the predictions arising from these screens. Using flotation assays, carbonate stripping of peripheral membrane proteins, mass spectrometry, immunogold labelling of purified synaptic vesicles, and immuno-organelle isolation, we found that Mover is indeed a peripheral synaptic vesicle membrane protein. In addition, by generating an antibody against phosphorylated Mover and Western blot analysis of fractionated rat brain, we found that Mover is a bona fide phospho-protein. The localization of Mover to synaptic vesicles is phosphorylation dependent; treatment with a phosphatase caused Mover to dissociate from synaptic vesicles. A yeast-2-hybrid screen, co-immunoprecipitation and cell-based optical assays of homomerization revealed that Mover undergoes homophilic interaction, and regions within both the N- and C- terminus of the protein are required for this interaction. Deleting a region required for homomeric interaction abolished presynaptic targeting of recombinant Mover in cultured neurons. Together, these data prove that Mover is associated with synaptic vesicles, and implicate phosphorylation and multimerization in targeting of Mover to synaptic vesicles and presynaptic sites. PMID:23723986

  4. Absolute Quantification of Middle- to High-Abundant Plasma Proteins via Targeted Proteomics.

    PubMed

    Dittrich, Julia; Ceglarek, Uta

    2017-01-01

    The increasing number of peptide and protein biomarker candidates requires expeditious and reliable quantification strategies. The utilization of liquid chromatography coupled to quadrupole tandem mass spectrometry (LC-MS/MS) for the absolute quantitation of plasma proteins and peptides facilitates the multiplexed verification of tens to hundreds of biomarkers from smallest sample quantities. Targeted proteomics assays derived from bottom-up proteomics principles rely on the identification and analysis of proteotypic peptides formed in an enzymatic digestion of the target protein. This protocol proposes a procedure for the establishment of a targeted absolute quantitation method for middle- to high-abundant plasma proteins waiving depletion or enrichment steps. Essential topics as proteotypic peptide identification and LC-MS/MS method development as well as sample preparation and calibration strategies are described in detail.

  5. Enhancing bioactive peptide release and identification using targeted enzymatic hydrolysis of milk proteins.

    PubMed

    Nongonierma, Alice B; FitzGerald, Richard J

    2018-06-01

    Milk proteins have been extensively studied for their ability to yield a range of bioactive peptides following enzymatic hydrolysis/digestion. However, many hurdles still exist regarding the widespread utilization of milk protein-derived bioactive peptides as health enhancing agents for humans. These mostly arise from the fact that most milk protein-derived bioactive peptides are not highly potent. In addition, they may be degraded during gastrointestinal digestion and/or have a low intestinal permeability. The targeted release of bioactive peptides during the enzymatic hydrolysis of milk proteins may allow the generation of particularly potent bioactive hydrolysates and peptides. Therefore, the development of milk protein hydrolysates capable of improving human health requires, in the first instance, optimized targeted release of specific bioactive peptides. The targeted hydrolysis of milk proteins has been aided by a range of in silico tools. These include peptide cutters and predictive modeling linking bioactivity to peptide structure [i.e., molecular docking, quantitative structure activity relationship (QSAR)], or hydrolysis parameters [design of experiments (DOE)]. Different targeted enzymatic release strategies employed during the generation of milk protein hydrolysates are reviewed herein and their limitations are outlined. In addition, specific examples are provided to demonstrate how in silico tools may help in the identification and discovery of potent milk protein-derived peptides. It is anticipated that the development of novel strategies employing a range of in silico tools may help in the generation of milk protein hydrolysates containing potent and bioavailable peptides, which in turn may be used to validate their health promoting effects in humans. Graphical abstract The targeted enzymatic hydrolysis of milk proteins may allow the generation of highly potent and bioavailable bioactive peptides.

  6. Increased CD69 Expression on Peripheral Eosinophils from Patients with Food Protein-Induced Enterocolitis Syndrome.

    PubMed

    Wada, Taizo; Matsuda, Yusuke; Toma, Tomoko; Koizumi, Eiko; Okamoto, Hiroyuki; Yachie, Akihiro

    2016-01-01

    Food protein-induced enterocolitis syndrome (FPIES) is an uncommon, non-IgE-mediated food allergy. We recently described a significant increase in fecal eosinophil-derived neurotoxin (EDN) after ingestion of the causative food. However, little is known about the activation status of circulating eosinophils in patients with an acute FPIES reaction. Surface CD69 expression was assessed by flow cytometry on peripheral eosinophils from 5 patients with FPIES before and after ingestion of the causative food. Fecal EDN was measured by enzyme-linked immunosorbent assay. No eosinophil activation was observed before ingestion; however, a significant increase in CD69 expression on eosinophils after an acute FIPES reaction was demonstrated in all of the patients. There was no significant change in absolute eosinophil counts in the peripheral blood. The levels of fecal EDN increased on the day after ingestion of the causative food in all patients. These results suggest that circulating eosinophils as well as eosinophils in the intestinal mucosal tissue are activated in acute FPIES reactions and might be associated with systemic immune events in FPIES. © 2016 S. Karger AG, Basel.

  7. The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation.

    PubMed

    Baumberger, Nicolas; Tsai, Ching-Hsui; Lie, Miranda; Havecker, Ericka; Baulcombe, David C

    2007-09-18

    Plant and animal viruses encode suppressor proteins of an adaptive immunity mechanism in which viral double-stranded RNA is processed into 21-25 nt short interfering (si)RNAs. The siRNAs guide ARGONAUTE (AGO) proteins so that they target viral RNA. Most viral suppressors bind long dsRNA or siRNAs and thereby prevent production of siRNA or binding of siRNA to AGO. The one exception is the 2b suppressor of Cucumoviruses that binds to and inhibits AGO1. Here we describe a novel suppressor mechanism in which a Polerovirus-encoded F box protein (P0) targets the PAZ motif and its adjacent upstream sequence in AGO1 and mediates its degradation. F box proteins are components of E3 ubiquitin ligase complexes that add polyubiquitin tracts on selected lysine residues and thereby mark a protein for proteasome-mediated degradation. With P0, however, the targeted degradation of AGO is insensitive to inhibition of the proteasome, indicating that the proteasome is not involved. We also show that P0 does not block a mobile signal of silencing, indicating that the signal molecule does not have AGO protein components. The ability of P0 to block silencing without affecting signal movement may contribute to the phloem restriction of viruses in the Polerovirus group.

  8. Human immune cell targeting of protein nanoparticles - caveospheres

    NASA Astrophysics Data System (ADS)

    Glass, Joshua J.; Yuen, Daniel; Rae, James; Johnston, Angus P. R.; Parton, Robert G.; Kent, Stephen J.; de Rose, Robert

    2016-04-01

    Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.

  9. Unusual Voltage-Gated Sodium Currents as Targets for Pain.

    PubMed

    Barbosa, C; Cummins, T R

    2016-01-01

    Pain is a serious health problem that impacts the lives of many individuals. Hyperexcitability of peripheral sensory neurons contributes to both acute and chronic pain syndromes. Because voltage-gated sodium currents are crucial to the transmission of electrical signals in peripheral sensory neurons, the channels that underlie these currents are attractive targets for pain therapeutics. Sodium currents and channels in peripheral sensory neurons are complex. Multiple-channel isoforms contribute to the macroscopic currents in nociceptive sensory neurons. These different isoforms exhibit substantial variations in their kinetics and pharmacology. Furthermore, sodium current complexity is enhanced by an array of interacting proteins that can substantially modify the properties of voltage-gated sodium channels. Resurgent sodium currents, atypical currents that can enhance recovery from inactivation and neuronal firing, are increasingly being recognized as playing potentially important roles in sensory neuron hyperexcitability and pain sensations. Here we discuss unusual sodium channels and currents that have been identified in nociceptive sensory neurons, describe what is known about the molecular determinants of the complex sodium currents in these neurons. Finally, we provide an overview of therapeutic strategies to target voltage-gated sodium currents in nociceptive neurons. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. A Universal Method for Fishing Target Proteins from Mixtures of Biomolecules using Isothermal Titration Calorimetry

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhou, X.; Sun, Q; Kini, R

    2008-01-01

    The most challenging tasks in biology include the identification of (1) the orphan receptor for a ligand, (2) the ligand for an orphan receptor protein, and (3) the target protein(s) for a given drug or a lead compound that are critical for the pharmacological or side effects. At present, several approaches are available, including cell- or animal-based assays, affinity labeling, solid-phase binding assays, surface plasmon resonance, and nuclear magnetic resonance. Most of these techniques are not easy to apply when the target protein is unknown and the compound is not amenable to labeling, chemical modification, or immobilization. Here we demonstratemore » a new universal method for fishing orphan target proteins from a complex mixture of biomolecules using isothermal titration calorimetry (ITC) as a tracking tool. We took snake venom, a crude mixture of several hundred proteins/peptides, as a model to demonstrate our proposed ITC method in tracking the isolation and purification of two distinct target proteins, a major component and a minor component. Identities of fished out target proteins were confirmed by amino acid sequencing and inhibition assays. This method has the potential to make a significant advancement in the area of identifying orphan target proteins and inhibitor screening in drug discovery and characterization.« less

  11. A Strategy Based on Protein-Protein Interface Motifs May Help in Identifying Drug Off-Targets

    PubMed Central

    Engin, H. Billur; Keskin, Ozlem; Nussinov, Ruth; Gursoy, Attila

    2014-01-01

    Networks are increasingly used to study the impact of drugs at the systems level. From the algorithmic standpoint, a drug can ‘attack’ nodes or edges of a protein-protein interaction network. In this work, we propose a new network strategy, “The Interface Attack”, based on protein-protein interfaces. Similar interface architectures can occur between unrelated proteins. Consequently, in principle, a drug that binds to one has a certain probability of binding others. The interface attack strategy simultaneously removes from the network all interactions that consist of similar interface motifs. This strategy is inspired by network pharmacology and allows inferring potential off-targets. We introduce a network model which we call “Protein Interface and Interaction Network (P2IN)”, which is the integration of protein-protein interface structures and protein interaction networks. This interface-based network organization clarifies which protein pairs have structurally similar interfaces, and which proteins may compete to bind the same surface region. We built the P2IN of p53 signaling network and performed network robustness analysis. We show that (1) ‘hitting’ frequent interfaces (a set of edges distributed around the network) might be as destructive as eleminating high degree proteins (hub nodes); (2) frequent interfaces are not always topologically critical elements in the network; and (3) interface attack may reveal functional changes in the system better than attack of single proteins. In the off-target detection case study, we found that drugs blocking the interface between CDK6 and CDKN2D may also affect the interaction between CDK4 and CDKN2D. PMID:22817115

  12. Localization and modulation of calcitonin gene-related peptide-receptor component protein-immunoreactive cells in the rat central and peripheral nervous systems.

    PubMed

    Ma, W; Chabot, J-G; Powell, K J; Jhamandas, K; Dickerson, I M; Quirion, R

    2003-01-01

    Calcitonin gene-related peptide (CGRP) is widely distributed in the central and peripheral nervous system. Its highly diverse biological activities are mediated via the G protein-coupled receptor that uniquely requires two accessory proteins for optimal function. CGRP receptor component protein (RCP) is a coupling protein necessary for CGRP-receptor signaling. In this study, we established the anatomical distribution of RCP in the rat central and peripheral nervous system and its relationship to CGRP immunoreactivity. RCP-immunoreactive (IR) perikarya are widely and selectively distributed in the cerebral cortex, septal nuclei, hippocampus, various hypothalamic nuclei, amygdala, nucleus colliculus, periaqueductal gray, parabrachial nuclei, locus coeruleus, cochlear nuclei, dorsal raphe nuclei, the solitary tractus nucleus and gracile nucleus, cerebellar cortex, various brainstem motor nuclei, the spinal dorsal and ventral horns. A sub-population of neurons in the dorsal root ganglia (DRG) and trigeminal ganglia were strongly RCP-IR. Overall, the localization of RCP-IR closely matched with that of CGRP-IR. We also determined whether RCP in DRG and dorsal horn neurons can be modulated by CGRP receptor blockade and pain-related pathological stimuli. The intrathecal injection of the antagonist CGRP(8-37) markedly increased RCP expression in the lumbar DRG and spinal dorsal horn. Carrageenan-induced plantar inflammation produced a dramatic bilateral increase in RCP expression in the dorsal horn while a partial sciatic nerve ligation reduced RCP expression in the ipsilateral superficial dorsal horn. Our data suggest that the distribution of RCP immunoreactivity is closely matched with CGRP immunoreactivity in most of central and peripheral nervous systems. The co-localization of RCP and CGRP in motoneurons and primary sensory neurons suggests that CGRP has an autocrine or paracrine effect on these neurons. Moreover, our data also suggest that RCP expression in DRG and

  13. Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms.

    PubMed Central

    Maeda, Kenji; Finnie, Christine; Svensson, Birte

    2004-01-01

    Barley thioredoxin h isoforms HvTrxh1 and HvTrxh2 differ in temporal and spatial distribution and in kinetic properties. Target proteins of HvTrxh1 and HvTrxh2 were identified in mature seeds and in seeds after 72 h of germination. Improvement of the established method for identification of thioredoxin-targeted proteins based on two-dimensional electrophoresis and fluorescence labelling of thiol groups was achieved by application of a highly sensitive Cy5 maleimide dye and large-format two-dimensional gels, resulting in a 10-fold increase in the observed number of labelled protein spots. The technique also provided information about accessible thiol groups in the proteins identified in the barley seed proteome. In total, 16 different putative target proteins were identified from 26 spots using tryptic in-gel digestion, matrix-assisted laser-desorption ionization-time-of-flight MS and database search. HvTrxh1 and HvTrxh2 were shown to have similar target specificity. Barley alpha-amylase/subtilisin inhibitor, previously demonstrated to be reduced by both HvTrxh1 and HvTrxh2, was among the identified target proteins, confirming the suitability of the method. Several alpha-amylase/trypsin inhibitors, some of which are already known as target proteins of thioredoxin h, and cyclophilin known as a target protein of m-type thioredoxin were also identified. Lipid transfer protein, embryospecific protein, three chitinase isoenzymes, a single-domain glyoxalase-like protein and superoxide dismutase were novel identifications of putative target proteins, suggesting new physiological roles of thioredoxin h in barley seeds. PMID:14636158

  14. A comparative study of disease genes and drug targets in the human protein interactome

    PubMed Central

    2015-01-01

    Background Disease genes cause or contribute genetically to the development of the most complex diseases. Drugs are the major approaches to treat the complex disease through interacting with their targets. Thus, drug targets are critical for treatment efficacy. However, the interrelationship between the disease genes and drug targets is not clear. Results In this study, we comprehensively compared the network properties of disease genes and drug targets for five major disease categories (cancer, cardiovascular disease, immune system disease, metabolic disease, and nervous system disease). We first collected disease genes from genome-wide association studies (GWAS) for five disease categories and collected their corresponding drugs based on drugs' Anatomical Therapeutic Chemical (ATC) classification. Then, we obtained the drug targets for these five different disease categories. We found that, though the intersections between disease genes and drug targets were small, disease genes were significantly enriched in targets compared to their enrichment in human protein-coding genes. We further compared network properties of the proteins encoded by disease genes and drug targets in human protein-protein interaction networks (interactome). The results showed that the drug targets tended to have higher degree, higher betweenness, and lower clustering coefficient in cancer Furthermore, we observed a clear fraction increase of disease proteins or drug targets in the near neighborhood compared with the randomized genes. Conclusions The study presents the first comprehensive comparison of the disease genes and drug targets in the context of interactome. The results provide some foundational network characteristics for further designing computational strategies to predict novel drug targets and drug repurposing. PMID:25861037

  15. A comparative study of disease genes and drug targets in the human protein interactome.

    PubMed

    Sun, Jingchun; Zhu, Kevin; Zheng, W; Xu, Hua

    2015-01-01

    Disease genes cause or contribute genetically to the development of the most complex diseases. Drugs are the major approaches to treat the complex disease through interacting with their targets. Thus, drug targets are critical for treatment efficacy. However, the interrelationship between the disease genes and drug targets is not clear. In this study, we comprehensively compared the network properties of disease genes and drug targets for five major disease categories (cancer, cardiovascular disease, immune system disease, metabolic disease, and nervous system disease). We first collected disease genes from genome-wide association studies (GWAS) for five disease categories and collected their corresponding drugs based on drugs' Anatomical Therapeutic Chemical (ATC) classification. Then, we obtained the drug targets for these five different disease categories. We found that, though the intersections between disease genes and drug targets were small, disease genes were significantly enriched in targets compared to their enrichment in human protein-coding genes. We further compared network properties of the proteins encoded by disease genes and drug targets in human protein-protein interaction networks (interactome). The results showed that the drug targets tended to have higher degree, higher betweenness, and lower clustering coefficient in cancer Furthermore, we observed a clear fraction increase of disease proteins or drug targets in the near neighborhood compared with the randomized genes. The study presents the first comprehensive comparison of the disease genes and drug targets in the context of interactome. The results provide some foundational network characteristics for further designing computational strategies to predict novel drug targets and drug repurposing.

  16. Purification of target proteins from intracellular inclusions mediated by intein cleavable polyhydroxyalkanoate synthase fusions.

    PubMed

    Du, Jinping; Rehm, Bernd H A

    2017-11-02

    Recombinant protein production and purification from Escherichia coli is often accompanied with expensive and complicated procedures, especially for therapeutic proteins. Here it was demonstrated that, by using an intein cleavable polyhydroxyalkanoate synthase fusion, recombinant proteins can be first produced and sequestered on a natural resin, the polyhydroxyalkanoate (PHA) inclusions, then separated from contaminating host proteins via simple PHA bead isolation steps, and finally purified by specific release into the soluble fraction induced by a pH reduction. By translationally fusing a target protein to PHA synthase using a self-cleaving intein as linker, intracellular production of PHA beads was achieved. Upon isolation of respective PHA beads the soluble pure target protein was released by a simple pH shift to 6. The utility of this approach was exemplified by producing six target proteins, including Aequorea victoria green fluorescent protein (GFP), Mycobacterium tuberculosis vaccine candidate Rv1626, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b). Here a new method for production and purification of a tag-less protein was developed through intein cleavable polyhydroxyalkanoate synthase fusion. Pure target protein could be easily obtained without laborious downstream processing.

  17. Protein S-nitrosylation as a therapeutic target for neurodegenerative diseases

    PubMed Central

    Nakamura, Tomohiro; Lipton, Stuart A.

    2015-01-01

    At physiological levels, nitric oxide (NO) contributes to the maintenance of normal neuronal activity and survival, thus serving as an important regulatory mechanism in the central nervous system. In contrast, accumulating evidence suggests that exposure to environmental toxins or the normal aging process can trigger excessive production of reactive oxygen/nitrogen species (such as NO), contributing to the etiology of several neurodegenerative diseases. Here we highlight protein S-nitrosylation, resulting from covalent attachment of an NO group to a cysteine thiol of the target protein, as a ubiquitous effector of NO signaling in both health and disease. We review our current understanding of this redox-dependent posttranslational modification under neurodegenerative conditions, and evaluate how targeting dysregulated protein S-nitrosylation can lead to novel therapeutics. PMID:26707925

  18. NMR-based investigations into target DNA search processes of proteins.

    PubMed

    Iwahara, Junji; Zandarashvili, Levani; Kemme, Catherine A; Esadze, Alexandre

    2018-05-10

    To perform their function, transcription factors and DNA-repair/modifying enzymes must first locate their targets in the vast presence of nonspecific, but structurally similar sites on genomic DNA. Before reaching their targets, these proteins stochastically scan DNA and dynamically move from one site to another on DNA. Solution NMR spectroscopy provides unique atomic-level insights into the dynamic DNA-scanning processes, which are difficult to gain by any other experimental means. In this review, we provide an introductory overview on the NMR methods for the structural, dynamic, and kinetic investigations of target DNA search by proteins. We also discuss advantages and disadvantages of these NMR methods over other methods such as single-molecule techniques and biochemical approaches. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Drug Target Validation Methods in Malaria - Protein Interference Assay (PIA) as a Tool for Highly Specific Drug Target Validation.

    PubMed

    Meissner, Kamila A; Lunev, Sergey; Wang, Yuan-Ze; Linzke, Marleen; de Assis Batista, Fernando; Wrenger, Carsten; Groves, Matthew R

    2017-01-01

    The validation of drug targets in malaria and other human diseases remains a highly difficult and laborious process. In the vast majority of cases, highly specific small molecule tools to inhibit a proteins function in vivo are simply not available. Additionally, the use of genetic tools in the analysis of malarial pathways is challenging. These issues result in difficulties in specifically modulating a hypothetical drug target's function in vivo. The current "toolbox" of various methods and techniques to identify a protein's function in vivo remains very limited and there is a pressing need for expansion. New approaches are urgently required to support target validation in the drug discovery process. Oligomerisation is the natural assembly of multiple copies of a single protein into one object and this self-assembly is present in more than half of all protein structures. Thus, oligomerisation plays a central role in the generation of functional biomolecules. A key feature of oligomerisation is that the oligomeric interfaces between the individual parts of the final assembly are highly specific. However, these interfaces have not yet been systematically explored or exploited to dissect biochemical pathways in vivo. This mini review will describe the current state of the antimalarial toolset as well as the potentially druggable malarial pathways. A specific focus is drawn to the initial efforts to exploit oligomerisation surfaces in drug target validation. As alternative to the conventional methods, Protein Interference Assay (PIA) can be used for specific distortion of the target protein function and pathway assessment in vivo. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Computer-aided identification of novel protein targets of bisphenol A.

    PubMed

    Montes-Grajales, Diana; Olivero-Verbel, Jesus

    2013-10-09

    The xenoestrogen bisphenol A (2,2-bis-(p-hydroxyphenyl)-2-propane, BPA) is a known endocrine-disrupting chemical used in the fabrication of plastics, resins and flame retardants, that can be found throughout the environment and in numerous every day products. Human exposure to this chemical is extensive and generally occurs via oral route because it leaches from the food and beverage containers that contain it. Although most of the effects related to BPA exposure have been linked to the activation of the estrogen receptor (ER), the mechanisms of the interaction of BPA with protein targets different from ER are still unknown. Therefore, the objective of this work was to use a bioinformatics approach to identify possible new targets for BPA. Docking studies were performed between the optimized structure of BPA and 271 proteins related to different biochemical processes, as selected by text-mining. Refinement docking experiments and conformational analyses were carried out using LigandScout 3.0 for the proteins selected through the affinity ranking (lower than -8.0kcal/mol). Several proteins including ERR gamma (-9.9kcal/mol), and dual specificity protein kinases CLK-4 (-9.5kcal/mol), CLK-1 (-9.1kcal/mol) and CLK-2 (-9.0kcal/mol) presented great in silico binding affinities for BPA. The interactions between those proteins and BPA were mostly hydrophobic with the presence of some hydrogen bonds formed by leucine and asparagine residues. Therefore, this study suggests that this endocrine disruptor may have other targets different from the ER. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  1. Role of Neuroactive Steroids in the Peripheral Nervous System

    PubMed Central

    Melcangi, Roberto Cosimo; Giatti, Silvia; Pesaresi, Marzia; Calabrese, Donato; Mitro, Nico; Caruso, Donatella; Garcia-Segura, Luis Miguel

    2011-01-01

    Several reviews have so far pointed out on the relevant physiological and pharmacological role exerted by neuroactive steroids in the central nervous system. In the present review we summarize observations indicating that synthesis and metabolism of neuroactive steroids also occur in the peripheral nerves. Interestingly, peripheral nervous system is also a target of their action. Indeed, as here reported neuroactive steroids are physiological regulators of peripheral nerve functions and they may also represent interesting therapeutic tools for different types of peripheral neuropathy. PMID:22654839

  2. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

    PubMed Central

    Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

    2015-01-01

    A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5’-end including the 5’-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer. PMID:26221730

  3. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

    PubMed

    Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

    2015-01-01

    A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

  4. Alkylation Damage by Lipid Electrophiles Targets Functional Protein Systems*

    PubMed Central

    Codreanu, Simona G.; Ullery, Jody C.; Zhu, Jing; Tallman, Keri A.; Beavers, William N.; Porter, Ned A.; Marnett, Lawrence J.; Zhang, Bing; Liebler, Daniel C.

    2014-01-01

    Protein alkylation by reactive electrophiles contributes to chemical toxicities and oxidative stress, but the functional impact of alkylation damage across proteomes is poorly understood. We used Click chemistry and shotgun proteomics to profile the accumulation of proteome damage in human cells treated with lipid electrophile probes. Protein target profiles revealed three damage susceptibility classes, as well as proteins that were highly resistant to alkylation. Damage occurred selectively across functional protein interaction networks, with the most highly alkylation-susceptible proteins mapping to networks involved in cytoskeletal regulation. Proteins with lower damage susceptibility mapped to networks involved in protein synthesis and turnover and were alkylated only at electrophile concentrations that caused significant toxicity. Hierarchical susceptibility of proteome systems to alkylation may allow cells to survive sublethal damage while protecting critical cell functions. PMID:24429493

  5. Urea transporter proteins as targets for small-molecule diuretics.

    PubMed

    Esteva-Font, Cristina; Anderson, Marc O; Verkman, Alan S

    2015-02-01

    Conventional diuretics such as furosemide and thiazides target salt transporters in kidney tubules, but urea transporters (UTs) have emerged as alternative targets. UTs are a family of transmembrane channels expressed in a variety of mammalian tissues, in particular the kidney. UT knockout mice and humans with UT mutations exhibit reduced maximal urinary osmolality, demonstrating that UTs are necessary for the concentration of urine. Small-molecule screening has identified potent and selective inhibitors of UT-A, the UT protein expressed in renal tubule epithelial cells, and UT-B, the UT protein expressed in vasa recta endothelial cells. Data from UT knockout mice and from rodents administered UT inhibitors support the diuretic action of UT inhibition. The kidney-specific expression of UT-A1, together with high selectivity of the small-molecule inhibitors, means that off-target effects of such small-molecule drugs should be minimal. This Review summarizes the structure, expression and function of UTs, and looks at the evidence supporting the validity of UTs as targets for the development of salt-sparing diuretics with a unique mechanism of action. UT-targeted inhibitors may be useful alone or in combination with conventional diuretics for therapy of various oedemas and hyponatraemias, potentially including those refractory to treatment with current diuretics.

  6. Genome Editing-Enabled HTS Assays Expand Drug Target Pathways for Charcot–Marie–Tooth Disease

    PubMed Central

    2015-01-01

    Copy number variation resulting in excess PMP22 protein causes the peripheral neuropathy Charcot–Marie–Tooth disease, type 1A. To broadly interrogate chemically sensitive transcriptional pathways controlling PMP22 protein levels, we used the targeting precision of TALEN-mediated genome editing to embed reporters within the genetic locus harboring the Peripheral Myelin Protein 22 (Pmp22) gene. Using a Schwann cell line with constitutively high endogenous levels of Pmp22, we obtained allelic insertion of secreted bioluminescent reporters with sufficient signal to enable a 1536-well assay. Our findings from the quantitative high-throughput screening (qHTS) of several thousand drugs and clinically investigated compounds using this assay design both overlapped and expanded results from a previous assay using a randomly inserted reporter gene controlled by a single regulatory element of the Pmp22 gene. A key difference was the identification of a kinase-controlled inhibitory pathway of Pmp22 transcription revealed by the activity of the Protein kinase C (PKC)-modulator bryostatin. PMID:25188731

  7. The similarity between N-terminal targeting signals for protein import into different organelles and its evolutionary relevance

    PubMed Central

    Kunze, Markus; Berger, Johannes

    2015-01-01

    The proper distribution of proteins between the cytosol and various membrane-bound compartments is crucial for the functionality of eukaryotic cells. This requires the cooperation between protein transport machineries that translocate diverse proteins from the cytosol into these compartments and targeting signal(s) encoded within the primary sequence of these proteins that define their cellular destination. The mechanisms exerting protein translocation differ remarkably between the compartments, but the predominant targeting signals for mitochondria, chloroplasts and the ER share the N-terminal position, an α-helical structural element and the removal from the core protein by intraorganellar cleavage. Interestingly, similar properties have been described for the peroxisomal targeting signal type 2 mediating the import of a fraction of soluble peroxisomal proteins, whereas other peroxisomal matrix proteins encode the type 1 targeting signal residing at the extreme C-terminus. The structural similarity of N-terminal targeting signals poses a challenge to the specificity of protein transport, but allows the generation of ambiguous targeting signals that mediate dual targeting of proteins into different compartments. Dual targeting might represent an advantage for adaptation processes that involve a redistribution of proteins, because it circumvents the hierarchy of targeting signals. Thus, the co-existence of two equally functional import pathways into peroxisomes might reflect a balance between evolutionary constant and flexible transport routes. PMID:26441678

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

    PubMed Central

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

    2014-01-01

    There is serious interest in understanding the dynamics of the receptor–receptor and receptor–protein interactions in space and time and their integration in GPCR heteroreceptor complexes of the CNS. Moonlighting proteins are special multifunctional proteins because they perform multiple autonomous, often unrelated, functions without partitioning into different protein domains. Moonlighting through receptor oligomerization can be operationally defined as an allosteric receptor–receptor interaction, which leads to novel functions of at least one receptor protomer. GPCR-mediated signaling is a more complicated process than previously described as every GPCR and GPCR heteroreceptor complex requires a set of G protein interacting proteins, which interacts with the receptor in an orchestrated spatio-temporal fashion. GPCR heteroreceptor complexes with allosteric receptor–receptor interactions operating through the receptor interface have become major integrative centers at the molecular level and their receptor protomers act as moonlighting proteins. The GPCR heteroreceptor complexes in the CNS have become exciting new targets for neurotherapeutics in Parkinson's disease, schizophrenia, drug addiction, and anxiety and depression opening a new field in neuropsychopharmacology. PMID:24105074

  9. A Role for Weak Electrostatic Interactions in Peripheral Membrane Protein Binding

    PubMed Central

    Khan, Hanif M.; He, Tao; Fuglebakk, Edvin; Grauffel, Cédric; Yang, Boqian; Roberts, Mary F.; Gershenson, Anne; Reuter, Nathalie

    2016-01-01

    Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is a secreted virulence factor that binds specifically to phosphatidylcholine (PC) bilayers containing negatively charged phospholipids. BtPI-PLC carries a negative net charge and its interfacial binding site has no obvious cluster of basic residues. Continuum electrostatic calculations show that, as expected, nonspecific electrostatic interactions between BtPI-PLC and membranes vary as a function of the fraction of anionic lipids present in the bilayers. Yet they are strikingly weak, with a calculated ΔGel below 1 kcal/mol, largely due to a single lysine (K44). When K44 is mutated to alanine, the equilibrium dissociation constant for small unilamellar vesicles increases more than 50 times (∼2.4 kcal/mol), suggesting that interactions between K44 and lipids are not merely electrostatic. Comparisons of molecular-dynamics simulations performed using different lipid compositions reveal that the bilayer composition does not affect either hydrogen bonds or hydrophobic contacts between the protein interfacial binding site and bilayers. However, the occupancies of cation-π interactions between PC choline headgroups and protein tyrosines vary as a function of PC content. The overall contribution of basic residues to binding affinity is also context dependent and cannot be approximated by a rule-of-thumb value because these residues can contribute to both nonspecific electrostatic and short-range protein-lipid interactions. Additionally, statistics on the distribution of basic amino acids in a data set of membrane-binding domains reveal that weak electrostatics, as observed for BtPI-PLC, might be a less unusual mechanism for peripheral membrane binding than is generally thought. PMID:27028646

  10. Small molecule therapeutics targeting F-box proteins in cancer.

    PubMed

    Liu, Yuan; Mallampalli, Rama K

    2016-02-01

    The ubiquitin proteasome system (UPS) plays vital roles in maintaining protein equilibrium mainly through proteolytic degradation of targeted substrates. The archetypical SCF ubiquitin E3 ligase complex contains a substrate recognition subunit F-box protein that recruits substrates to the catalytic ligase core for its polyubiquitylation and subsequent proteasomal degradation. Several well-characterized F-box proteins have been demonstrated that are tightly linked to neoplasia. There is mounting information characterizing F-box protein-substrate interactions with the rationale to develop unique therapeutics for cancer treatment. Here we review that how F-box proteins function in cancer and summarize potential small molecule inhibitors for cancer therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

    PubMed Central

    Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

    2013-01-01

    Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity. PMID:24349308

  12. Advances in the Study of Aptamer-Protein Target Identification Using the Chromatographic Approach.

    PubMed

    Drabik, Anna; Ner-Kluza, Joanna; Mielczarek, Przemyslaw; Civit, Laia; Mayer, Günter; Silberring, Jerzy

    2018-06-01

    Ever since the development of the process known as the systematic evolution of ligands by exponential enrichment (SELEX), aptamers have been widely used in a variety of studies, including the exploration of new diagnostic tools and the discovery of new treatment methods. Aptamers' ability to bind to proteins with high affinity and specificity, often compared to that of antibodies, enables the search for potential cancer biomarkers and helps us understand the mechanisms of carcinogenesis. The blind spot of those investigations is usually the difficulty in the selective extraction of targets attached to the aptamer. There are many studies describing the cell SELEX for the prime choice of aptamers toward living cancer cells or even whole tumors in the animal models. However, a dilemma arises when a large number of proteins are being identified as potential targets, which is often the case. In this article, we present a new analytical approach designed to selectively target proteins bound to aptamers. During studies, we have focused on the unambiguous identification of the molecular targets of aptamers characterized by high specificity to the prostate cancer cells. We have compared four assay approaches using electrophoretic and chromatographic methods for "fishing out" aptamer protein targets followed by mass spectrometry identification. We have established a new methodology, based on the fluorescent-tagged oligonucleotides commonly used for flow-cytometry experiments or as optic aptasensors, that allowed the detection of specific aptamer-protein interactions by mass spectrometry. The use of atto488-labeled aptamers for the tracking of the formation of specific aptamer-target complexes provides the possibility of studying putative protein counterparts without needing to apply enrichment techniques. Significantly, changes in the hydrophobic properties of atto488-labeled aptamer-protein complexes facilitate their separation by reverse-phase chromatography combined with

  13. Advances in identification and validation of protein targets of natural products without chemical modification.

    PubMed

    Chang, J; Kim, Y; Kwon, H J

    2016-05-04

    Covering: up to February 2016Identification of the target proteins of natural products is pivotal to understanding the mechanisms of action to develop natural products for use as molecular probes and potential therapeutic drugs. Affinity chromatography of immobilized natural products has been conventionally used to identify target proteins, and has yielded good results. However, this method has limitations, in that labeling or tagging for immobilization and affinity purification often result in reduced or altered activity of the natural product. New strategies have recently been developed and applied to identify the target proteins of natural products and synthetic small molecules without chemical modification of the natural product. These direct and indirect methods for target identification of label-free natural products include drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), thermal proteome profiling (TPP), and bioinformatics-based analysis of connectivity. This review focuses on and reports case studies of the latest advances in target protein identification methods for label-free natural products. The integration of newly developed technologies will provide new insights and highlight the value of natural products for use as biological probes and new drug candidates.

  14. Development of Cell-Permeable, Non-Helical Constrained Peptides to Target a Key Protein-Protein Interaction in Ovarian Cancer.

    PubMed

    Wiedmann, Mareike M; Tan, Yaw Sing; Wu, Yuteng; Aibara, Shintaro; Xu, Wenshu; Sore, Hannah F; Verma, Chandra S; Itzhaki, Laura; Stewart, Murray; Brenton, James D; Spring, David R

    2017-01-09

    There is a lack of current treatment options for ovarian clear cell carcinoma (CCC) and the cancer is often resistant to platinum-based chemotherapy. Hence there is an urgent need for novel therapeutics. The transcription factor hepatocyte nuclear factor 1β (HNF1β) is ubiquitously overexpressed in CCC and is seen as an attractive therapeutic target. This was validated through shRNA-mediated knockdown of the target protein, HNF1β, in five high- and low-HNF1β-expressing CCC lines. To inhibit the protein function, cell-permeable, non-helical constrained proteomimetics to target the HNF1β-importin α protein-protein interaction were designed, guided by X-ray crystallographic data and molecular dynamics simulations. In this way, we developed the first reported series of constrained peptide nuclear import inhibitors. Importantly, this general approach may be extended to other transcription factors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Update on peripheral mechanisms of pain: beyond prostaglandins and cytokines

    PubMed Central

    2011-01-01

    The peripheral nociceptor is an important target of pain therapy because many pathological conditions such as inflammation excite and sensitize peripheral nociceptors. Numerous ion channels and receptors for inflammatory mediators were identified in nociceptors that are involved in neuronal excitation and sensitization, and new targets, beyond prostaglandins and cytokines, emerged for pain therapy. This review addresses mechanisms of nociception and focuses on molecules that are currently favored as new targets in drug development or that are already targeted by new compounds at the stage of clinical trials - namely the transient receptor potential V1 receptor, nerve growth factor, and voltage-gated sodium channels - or both. PMID:21542894

  16. A targeted IL-15 fusion protein with potent anti-tumor activity

    PubMed Central

    Chen, Siqi; Huang, Qiang; Liu, Jiayu; Xing, Jieyu; Zhang, Ning; Liu, Yawei; Wang, Zhong; Li, Qing

    2015-01-01

    IL-15 has been actively investigated for its potential in tumor immunotherapy. To enhance the anti-tumor activity of IL-15, the novel PFC-1 construct was designed, which comprises the following 3 parts: (1) IL-15Rα fused with IL-15 to enhance IL-15 activity, (2) an Fc fragment to increase protein half-life, and (3) an integrin-targeting RGD peptide to enhance tumor targeting. PFC-1 showed tumor cell targeting without compromising IL-15 activity. PFC-1 also had potent anti-tumor activities in xenograft models, suggesting the potential application of this multi-functional fusion protein in tumor therapy. PMID:26176990

  17. Amoxicillin haptenates intracellular proteins that can be transported in exosomes to target cells.

    PubMed

    Sánchez-Gómez, F J; González-Morena, J M; Vida, Y; Pérez-Inestrosa, E; Blanca, M; Torres, M J; Pérez-Sala, D

    2017-03-01

    Allergic reactions to β-lactams are among the most frequent causes of drug allergy and constitute an important clinical problem. Drug covalent binding to endogenous proteins (haptenation) is thought to be required for activation of the immune system. Nevertheless, neither the nature nor the role of the drug protein targets involved in this process is fully understood. Here, we aim to identify novel intracellular targets for haptenation by amoxicillin (AX) and their cellular fate. We have treated B lymphocytes with either AX or a biotinylated analog (AX-B). The identification of protein targets for haptenation by AX has been approached by mass spectrometry and immunoaffinity techniques. In addition, intercellular communication mediated by the delivery of vesicles loaded with AX-B-protein adducts has been explored by microscopy techniques. We have observed a complex pattern of AX-haptenated proteins. Several novel targets for haptenation by AX in B lymphocytes have been identified. AX-haptenated proteins were detected in cell lysates and extracellularly, either as soluble proteins or in lymphocyte-derived extracellular vesicles. Interestingly, exosomes from AX-B-treated cells showed a positive biotin signal in electron microscopy. Moreover, they were internalized by endothelial cells, thus supporting their involvement in intercellular transfer of haptenated proteins. These results represent the first identification of AX-mediated haptenation of intracellular proteins. Moreover, they show that exosomes can constitute a novel vehicle for haptenated proteins, and raise the hypothesis that they could provide antigens for activation of the immune system during the allergic response. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Protein targeting in the analysis of learning and memory: a potential alternative to gene targeting.

    PubMed

    Gerlai, R; Williams, S P; Cairns, B; Van Bruggen, N; Moran, P; Shih, A; Caras, I; Sauer, H; Phillips, H S; Winslow, J W

    1998-11-01

    Gene targeting using homologous recombination in embryonic stem (ES) cells offers unprecedented precision with which one may manipulate single genes and investigate the in vivo effects of defined mutations in the mouse. Geneticists argue that this technique abrogates the lack of highly specific pharmacological tools in the study of brain function and behavior. However, by now it has become clear that gene targeting has some limitations too. One problem is spatial and temporal specificity of the generated mutation, which may appear in multiple brain regions or even in other organs and may also be present throughout development, giving rise to complex, secondary phenotypical alterations. This may be a disadvantage in the functional analysis of a number of genes associated with learning and memory processes. For example, several proteins, including neurotrophins--cell-adhesion molecules--and protein kinases, that play a significant developmental role have recently been suggested to be also involved in neural and behavioral plasticity. Knocking out genes of such proteins may lead to developmental alterations or even embryonic lethality in the mouse, making it difficult to study their function in neural plasticity, learning, and memory. Therefore, alternative strategies to gene targeting may be needed. Here, we suggest a potentially useful in vivo strategy based on systemic application of immunoadhesins, genetically engineered fusion proteins possessing the Fc portion of the human IgG molecule and, for example, a binding domain of a receptor of interest. These proteins are stable in vivo and exhibit high binding specificity and affinity for the endogenous ligand of the receptor, but lack the ability to signal. Thus, if delivered to the brain, immunoadhesins may specifically block signalling of the receptor of interest. Using osmotic minipumps, the protein can be infused in a localized region of the brain for a specified period of time (days or weeks). Thus, the location

  19. Characteristics of viral protein expression by Epstein-Barr virus-infected B cells in peripheral blood of patients with infectious mononucleosis.

    PubMed

    Wagner, H J; Hornef, M; Middeldorp, J; Kirchner, H

    1995-11-01

    The frequency of Epstein-Barr virus (EBV) antigen-positive B cells in the peripheral blood of patients with infectious mononucleosis compared with that for latently EBV-infected individuals was examined by immunocytochemistry. B cells positive for Epstein-Barr nuclear antigen (EBNA) 1, EBNA2, and latent membrane protein were frequently found in all peripheral B lymphocyte preparations from 25 patients suffering for 3 to 28 days from infectious mononucleosis by using monoclonal antibodies and the alkaline phosphatase anti-alkaline technique. There was a significant decrease in the number of positive B cells during the course of disease. EBNA1-positive B cells were detected in 0.01 to 2.5% of total B cells (median, 0.8%), EBNA2-positive B cells were detected in 0.01 to 4.5% of total B cells (median, 0.9%), and latent membrane protein-positive B cells were detected in 0.01 to 1.8% of total B cells (median, 0.5%), depending on the duration of clinical signs. In contrast, we did not find any EBNA1- or EBNA2-positive B cells in 2 x 10(6) peripheral blood B lymphocytes of 10 latently EBV-infected individuals, whereas aliquots of the same cell preparations were EBV DNA positive by a PCR assay. Therefore, it appears to be possible to detect infectious mononucleosis by immunocytochemical determination of latent EBV products, which might be of relevance for the diagnosis of EBV reactivations in immunosuppressed patients.

  20. Integrin-mediated targeting of protein polymer nanoparticles carrying a cytostatic macrolide

    NASA Astrophysics Data System (ADS)

    Shi, Pu

    Cytotoxicity, low water solubility, rapid clearance from circulation, and offtarget side-effects are common drawbacks of conventional small-molecule drugs. To overcome these shortcomings, many multifunctional nanocarriers have been proposed to enhance drug delivery. In concept, multifunctional nanoparticles might carry multiple agents, control release rate, biodegrade, and utilize target-mediated drug delivery; however, the design of these particles presents many challenges at the stage of pharmaceutical development. An emerging solution to improve control over these particles is to turn to genetic engineering. Genetically engineered nanocarriers are precisely controlled in size and structure and can provide specific control over sites for chemical attachment of drugs. Genetically engineered drug carriers that assemble nanostructures including nanoparticles and nanofibers can be polymeric or nonpolymeric. This chapter summarizes the recent development of applications in drug and gene delivery utilizing nanostructures of polymeric genetically engineered drug carriers such as elastin-like polypeptides, silk-like polypeptides, and silk-elastin-like protein polymers, and non-polymeric genetically engineered drug carriers such as vault proteins and viral proteins. This chapter explores an alternative encapsulation strategy based on high-specificity avidity between a small molecule drug and its cognate protein target fused to the corona of protein polymer nanoparticles. With the new strategy, the drug associates tightly to the carrier and releases slowly, which may decrease toxicity and promote tumor accumulation via the enhanced permeability and retention effect. To test this hypothesis, the drug Rapamycin (Rapa) was selected for its potent anti-proliferative properties, which give it immunosuppressant and anti-tumor activity. Despite its potency, Rapa has low solubility, low oral bioavailability, and rapid systemic clearance, which make it an excellent candidate for

  1. [EFFECT OF RECOMBINANT ADENOVIRUS-BONE MORPHOGENETIC PROTEIN 12 TRANSFECTION ON DIFFERENTIATION OF PERIPHERAL BLOOD MESENCHYMAL STEM CELLS INTO TENDON/LIGAMENT CELLS].

    PubMed

    Fu, Weili; Chen, Gang; Tang, Xin; Li, Qi; Ll, Jian

    2015-04-01

    To research the effect of recombinant adenovirus-bone morphogenetic protein 12 (Ad-BMP-12) transfection on the differentiation of peripheral blood mesenchymal stem cells (MSCs) into tendon/ligament cells. Peripheral blood MSCs were isolated from New Zealand rabbits (3-4 months old) and cultured in vitro until passage 3. The recombinant adenoviral vector system was prepared using AdEasy system, then transfected into MSCs at passage 3 (transfected group); untransfected MSCs served as control (untransfected group). The morphological characteristics and growth of transfected cells were observed under inverted phase contrast microscope. The transfection efficiency and green fluorescent protein (GFP) expression were detected by flow cytometry (FCM) and fluorescence microscopy. After cultured for 14 days in vitro, the expressions of tendon/ligament-specific markers were determined by immunohistochemistry and real-time fluorescent quantitative PCR. GFP expression could be observed in peripheral blood MSCs at 8 hours after transfection. At 24 hours after transfection, the cells had clear morphology and grew slowly under inverted phase contrast microscope and almost all expressed GFP at the same field under fluorescence microscopy. FCM analysis showed that the transfection efficiency of the transfected group was 99.57%, while it was 2.46% in the untransfected group. The immunohistochemistry showed that the expression of collagen type I gradually increased with culture time in vitro. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of the tendon/ligament-specific genes (Tenomodulin, Tenascin-C, and Decorin) in the transfected group were significantly higher than those in untransfected group (0.061+/- 0.013 vs. 0.004 +/- 0.002, t = -7.700, P=0.031; 0.029 +/- 0.008 vs. 0.003 +/- 0.001, t = -5.741, P=0.020; 0.679 +/- 0.067 vs. 0.142 +/- 0.024, t = -12.998, P=0.000). Ad-BMP-12 can significantly promote differentiation of peripheral blood MSCs into

  2. Targeted delivery of siRNA into breast cancer cells via phage fusion proteins.

    PubMed

    Bedi, Deepa; Gillespie, James W; Petrenko, Vasily A; Ebner, Andreas; Leitner, Michael; Hinterdorfer, Peter; Petrenko, Valery A

    2013-02-04

    Nucleic acids, including antisense oligonucleotides, small interfering RNA (siRNA), aptamers, and rybozymes, emerged as versatile therapeutics due to their ability to interfere in a well-planned manner with the flow of genetic information from DNA to protein. However, a systemic use of NAs is hindered by their instability in physiological liquids and inability of intracellular accumulation in the site of action. We first evaluated the potential of cancer specific phage fusion proteins as targeting ligands that provide encapsulation, protection, and navigation of siRNA to the target cell. The tumor-specific proteins were isolated from phages that were affinity selected from a landscape phage library against target breast cancer cells. It was found that fusion phage coat protein fpVIII displaying cancer-targeting peptides can effectively encapsulate siRNAs and deliver them into the cells leading to specific silencing of the model gene GAPDH. Complexes of siRNA and phage protein form nanoparticles (nanophages), which were characterized by atomic force microscopy and ELISA, and their stability was demonstrated by resistance of encapsulated siRNA to degradation by serum nucleases. The phage protein/siRNA complexes can make a new type of highly selective, stable, active, and physiologically acceptable cancer nanomedicine.

  3. Proteoform profiling of peripheral blood serum proteins from pregnant women provides a molecular IUGR signature.

    PubMed

    Wölter, M; Röwer, C; Koy, C; Rath, W; Pecks, U; Glocker, M O

    2016-10-21

    Intrauterine growth restriction (IUGR) is an important cause of perinatal morbidity and mortality and contributes substantially to medically indicated preterm birth; preventing fetal death. Molecular profiling of the mothers' peripheral blood was desired to monitor the health conditions of the fetuses. To develop such a minimally invasive assay, we applied a protein affinity fractionation method to peripheral blood serum samples from pregnant women belonging to either the IUGR or to the control group. Proof-of-principle was shown by relative quantitation analysis of mixtures of intact proteoforms using MALDI-ToF mass spectrometry. The two best differentiating proteins and proteoforms, respectively, were apolipoprotein C-II and apolipoprotein C-III 0 . Together with three robustly expressed protein proteoforms proapolipoprotein C-II, apolipoprotein C-III 1 , and apolipoprotein C-III 2 , which served as landmarks for relative quantitation analysis, they constituted the maternal IUGR proteome signature. Separation confidence of our IUGR proteoform signature reached a sensitivity of 0.73 and a specificity of 0.87 with an area under curve of 0.86 in receiver operator characteristics. Identification of IUGR newborns in the case room is required as children are severely diseased and need specialized care during infancy. Yet, at time of birth there is no readily applicable clinical test available. Hence, a molecular profiling assay is highly desired. It needs to be mentioned that current clinical definitions and recommendations for IUGR are unfortunately misleading and are not universally applicable. The most commonly adopted definition is an abdominal circumference (AC) or estimated fetal weight measurement <10th percentile. Although both, the American College of Obstetricians and Gynecologists (ACOG) and the Royal College of Obstetricians and Gynecologists (RCOG) agree that at this cut-off the risk of perinatal morbidity and mortality increases, this definition does not

  4. Intracellular Protein Delivery System Using a Target-Specific Repebody and Translocation Domain of Bacterial Exotoxin.

    PubMed

    Kim, Hee-Yeon; Kang, Jung Ae; Ryou, Jeong-Hyun; Lee, Gyeong Hee; Choi, Dae Seong; Lee, Dong Eun; Kim, Hak-Sung

    2017-11-17

    With the high efficacy of protein-based therapeutics and plenty of intracellular drug targets, cytosolic protein delivery in a cell-specific manner has attracted considerable attention in the field of precision medicine. Herein, we present an intracellular protein delivery system based on a target-specific repebody and the translocation domain of Pseudomonas aeruginosa exotoxin A. The delivery platform was constructed by genetically fusing an EGFR-specific repebody as a targeting moiety to the translocation domain, while a protein cargo was fused to the C-terminal end of the delivery platform. The delivery platform was revealed to efficiently translocate a protein cargo to the cytosol in a target-specific manner. We demonstrate the utility and potential of the delivery platform by showing a remarkable tumor regression with negligible toxicity in a xenograft mice model when gelonin was used as the cytotoxic protein cargo. The present platform can find wide applications to the cell-selective cytosolic delivery of diverse proteins in many areas.

  5. Target Identification of Grape Seed Extract in Colorectal Cancer using Drug Affinity Responsive Target Stability (DARTS) Technique: Role of Endoplasmic Reticulum Stress Response Proteins

    PubMed Central

    Derry, Molly M.; Somasagara, Ranganatha; Raina, Komal; Kumar, Sushil; Gomez, Joe; Patel, Manisha; Agarwal, Rajesh; Agarwal, Chapla

    2014-01-01

    Various natural agents, including grape seed extract (GSE), have shown considerable chemopreventive and anti-cancer efficacy against different cancers in pre-clinical studies; however, their specific protein targets are largely unknown and thus, their clinical usefulness is marred by limited scientific evidences about their direct cellular targets. Accordingly, herein, employing, for the first time, the recently developed drug affinity responsive target stability (DARTS) technique, we aimed to profile the potential protein targets of GSE in human colorectal cancer (CRC) cells. Unlike other methods, which can cause chemical alteration of the drug components to allow for detection, this approach relies on the fact that a drug bound protein may become less susceptible to proteolysis and hence the enriched proteins can be detected by Mass Spectroscopy methods. Our results, utilizing the DARTS technique followed by examination of the spectral output by LC/MS and the MASCOT data, revealed that GSE targets endoplasmic reticulum (ER) stress response proteins resulting in overall down regulation of proteins involved in translation and that GSE also causes oxidative protein modifications, specifically on methionine amino acids residues on its protein targets. Corroborating these findings, mechanistic studies revealed that GSE indeed caused ER stress and strongly inhibited PI3k-Akt–mTOR pathway for its biological effects in CRC cells. Furthermore, bioenergetics studies indicated that GSE also interferes with glycolysis and mitochondrial metabolism in CRC cells. Together, the present study identifying GSE molecular targets in CRC cells, combined with its efficacy in vast pre-clinical CRC models, further supports its usefulness for CRC prevention and treatment. PMID:24724981

  6. Expression patterns and role of PTEN in rat peripheral nerve development and injury.

    PubMed

    Chen, Hui; Xiang, Jianping; Wu, Junxia; He, Bo; Lin, Tao; Zhu, Qingtang; Liu, Xiaolin; Zheng, Canbin

    2018-05-29

    Studies have suggested that phosphatase and tensin homolog (PTEN) plays an important role in neuroprotection and neuronal regeneration. To better understand the potential role of PTEN with respect to peripheral nerve development and injury, we investigated the expression pattern of PTEN at different stages of rat peripheral nerve development and injury and subsequently assessed the effect of pharmacological inhibition of PTEN using bpV(pic) on axonal regeneration in a rat sciatic nerve crush injury model. During the early stages of development, PTEN exhibits low expression in neuronal cell bodies and axons. From embryonic day (E) 18.5 and postnatal day (P)5 to adult, PTEN protein becomes more detectable, with high expression in the dorsal root ganglia (DRG) and axons. PTEN expression is inhibited in peripheral nerves, preceding myelination during neuronal development and remyelination after acute nerve injury. Low PTEN expression after nerve injury promotes Akt/mammalian target of rapamycin (mTOR) signaling pathway activity. In vivo pharmacological inhibition of PTEN using bpV(pic) promoted axonal regrowth, increased the number of myelinated nerve fibers, improved locomotive recovery and enhanced the amplitude response and nerve conduction velocity following stimulation in a rat sciatic nerve crush injury model. Thus, we suggest that PTEN may play potential roles in peripheral nerve development and regeneration and that inhibition of PTEN expression is beneficial for nerve regeneration and functional recovery after peripheral nerve injury. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. 5-HT2A SEROTONIN RECEPTOR BIOLOGY: Interacting proteins, kinases and paradoxical regulation

    PubMed Central

    Roth, Bryan L

    2011-01-01

    5-hydroxytryptamine2A (5-HT2A) serotonin receptors are important pharmacological targets for a large number of central nervous system and peripheral serotonergic medications. In this review article I summarize work mainly from my lab regarding serotonin receptor anatomy, pharmacology, signaling and regulation. I highlight the role of serotonin receptor interacting proteins and the emerging paradigm of G-protein coupled receptor functional selectivity. PMID:21288474

  8. Quantification of proteins in urine samples using targeted mass spectrometry methods.

    PubMed

    Khristenko, Nina; Domon, Bruno

    2015-01-01

    Numerous clinical proteomics studies are focused on the development of biomarkers to improve either diagnostics for early disease detection or the monitoring of the response to the treatment. Although, a wealth of biomarker candidates are available, their evaluation and validation in a true clinical setup remains challenging. In biomarkers evaluation studies, a panel of proteins of interest are systematically analyzed in a large cohort of samples. However, in spite of the latest progresses in mass spectrometry, the consistent detection of pertinent proteins in high complex biological samples is still a challenging task. Thus, targeted LC-MS/MS methods are better suited for the systematic analysis of biomarkers rather than shotgun approaches. This chapter describes the workflow used to perform targeted quantitative analyses of proteins in urinary samples. The peptides, as surrogates of the protein of interest, are commonly measured using a triple quadrupole mass spectrometers operated in selected reaction monitoring (SRM) mode. More recently, the advances in targeted LC-MS/MS analysis based on parallel reaction monitoring (PRM) performed on a quadrupole-orbitrap instrument have allowed to increase the specificity and selectivity of the measurements.

  9. The peripheral clock regulates human pigmentation.

    PubMed

    Hardman, Jonathan A; Tobin, Desmond J; Haslam, Iain S; Farjo, Nilofer; Farjo, Bessam; Al-Nuaimi, Yusur; Grimaldi, Benedetto; Paus, Ralf

    2015-04-01

    Although the regulation of pigmentation is well characterized, it remains unclear whether cell-autonomous controls regulate the cyclic on-off switching of pigmentation in the hair follicle (HF). As human HFs and epidermal melanocytes express clock genes and proteins, and given that core clock genes (PER1, BMAL1) modulate human HF cycling, we investigated whether peripheral clock activity influences human HF pigmentation. We found that silencing BMAL1 or PER1 in human HFs increased HF melanin content. Furthermore, tyrosinase expression and activity, as well as TYRP1 and TYRP2 mRNA levels, gp100 protein expression, melanocyte dendricity, and the number gp100+ HF melanocytes, were all significantly increased in BMAL1 and/or PER1-silenced HFs. BMAL1 or PER1 silencing also increased epidermal melanin content, gp100 protein expression, and tyrosinase activity in human skin. These effects reflect direct modulation of melanocytes, as BMAL1 and/or PER1 silencing in isolated melanocytes increased tyrosinase activity and TYRP1/2 expression. Mechanistically, BMAL1 knockdown reduces PER1 transcription, and PER1 silencing induces phosphorylation of the master regulator of melanogenesis, microphthalmia-associated transcription factor, thus stimulating human melanogenesis and melanocyte activity in situ and in vitro. Therefore, the molecular clock operates as a cell-autonomous modulator of human pigmentation and may be targeted for future therapeutic strategies.

  10. Peripheral elastic and inelastic scattering of O17,18 on light targets at 12 MeV/nucleon

    NASA Astrophysics Data System (ADS)

    Al-Abdullah, T.; Carstoiu, F.; Gagliardi, C. A.; Tabacaru, G.; Trache, L.; Tribble, R. E.

    2014-06-01

    A study of interaction of neutron-rich oxygen isotopes O17,18 with light targets has been undertaken in order to determine the optical potentials needed for the transfer reaction C13(O17,O18)C12. Optical potentials in both incoming and outgoing channels have been determined in a single experiment. This transfer reaction was used to infer the direct capture rate to the F17(p,γ)Ne18 which is essential to estimate the production of F18 at stellar energies in ONe novae. The success of the asymptotic normalization coefficient (ANC) as indirect method for astrophysics is guaranteed if the reaction mechanism is peripheral and the distorted wave Born approximation cross-section calculations are warranted and stable against the optical model potential (OMP) used. We demonstrate the stability of the ANC method and the OMP results by using good-quality elastic and inelastic-scattering data with stable beams before extending the procedures to rare-ion beams. The peripherality of our reaction is inferred from a semiclassical decomposition of the total-scattering amplitude into barrier and internal barrier components. Comparison between elastic scattering of O17, O18, and O16 projectiles is made.

  11. Affinity resins as new tools for identifying target proteins of ascorbic acid.

    PubMed

    Iwaoka, Yuji; Nishino, Kohei; Ishikawa, Takahiro; Ito, Hideyuki; Sawa, Yoshihiro; Tai, Akihiro

    2018-02-12

    l-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.

  12. Protein targets for anticancer gold compounds: mechanistic inferences.

    PubMed

    Gabbiani, Chiara; Messori, Luigi

    2011-12-01

    Gold compounds form an interesting class of antiproliferative agents of potential pharmacological use in cancer treatment. Indeed, a number of gold compounds, either gold(III) or gold(I), were recently described and characterised that manifested remarkable cytotoxic properties in vitro against cultured cancer cells; for some of them encouraging in vivo results were also reported toward a few relevant animal models of cancer. The molecular mechanisms through which gold compounds exert their biological effects are still largely unknown and the subject of intense investigations. Recent studies point out that the modes of action of cytotoxic gold compounds are essentially DNA-independent and cisplatin-unrelated, relying -most likely- on gold interactions with a variety of protein targets. Notably, a few cellular proteins playing relevant functional roles were proposed to represent effective targets for cytotoxic gold compounds but these hypotheses need adequate validation. The state of the art of this research area and the perspectives for future studies are herein critically analysed and discussed.

  13. Cholesterol regulates the endoplasmic reticulum exit of the major membrane protein P0 required for peripheral myelin compaction.

    PubMed

    Saher, Gesine; Quintes, Susanne; Möbius, Wiebke; Wehr, Michael C; Krämer-Albers, Eva-Maria; Brügger, Britta; Nave, Klaus-Armin

    2009-05-13

    Rapid impulse conduction requires electrical insulation of axons by myelin, a cholesterol-rich extension of the glial cell membrane with a characteristic composition of proteins and lipids. Mutations in several myelin protein genes cause endoplasmic reticulum (ER) retention and disease, presumably attributable to failure of misfolded proteins to pass the ER quality control. Because many myelin proteins partition into cholesterol-rich membrane rafts, their interaction with cholesterol could potentially be part of the ER quality control system. Here, we provide in vitro and in vivo evidence that the major peripheral myelin protein P0 requires cholesterol for exiting the ER and reaching the myelin compartment. Cholesterol dependency of P0 trafficking in heterologous cells is mediated by a cholesterol recognition/interaction amino acid consensus (CRAC) motif. Mutant mice lacking cholesterol biosynthesis in Schwann cells suffer from severe hypomyelination with numerous uncompacted myelin stretches. This demonstrates that high-level cholesterol coordinates P0 export with myelin membrane synthesis, which is required for the correct stoichiometry of myelin components and for myelin compaction.

  14. Facilitated Protein Association via Engineered Target Search Pathways Visualized by Paramagnetic NMR Spectroscopy.

    PubMed

    An, So Young; Kim, Eun-Hee; Suh, Jeong-Yong

    2018-06-05

    Proteins assemble to form functional complexes via the progressive evolution of nonspecific complexes formed by transient encounters. This target search process generally involves multiple routes that lead the initial encounters to the final complex. In this study, we have employed NMR paramagnetic relaxation enhancement to visualize the encounter complexes between histidine-containing phosphocarrier protein and the N-terminal domain of enzyme I and demonstrate that protein association can be significantly enhanced by engineering on-pathways. Specifically, mutations in surface charges away from the binding interface can elicit new on-pathway encounter complexes, increasing their binding affinity by an order of magnitude. The structure of these encounter complexes indicates that such on-pathways extend the built-in target search process of the native protein complex. Furthermore, blocking on-pathways by countering mutations reverts their binding affinity. Our study thus illustrates that protein interactions can be engineered by rewiring the target search process. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Multisystemic Disease Modeling of Liver-Derived Protein Folding Disorders Using Induced Pluripotent Stem Cells (iPSCs).

    PubMed

    Leung, Amy; Murphy, George J

    2016-01-01

    Familial transthyretin amyloidosis (ATTR) is an autosomal dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR, protein secreted from the liver aggregates and forms fibrils in target organs, chiefly the heart and peripheral nervous system, highlighting the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Here, we describe detailed methodologies for the directed differentiation of protein folding disease-specific iPSCs into hepatocytes that produce mutant protein, and neural-lineage cells often targeted in disease. Methodologies are also described for the construction of multisystem models and drug screening using iPSCs.

  16. 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.

  17. Fusion Protein Vaccines Targeting Two Tumor Antigens Generate Synergistic Anti-Tumor Effects

    PubMed Central

    Cheng, Wen-Fang; Chang, Ming-Cheng; Sun, Wei-Zen; Jen, Yu-Wei; Liao, Chao-Wei; Chen, Yun-Yuan; Chen, Chi-An

    2013-01-01

    Introduction Human papillomavirus (HPV) has been consistently implicated in causing several kinds of malignancies, and two HPV oncogenes, E6 and E7, represent two potential target antigens for cancer vaccines. We developed two fusion protein vaccines, PE(ΔIII)/E6 and PE(ΔIII)/E7 by targeting these two tumor antigens to test whether a combination of two fusion proteins can generate more potent anti-tumor effects than a single fusion protein. Materials and Methods In vivo antitumor effects including preventive, therapeutic, and antibody depletion experiments were performed. In vitro assays including intracellular cytokine staining and ELISA for Ab responses were also performed. Results PE(ΔIII)/E6+PE(ΔIII)/E7 generated both stronger E6 and E7-specific immunity. Only 60% of the tumor protective effect was observed in the PE(ΔIII)/E6 group compared to 100% in the PE(ΔIII)/E7 and PE(ΔIII)/E6+PE(ΔIII)/E7 groups. Mice vaccinated with the PE(ΔIII)/E6+PE(ΔIII)/E7 fusion proteins had a smaller subcutaneous tumor size than those vaccinated with PE(ΔIII)/E6 or PE(ΔIII)/E7 fusion proteins alone. Conclusion Fusion protein vaccines targeting both E6 and E7 tumor antigens generated more potent immunotherapeutic effects than E6 or E7 tumor antigens alone. This novel strategy of targeting two tumor antigens together can promote the development of cancer vaccines and immunotherapy in HPV-related malignancies. PMID:24058440

  18. Protein kinases: mechanisms and downstream targets in inflammation-mediated obesity and insulin resistance.

    PubMed

    Nandipati, Kalyana C; Subramanian, Saravanan; Agrawal, Devendra K

    2017-02-01

    Obesity-induced low-grade inflammation (metaflammation) impairs insulin receptor signaling. This has been implicated in the development of insulin resistance. Insulin signaling in the target tissues is mediated by stress kinases such as p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, inhibitor of NF-kB kinase complex β (IKKβ), AMP-activated protein kinase, protein kinase C, Rho-associated coiled-coil containing protein kinase, and RNA-activated protein kinase. Most of these kinases phosphorylate several key regulators in glucose homeostasis. The phosphorylation of serine residues in the insulin receptor and IRS-1 molecule results in diminished enzymatic activity in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. This has been one of the key mechanisms observed in the tissues that are implicated in insulin resistance especially in type 2 diabetes mellitus (T2-DM). Identifying the specific protein kinases involved in obesity-induced chronic inflammation may help in developing the targeted drug therapies to minimize the insulin resistance. This review is focused on the protein kinases involved in the inflammatory cascade and molecular mechanisms and their downstream targets with special reference to obesity-induced T2-DM.

  19. Pharmacoperone drugs: targeting misfolded proteins causing lysosomal storage-, ion channels-, and G protein-coupled receptors-associated conformational disorders.

    PubMed

    Hou, Zhi-Shuai; Ulloa-Aguirre, Alfredo; Tao, Ya-Xiong

    2018-06-01

    Conformational diseases are caused by structurally abnormal proteins that cannot fold properly and achieve their native conformation. Misfolded proteins frequently originate from genetic mutations that may lead to loss-of-function diseases involving a variety of structurally diverse proteins including enzymes, ion channels, and membrane receptors. Pharmacoperones are small molecules that cross the cell surface plasma membrane and reach their target proteins within the cell, serving as molecular scaffolds to stabilize the native conformation of misfolded or well-folded but destabilized proteins, to prevent their degradation and promote correct trafficking to their functional site of action. Because of their high specificity toward the target protein, pharmacoperones are currently the focus of intense investigation as therapy for several conformational diseases. Areas covered: This review summarizes data on the mechanisms leading to protein misfolding and the use of pharmacoperone drugs as an experimental approach to rescue function of distinct misfolded/misrouted proteins associated with a variety of diseases, such as lysosomal storage diseases, channelopathies, and G protein-coupled receptor misfolding diseases. Expert commentary: The fact that many misfolded proteins may retain function, offers a unique therapeutic opportunity to cure disease by directly correcting misrouting through administering pharmacoperone drugs thereby rescuing function of disease-causing, conformationally abnormal proteins.

  20. Y-Trap Cancer Immunotherapy Drug Targets Two Proteins

    Cancer.gov

    Two groups of researchers, working independently, have fused a TGF-beta receptor to a monoclonal antibody that targets a checkpoint protein. The result, this Cancer Currents blog describes, is a single hybrid molecule called a Y-trap that blocks two pathways used by tumors to evade the immune system.

  1. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

    PubMed Central

    Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

    2012-01-01

    Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

  2. Prioritization of potential drug targets against P. aeruginosa by core proteomic analysis using computational subtractive genomics and Protein-Protein interaction network.

    PubMed

    Uddin, Reaz; Jamil, Faiza

    2018-06-01

    Pseudomonas aeruginosa is an opportunistic gram-negative bacterium that has the capability to acquire resistance under hostile conditions and become a threat worldwide. It is involved in nosocomial infections. In the current study, potential novel drug targets against P. aeruginosa have been identified using core proteomic analysis and Protein-Protein Interactions (PPIs) studies. The non-redundant reference proteome of 68 strains having complete genome and latest assembly version of P. aeruginosa were downloaded from ftp NCBI RefSeq server in October 2016. The standalone CD-HIT tool was used to cluster ortholog proteins (having >=80% amino acid identity) present in all strains. The pan-proteome was clustered in 12,380 Clusters of Orthologous Proteins (COPs). By using in-house shell scripts, 3252 common COPs were extracted out and designated as clusters of core proteome. The core proteome of PAO1 strain was selected by fetching PAO1's proteome from common COPs. As a result, 1212 proteins were shortlisted that are non-homologous to the human but essential for the survival of the pathogen. Among these 1212 proteins, 321 proteins are conserved hypothetical proteins. Considering their potential as drug target, those 321 hypothetical proteins were selected and their probable functions were characterized. Based on the druggability criteria, 18 proteins were shortlisted. The interacting partners were identified by investigating the PPIs network using STRING v10 database. Subsequently, 8 proteins were shortlisted as 'hub proteins' and proposed as potential novel drug targets against P. aeruginosa. The study is interesting for the scientific community working to identify novel drug targets against MDR pathogens particularly P. aeruginosa. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. TargetCrys: protein crystallization prediction by fusing multi-view features with two-layered SVM.

    PubMed

    Hu, Jun; Han, Ke; Li, Yang; Yang, Jing-Yu; Shen, Hong-Bin; Yu, Dong-Jun

    2016-11-01

    The accurate prediction of whether a protein will crystallize plays a crucial role in improving the success rate of protein crystallization projects. A common critical problem in the development of machine-learning-based protein crystallization predictors is how to effectively utilize protein features extracted from different views. In this study, we aimed to improve the efficiency of fusing multi-view protein features by proposing a new two-layered SVM (2L-SVM) which switches the feature-level fusion problem to a decision-level fusion problem: the SVMs in the 1st layer of the 2L-SVM are trained on each of the multi-view feature sets; then, the outputs of the 1st layer SVMs, which are the "intermediate" decisions made based on the respective feature sets, are further ensembled by a 2nd layer SVM. Based on the proposed 2L-SVM, we implemented a sequence-based protein crystallization predictor called TargetCrys. Experimental results on several benchmark datasets demonstrated the efficacy of the proposed 2L-SVM for fusing multi-view features. We also compared TargetCrys with existing sequence-based protein crystallization predictors and demonstrated that the proposed TargetCrys outperformed most of the existing predictors and is competitive with the state-of-the-art predictors. The TargetCrys webserver and datasets used in this study are freely available for academic use at: http://csbio.njust.edu.cn/bioinf/TargetCrys .

  4. Microfibrillar-associated protein 4 variation in symptomatic peripheral artery disease.

    PubMed

    Hemstra, Line Ea; Schlosser, Anders; Lindholt, Jes Sanddal; Sorensen, Grith L

    2018-06-08

    Symptomatic peripheral artery disease (PAD) is an atherosclerotic occlusive disease affecting the lower extremities. The cause of symptomatic PAD is atherosclerosis, vascular dysfunctions, impaired angiogenesis and neointima formation. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein, which is highly expressed in the heart and arteries and recently introduced as a potential mediator of pathological vascular remodeling and neointima formation. We aimed to investigate the relationship between serum MFAP4 (sMFAP4) and symptomatic PAD outcomes. A total of 286 PAD patients were analyzed if they had either intermittent claudication or critical lower-extremity ischemia (CLI) and followed for 7 years. The level of serum MFAP4 (sMFAP4) was measured by alphaLISA. Kaplan-Meier, Cox proportional hazard and logistic regression analysis were used to analyze the associations between upper tertile sMFAP4 and symptomatic PAD outcomes. Patients with upper tertile sMFAP4 had an odds ratio (OR) of 2.65 (p < 0.001) for having CLI diagnosis. Further analysis indicated that patients with upper tertile sMFAP4 had a hazard ratio (HR) of 1.97 (p = 0.04) for cardiovascular death during the 7-years follow-up. However, analysis of 2-year primary patency showed that patients with upper tertile sMFAP4 had decreased risk of vascular occlusion after reconstructive surgery with HR of 0.15 (p = 0.02). sMFAP4 has potential as a prognostic marker for cardiovascular death, primary patency of reconstructed vessels and CLI diagnosis in symptomatic PAD patients. Confirmation of observations in larger cohorts is warranted.

  5. Efficient protein targeting to the inner nuclear membrane requires Atlastin-dependent maintenance of ER topology

    PubMed Central

    Pawar, Sumit; Ungricht, Rosemarie; Tiefenboeck, Peter; Leroux, Jean-Christophe

    2017-01-01

    Newly synthesized membrane proteins are targeted to the inner nuclear membrane (INM) by diffusion within the membrane system of the endoplasmic reticulum (ER), translocation through nuclear pore complexes (NPCs) and retention on nuclear partners. Using a visual in vitro assay we previously showed that efficient protein targeting to the INM depends on nucleotide hydrolysis. We now reveal that INM targeting is GTP-dependent. Exploiting in vitro reconstitution and in vivo analysis of INM targeting, we establish that Atlastins, membrane-bound GTPases of the ER, sustain the efficient targeting of proteins to the INM by their continued activity in preserving ER topology. When ER topology is altered, the long-range diffusional exchange of proteins in the ER network and targeting efficiency to the INM are diminished. Highlighting the general importance of proper ER topology, we show that Atlastins also influence NPC biogenesis and timely exit of secretory cargo from the ER. PMID:28826471

  6. RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses

    PubMed Central

    Baker, Oliver; Gupta, Ashish; Obst, Mandy; Zhang, Youming; Anastassiadis, Konstantinos; Fu, Jun; Stewart, A. Francis

    2016-01-01

    A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated recombineering pipeline for fluent generation of long arm targeting constructs is also presented. PMID:27216209

  7. Targeting RNA–Protein Interactions within the Human Immunodeficiency Virus Type 1 Lifecycle

    PubMed Central

    2013-01-01

    RNA–protein interactions are vital throughout the HIV-1 life cycle for the successful production of infectious virus particles. One such essential RNA–protein interaction occurs between the full-length genomic viral RNA and the major structural protein of the virus. The initial interaction is between the Gag polyprotein and the viral RNA packaging signal (psi or Ψ), a highly conserved RNA structural element within the 5′-UTR of the HIV-1 genome, which has gained attention as a potential therapeutic target. Here, we report the application of a target-based assay to identify small molecules, which modulate the interaction between Gag and Ψ. We then demonstrate that one such molecule exhibits potent inhibitory activity in a viral replication assay. The mode of binding of the lead molecules to the RNA target was characterized by 1H NMR spectroscopy. PMID:24358934

  8. Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

    PubMed Central

    Navarre, William Wiley; Schneewind, Olaf

    1999-01-01

    The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins. PMID:10066836

  9. The influenza virus NS1 protein as a therapeutic target.

    PubMed

    Engel, Daniel A

    2013-09-01

    Nonstructural protein 1 (NS1) of influenza A virus plays a central role in virus replication and blockade of the host innate immune response, and is therefore being considered as a potential therapeutic target. The primary function of NS1 is to dampen the host interferon (IFN) response through several distinct molecular mechanisms that are triggered by interactions with dsRNA or specific cellular proteins. Sequestration of dsRNA by NS1 results in inhibition of the 2'-5' oligoadenylate synthetase/RNase L antiviral pathway, and also inhibition of dsRNA-dependent signaling required for new IFN production. Binding of NS1 to the E3 ubiquitin ligase TRIM25 prevents activation of RIG-I signaling and subsequent IFN induction. Cellular RNA processing is also targeted by NS1, through recognition of cleavage and polyadenylation specificity factor 30 (CPSF30), leading to inhibition of IFN-β mRNA processing as well as that of other cellular mRNAs. In addition NS1 binds to and inhibits cellular protein kinase R (PKR), thus blocking an important arm of the IFN system. Many additional proteins have been reported to interact with NS1, either directly or indirectly, which may serve its anti-IFN and additional functions, including the regulation of viral and host gene expression, signaling pathways and viral pathogenesis. Many of these interactions are potential targets for small-molecule intervention. Structural, biochemical and functional studies have resulted in hypotheses for drug discovery approaches that are beginning to bear experimental fruit, such as targeting the dsRNA-NS1 interaction, which could lead to restoration of innate immune function and inhibition of virus replication. This review describes biochemical, cell-based and nucleic acid-based approaches to identifying NS1 antagonists. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  10. The influenza virus NS1 protein as a therapeutic target

    PubMed Central

    Engel, Daniel A.

    2015-01-01

    Nonstructural protein 1 (NS1) of influenza A virus plays a central role in virus replication and blockade of the host innate immune response, and is therefore being considered as a potential therapeutic target. The primary function of NS1 is to dampen the host interferon (IFN) response through several distinct molecular mechanisms that are triggered by interactions with dsRNA or specific cellular proteins. Sequestration of dsRNA by NS1 results in inhibition of the 2’-5’ oligoadenylate synthetase/RNase L antiviral pathway, and also inhibition of dsRNA-dependent signaling required for new IFN production. Binding of NS1 to the E3 ubiquitin ligase TRIM25 prevents activation of RIG-I signaling and subsequent IFN induction. Cellular RNA processing is also targeted by NS1, through recognition of cleavage and polyadenylation specificity factor 30 (CPSF30), leading to inhibition of IFN- mRNA processing as well as that of other cellular mRNAs. In addition NS1 binds to and inhibits cellular protein kinase R (PKR), thus blocking an important arm of the IFN system. Many additional proteins have been reported to interact with NS1, either directly or indirectly, which may serve its anti-IFN and additional functions, including the regulation of viral and host gene expression, signaling pathways and viral pathogenesis. Many of these interactions are potential targets for small-molecule intervention. Structural, biochemical and functional studies have resulted in hypotheses for drug discovery approaches that are beginning to bear experimental fruit, such as targeting the dsRNA-NS1 interaction, which could lead to restoration of innate immune function and inhibition of virus replication. This review describes biochemical, cell-based and nucleic acid-based approaches to identifying NS1 antagonists. PMID:23796981

  11. Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Strauch, Eva-Maria; Bernard, Steffen M.; La, David

    Many viral surface glycoproteins and cell surface receptors are homo-oligomers1, 2, 3, 4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5, 6, 7, 8. In the first step, a small protein ismore » designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.« less

  12. The past and presence of gene targeting: from chemicals and DNA via proteins to RNA.

    PubMed

    Geel, T M; Ruiters, M H J; Cool, R H; Halby, L; Voshart, D C; Andrade Ruiz, L; Niezen-Koning, K E; Arimondo, P B; Rots, M G

    2018-06-05

    The ability to target DNA specifically at any given position within the genome allows many intriguing possibilities and has inspired scientists for decades. Early gene-targeting efforts exploited chemicals or DNA oligonucleotides to interfere with the DNA at a given location in order to inactivate a gene or to correct mutations. We here describe an example towards correcting a genetic mutation underlying Pompe's disease using a nucleotide-fused nuclease (TFO-MunI). In addition to the promise of gene correction, scientists soon realized that genes could be inactivated or even re-activated without inducing potentially harmful DNA damage by targeting transcriptional modulators to a particular gene. However, it proved difficult to fuse protein effector domains to the first generation of programmable DNA-binding agents. The engineering of gene-targeting proteins (zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs)) circumvented this problem. The disadvantage of protein-based gene targeting is that a fusion protein needs to be engineered for every locus. The recent introduction of CRISPR/Cas offers a flexible approach to target a (fusion) protein to the locus of interest using cheap designer RNA molecules. Many research groups now exploit this platform and the first human clinical trials have been initiated: CRISPR/Cas has kicked off a new era of gene targeting and is revolutionizing biomedical sciences.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'. © 2018 The Author(s).

  13. Chemical approaches to targeted protein degradation through modulation of the ubiquitin-proteasome pathway.

    PubMed

    Collins, Ian; Wang, Hannah; Caldwell, John J; Chopra, Raj

    2017-03-15

    Manipulation of the ubiquitin-proteasome system to achieve targeted degradation of proteins within cells using chemical tools and drugs has the potential to transform pharmacological and therapeutic approaches in cancer and other diseases. An increased understanding of the molecular mechanism of thalidomide and its analogues following their clinical use has unlocked small-molecule modulation of the substrate specificity of the E3 ligase cereblon (CRBN), which in turn has resulted in the advancement of new immunomodulatory drugs (IMiDs) into the clinic. The degradation of multiple context-specific proteins by these pleiotropic small molecules provides a means to uncover new cell biology and to generate future drug molecules against currently undruggable targets. In parallel, the development of larger bifunctional molecules that bring together highly specific protein targets in complexes with CRBN, von Hippel-Lindau, or other E3 ligases to promote ubiquitin-dependent degradation has progressed to generate selective chemical compounds with potent effects in cells and in vivo models, providing valuable tools for biological target validation and with future potential for therapeutic use. In this review, we survey recent breakthroughs achieved in these two complementary methods and the discovery of new modes of direct and indirect engagement of target proteins with the proteasome. We discuss the experimental characterisation that validates the use of molecules that promote protein degradation as chemical tools, the preclinical and clinical examples disclosed to date, and the future prospects for this exciting area of chemical biology. © 2017 The Author(s).

  14. Role of protein kinase D in Golgi exit and lysosomal targeting of the transmembrane protein, Mcoln1

    PubMed Central

    Marks, David L.; Holicky, Eileen L.; Wheatley, Christine L.; Frumkin, Ayala; Bach, Gideon; Pagano, Richard E.

    2012-01-01

    The targeting of lysosomal transmembrane proteins from the Golgi apparatus to lysosomes is a complex process that is only beginning to be understood. Here, the lysosomal targeting of Mcoln1, the transmembrane protein defective in the autosomal recessive disease, Mucolipidosis, type IV, was studied by over-expressing full length and truncated forms of the protein in human cells, followed by detection using immunofluorescence and immunoblotting. We demonstrated that a 53 amino acid C-terminal region of Mcoln1 is required for efficient exit from the Golgi. Truncations lacking this region exhibited reduced delivery to lysosomes and decreased proteolytic cleavage of Mcoln1 into characteristic ~35 kDa fragments, suggesting that this cleavage occurs in lysosomes. In addition, we found that co-expression of full length Mcoln1 with kinase-inactive protein kinase D (PKD) 1 or 2 inhibited Mcoln1 Golgi exit and transport to lysosomes and decreased Mcoln1 cleavage. These studies suggest that PKDs play a role in the delivery of some lysosomal resident transmembrane proteins from the Golgi to the lysosomes. PMID:22268962

  15. Plasmids for variable expression of proteins targeted to the mitochondrial matrix or intermembrane space.

    PubMed

    Newman, Laura E; Schiavon, Cara; Kahn, Richard A

    2016-01-01

    We describe the construction and uses of a series of plasmids for directing expression to varied levels of exogenous proteins targeted to the mitochondrial matrix or intermembrane space. We found that the level of protein expression achieved, the kinetics of expression and mitochondrial import, and half-life after import can each vary with the protein examined. These factors should be considered when directing localization of an exogenous protein to mitochondria for rescue, proteomics, or other approaches. We describe the construction of a collection of plasmids for varied expression of proteins targeted to the mitochondrial matrix or intermembrane space, using previously defined targeting sequences and strength CMV promoters. The limited size of these compartments makes them particularly vulnerable to artifacts from over-expression. We found that different proteins display different kinetics of expression and import that should be considered when analyzing results from this approach. Finally, this collection of plasmids has been deposited in the Addgene plasmid repository to facilitate the ready access and use of these tools.

  16. Peripheral prism glasses: effects of moving and stationary backgrounds.

    PubMed

    Shen, Jieming; Peli, Eli; Bowers, Alex R

    2015-04-01

    Unilateral peripheral prisms for homonymous hemianopia (HH) expand the visual field through peripheral binocular visual confusion, a stimulus for binocular rivalry that could lead to reduced predominance and partial suppression of the prism image, thereby limiting device functionality. Using natural-scene images and motion videos, we evaluated whether detection was reduced in binocular compared with monocular viewing. Detection rates of nine participants with HH or quadranopia and normal binocularity wearing peripheral prisms were determined for static checkerboard perimetry targets briefly presented in the prism expansion area and the seeing hemifield. Perimetry was conducted under monocular and binocular viewing with targets presented over videos of real-world driving scenes and still frame images derived from those videos. With unilateral prisms, detection rates in the prism expansion area were significantly lower in binocular than in monocular (prism eye) viewing on the motion background (medians, 13 and 58%, respectively, p = 0.008) but not the still frame background (medians, 63 and 68%, p = 0.123). When the stimulus for binocular rivalry was reduced by fitting prisms bilaterally in one HH and one normally sighted subject with simulated HH, prism-area detection rates on the motion background were not significantly different (p > 0.6) in binocular and monocular viewing. Conflicting binocular motion appears to be a stimulus for reduced predominance of the prism image in binocular viewing when using unilateral peripheral prisms. However, the effect was only found for relatively small targets. Further testing is needed to determine the extent to which this phenomenon might affect the functionality of unilateral peripheral prisms in more real-world situations.

  17. Immunological Reactivity Using Monoclonal and Polyclonal Antibodies of Autoimmune Thyroid Target Sites with Dietary Proteins

    PubMed Central

    Herbert, Martha

    2017-01-01

    Many hypothyroid and autoimmune thyroid patients experience reactions with specific foods. Additionally, food interactions may play a role in a subset of individuals who have difficulty finding a suitable thyroid hormone dosage. Our study was designed to investigate the potential role of dietary protein immune reactivity with thyroid hormones and thyroid axis target sites. We identified immune reactivity between dietary proteins and target sites on the thyroid axis that includes thyroid hormones, thyroid receptors, enzymes, and transport proteins. We also measured immune reactivity of either target specific monoclonal or polyclonal antibodies for thyroid-stimulating hormone (TSH) receptor, 5′deiodinase, thyroid peroxidase, thyroglobulin, thyroxine-binding globulin, thyroxine, and triiodothyronine against 204 purified dietary proteins commonly consumed in cooked and raw forms. Dietary protein determinants included unmodified (raw) and modified (cooked and roasted) foods, herbs, spices, food gums, brewed beverages, and additives. There were no dietary protein immune reactions with TSH receptor, thyroid peroxidase, and thyroxine-binding globulin. However, specific antigen-antibody immune reactivity was identified with several purified food proteins with triiodothyronine, thyroxine, thyroglobulin, and 5′deiodinase. Laboratory analysis of immunological cross-reactivity between thyroid target sites and dietary proteins is the initial step necessary in determining whether dietary proteins may play a potential immunoreactive role in autoimmune thyroid disease. PMID:28894619

  18. Progranulin promotes peripheral nerve regeneration and reinnervation: role of notch signaling.

    PubMed

    Altmann, Christine; Vasic, Verica; Hardt, Stefanie; Heidler, Juliana; Häussler, Annett; Wittig, Ilka; Schmidt, Mirko H H; Tegeder, Irmgard

    2016-10-22

    Peripheral nerve injury is a frequent cause of lasting motor deficits and chronic pain. Although peripheral nerves are capable of regrowth they often fail to re-innervate target tissues. Using newly generated transgenic mice with inducible neuronal progranulin overexpression we show that progranulin accelerates axonal regrowth, restoration of neuromuscular synapses and recovery of sensory and motor functions after injury of the sciatic nerve. Oppositely, progranulin deficient mice have long-lasting deficits in motor function tests after nerve injury due to enhanced losses of motor neurons and stronger microglia activation in the ventral horn of the spinal cord. Deep proteome and gene ontology (GO) enrichment analysis revealed that the proteins upregulated in progranulin overexpressing mice were involved in 'regulation of transcription' and 'response to insulin' (GO terms). Transcription factor prediction pointed to activation of Notch signaling and indeed, co-immunoprecipitation studies revealed that progranulin bound to the extracellular domain of Notch receptors, and this was functionally associated with higher expression of Notch target genes in the dorsal root ganglia of transgenic mice with neuronal progranulin overexpression. Functionally, these transgenic mice recovered normal gait and running, which was not achieved by controls and was stronger impaired in progranulin deficient mice. We infer that progranulin activates Notch signaling pathways, enhancing thereby the regenerative capacity of partially injured neurons, which leads to improved motor function recovery.

  19. Protein kinases: mechanisms and downstream targets in inflammation mediated obesity and insulin resistance

    PubMed Central

    Nandipati, Kalyana C; Subramanian, Saravanan; Agrawal, Devendra K

    2016-01-01

    Obesity induced low-grade inflammation (metaflammation) impairs insulin receptor signaling (IRS). This has been implicated in the development of insulin resistance. Insulin signaling in the target tissues is mediated by stress kinases such as p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), inhibitor of NF-kB kinase complex beta (IKKβ), AMP activated protein kinase (AMPK), protein kinase C (PKC), Rho associated coiled-coil containing protein kinase (ROCK) and RNA-activated protein kinase (PKR), etc. Most of these kinases phosphorylate several key regulators in glucose homeostasis. The phosphorylation of serine residues in the insulin receptor (IR) and IRS-1 molecule results in diminished enzymatic activity in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. This has been one of the key mechanisms observed in the tissues that are implicated in insulin resistance especially in Type II Diabetes Mellitus (T2-DM). Identifying the specific protein kinases involved in obesity induced chronic inflammation may help in developing the targeted drug therapies to minimize the insulin resistance. This review is focused on the protein kinases involved in the inflammatory cascade and molecular mechanisms and their downstream targets with special reference to obesity induced T2-DM. PMID:27868170

  20. Mass spectrometry-based targeted quantitative proteomics: achieving sensitive and reproducible detection of proteins.

    PubMed

    Boja, Emily S; Rodriguez, Henry

    2012-04-01

    Traditional shotgun proteomics used to detect a mixture of hundreds to thousands of proteins through mass spectrometric analysis, has been the standard approach in research to profile protein content in a biological sample which could lead to the discovery of new (and all) protein candidates with diagnostic, prognostic, and therapeutic values. In practice, this approach requires significant resources and time, and does not necessarily represent the goal of the researcher who would rather study a subset of such discovered proteins (including their variations or posttranslational modifications) under different biological conditions. In this context, targeted proteomics is playing an increasingly important role in the accurate measurement of protein targets in biological samples in the hope of elucidating the molecular mechanism of cellular function via the understanding of intricate protein networks and pathways. One such (targeted) approach, selected reaction monitoring (or multiple reaction monitoring) mass spectrometry (MRM-MS), offers the capability of measuring multiple proteins with higher sensitivity and throughput than shotgun proteomics. Developing and validating MRM-MS-based assays, however, is an extensive and iterative process, requiring a coordinated and collaborative effort by the scientific community through the sharing of publicly accessible data and datasets, bioinformatic tools, standard operating procedures, and well characterized reagents. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766)

    PubMed Central

    2017-01-01

    Demonstrating intracellular protein target engagement is an essential step in the development and progression of new chemical probes and potential small molecule therapeutics. However, this can be particularly challenging for poorly studied and noncatalytic proteins, as robust proximal biomarkers are rarely known. To confirm that our recently discovered chemical probe 1 (CCT251236) binds the putative transcription factor regulator pirin in living cells, we developed a heterobifunctional protein degradation probe. Focusing on linker design and physicochemical properties, we generated a highly active probe 16 (CCT367766) in only three iterations, validating our efficient strategy for degradation probe design against nonvalidated protein targets. PMID:29240418

  2. Strategic Protein Target Analysis for Developing Drugs to Stop Dental Caries

    PubMed Central

    Horst, J.A.; Pieper, U.; Sali, A.; Zhan, L.; Chopra, G.; Samudrala, R.; Featherstone, J.D.B.

    2012-01-01

    Dental caries is the most common disease to cause irreversible damage in humans. Several therapeutic agents are available to treat or prevent dental caries, but none besides fluoride has significantly influenced the disease burden globally. Etiologic mechanisms of the mutans group streptococci and specific Lactobacillus species have been characterized to various degrees of detail, from identification of physiologic processes to specific proteins. Here, we analyze the entire Streptococcus mutans proteome for potential drug targets by investigating their uniqueness with respect to non-cariogenic dental plaque bacteria, quality of protein structure models, and the likelihood of finding a drug for the active site. Our results suggest specific targets for rational drug discovery, including 15 known virulence factors, 16 proteins for which crystallographic structures are available, and 84 previously uncharacterized proteins, with various levels of similarity to homologs in dental plaque bacteria. This analysis provides a map to streamline the process of clinical development of effective multispecies pharmacologic interventions for dental caries. PMID:22899687

  3. Protein sorting, targeting and trafficking in photoreceptor cells

    PubMed Central

    Pearring, Jillian N.; Salinas, Raquel Y.; Baker, Sheila A.; Arshavsky, Vadim Y.

    2013-01-01

    Vision is the most fundamental of our senses initiated when photons are absorbed by the rod and cone photoreceptor neurons of the retina. At the distal end of each photoreceptor resides a light-sensing organelle, called the outer segment, which is a modified primary cilium highly enriched with proteins involved in visual signal transduction. At the proximal end, each photoreceptor has a synaptic terminal, which connects this cell to the downstream neurons for further processing of the visual information. Understanding the mechanisms involved in creating and maintaining functional compartmentalization of photoreceptor cells remains among the most fascinating topics in ocular cell biology. This review will discuss how photoreceptor compartmentalization is supported by protein sorting, targeting and trafficking, with an emphasis on the best-studied cases of outer segment-resident proteins. PMID:23562855

  4. The therapeutic potential of targeting the peripheral endocannabinoid/CB1 receptor system.

    PubMed

    Tam, Joseph; Hinden, Liad; Drori, Adi; Udi, Shiran; Azar, Shahar; Baraghithy, Saja

    2018-03-01

    Endocannabinoids (eCBs) are internal lipid mediators recognized by the cannabinoid-1 and -2 receptors (CB 1 R and CB 2 R, respectively), which also mediate the different physiological effects of marijuana. The endocannabinoid system, consisting of eCBs, their receptors, and the enzymes involved in their biosynthesis and degradation, is present in a vast number of peripheral organs. In this review we describe the role of the eCB/CB 1 R system in modulating the metabolism in several peripheral organs. We assess how eCBs, via activating the CB 1 R, contribute to obesity and regulate food intake. In addition, we describe their roles in modulating liver and kidney functions, as well as bone remodeling and mass. Special importance is given to emphasizing the efficacy of the recently developed peripherally restricted CB 1 R antagonists, which were pre-clinically tested in the management of energy homeostasis, and in ameliorating both obesity- and diabetes-induced metabolic complications. Copyright © 2018 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

  5. Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

    PubMed Central

    Tan, Siyuan; Guschin, Dmitry; Davalos, Albert; Lee, Ya-Li; Snowden, Andrew W.; Jouvenot, Yann; Zhang, H. Steven; Howes, Katherine; McNamara, Andrew R.; Lai, Albert; Ullman, Chris; Reynolds, Lindsey; Moore, Michael; Isalan, Mark; Berg, Lutz-Peter; Campos, Bradley; Qi, Hong; Spratt, S. Kaye; Case, Casey C.; Pabo, Carl O.; Campisi, Judith; Gregory, Philip D.

    2003-01-01

    Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics. PMID:14514889

  6. Identification of human microRNA targets from isolated argonaute protein complexes.

    PubMed

    Beitzinger, Michaela; Peters, Lasse; Zhu, Jia Yun; Kremmer, Elisabeth; Meister, Gunter

    2007-06-01

    MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that regulate gene expression on the level of translation and/or mRNA stability. Mammalian miRNAs associate with members of the Argonaute (Ago) protein family and bind to partially complementary sequences in the 3' untranslated region (UTR) of specific target mRNAs. Computer algorithms based on factors such as free binding energy or sequence conservation have been used to predict miRNA target mRNAs. Based on such predictions, up to one third of all mammalian mRNAs seem to be under miRNA regulation. However, due to the low degree of complementarity between the miRNA and its target, such computer programs are often imprecise and therefore not very reliable. Here we report the first biochemical identification approach of miRNA targets from human cells. Using highly specific monoclonal antibodies against members of the Ago protein family, we co-immunoprecipitate Ago-bound mRNAs and identify them by cloning. Interestingly, most of the identified targets are also predicted by different computer programs. Moreover, we randomly analyzed six different target candidates and were able to experimentally validate five as miRNA targets. Our data clearly indicate that miRNA targets can be experimentally identified from Ago complexes and therefore provide a new tool to directly analyze miRNA function.

  7. Combinatorial Approaches for the Identification of Brain Drug Delivery Targets

    PubMed Central

    Stutz, Charles C.; Zhang, Xiaobin; Shusta, Eric V.

    2018-01-01

    The blood-brain barrier (BBB) represents a large obstacle for the treatment of central nervous system diseases. Targeting endogenous nutrient transporters that transcytose the BBB is one promising approach to selectively and noninvasively deliver a drug payload to the brain. The main limitations of the currently employed transcytosing receptors are their ubiquitous expression in the peripheral vasculature and the inherent low levels of transcytosis mediated by such systems. In this review, approaches designed to increase the repertoire of transcytosing receptors which can be targeted for the purpose of drug delivery are discussed. In particular, combinatorial protein libraries can be screened on BBB cells in vitro or in vivo to isolate targeting peptides or antibodies that can trigger transcytosis. Once these targeting reagents are discovered, the cognate BBB transcytosis system can be identified using techniques such as expression cloning or immunoprecipitation coupled with mass spectrometry. Continued technological advances in BBB genomics and proteomics, membrane protein manipulation, and in vitro BBB technology promise to further advance the capability to identify and optimize peptides and antibodies capable of mediating drug transport across the BBB. PMID:23789958

  8. Effect of Ca2+ on the promiscuous target-protein binding of calmodulin.

    PubMed

    Westerlund, Annie M; Delemotte, Lucie

    2018-04-01

    Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.

  9. Effect of Ca2+ on the promiscuous target-protein binding of calmodulin

    PubMed Central

    Westerlund, Annie M.

    2018-01-01

    Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity. PMID:29614072

  10. AlphaSpace: Fragment-Centric Topographical Mapping To Target Protein–Protein Interaction Interfaces

    PubMed Central

    2016-01-01

    Inhibition of protein–protein interactions (PPIs) is emerging as a promising therapeutic strategy despite the difficulty in targeting such interfaces with drug-like small molecules. PPIs generally feature large and flat binding surfaces as compared to typical drug targets. These features pose a challenge for structural characterization of the surface using geometry-based pocket-detection methods. An attractive mapping strategy—that builds on the principles of fragment-based drug discovery (FBDD)—is to detect the fragment-centric modularity at the protein surface and then characterize the large PPI interface as a set of localized, fragment-targetable interaction regions. Here, we introduce AlphaSpace, a computational analysis tool designed for fragment-centric topographical mapping (FCTM) of PPI interfaces. Our approach uses the alpha sphere construct, a geometric feature of a protein’s Voronoi diagram, to map out concave interaction space at the protein surface. We introduce two new features—alpha-atom and alpha-space—and the concept of the alpha-atom/alpha-space pair to rank pockets for fragment-targetability and to facilitate the evaluation of pocket/fragment complementarity. The resulting high-resolution interfacial map of targetable pocket space can be used to guide the rational design and optimization of small molecule or biomimetic PPI inhibitors. PMID:26225450

  11. Targeting protein homeostasis in sporadic inclusion body myositis.

    PubMed

    Ahmed, Mhoriam; Machado, Pedro M; Miller, Adrian; Spicer, Charlotte; Herbelin, Laura; He, Jianghua; Noel, Janelle; Wang, Yunxia; McVey, April L; Pasnoor, Mamatha; Gallagher, Philip; Statland, Jeffrey; Lu, Ching-Hua; Kalmar, Bernadett; Brady, Stefen; Sethi, Huma; Samandouras, George; Parton, Matt; Holton, Janice L; Weston, Anne; Collinson, Lucy; Taylor, J Paul; Schiavo, Giampietro; Hanna, Michael G; Barohn, Richard J; Dimachkie, Mazen M; Greensmith, Linda

    2016-03-23

    Sporadic inclusion body myositis (sIBM) is the commonest severe myopathy in patients more than 50 years of age. Previous therapeutic trials have targeted the inflammatory features of sIBM but all have failed. Because protein dyshomeostasis may also play a role in sIBM, we tested the effects of targeting this feature of the disease. Using rat myoblast cultures, we found that up-regulation of the heat shock response with arimoclomol reduced key pathological markers of sIBM in vitro. Furthermore, in mutant valosin-containing protein (VCP) mice, which develop an inclusion body myopathy, treatment with arimoclomol ameliorated disease pathology and improved muscle function. We therefore evaluated arimoclomol in an investigator-led, randomized, double-blind, placebo-controlled, proof-of-concept trial in sIBM patients and showed that arimoclomol was safe and well tolerated. Although arimoclomol improved some IBM-like pathology in the mutant VCP mouse, we did not see statistically significant evidence of efficacy in the proof-of-concept patient trial. Copyright © 2016, American Association for the Advancement of Science.

  12. Targeting Protein Homeostasis in Sporadic Inclusion Body Myositis

    PubMed Central

    Ahmed, Mhoriam; Machado, Pedro M.; Miller, Adrian; Spicer, Charlotte; Herbelin, Laura; He, Jianghua; Noel, Janelle; Wang, Yunxia; McVey, April L.; Pasnoor, Mamatha; Gallagher, Philip; Statland, Jeffrey; Lu, Ching-Hua; Kalmar, Bernadett; Brady, Stefen; Sethi, Huma; Samandouras, George; Parton, Matt; Holton, Janice L.; Weston, Anne; Collinson, Lucy; Taylor, J. Paul; Schiavo, Giampietro; Hanna, Michael G.; Barohn, Richard J.; Dimachkie, Mazen M.; Greensmith, Linda

    2016-01-01

    Sporadic inclusion body myositis (sIBM) is the commonest severe myopathy in patients over age 50. Previous therapeutic trials have targeted the inflammatory features of sIBM, but all have failed. Since protein dyshomeostasis may also play a role in sIBM, we tested the effects of targeting this feature of the disease. Using rat myoblast cultures, we found that up-regulation of the heat shock response with Arimoclomol reduced key pathological markers of sIBM in vitro. Furthermore, in mutant valosin-containing protein VCP mice, which develop an inclusion body myopathy (IBM), treatment with Arimoclomol ameliorated disease pathology and improved muscle function. We therefore evaluated the safety and tolerability of Arimoclomol in an investigator-lead, randomised, double-blind, placebo-controlled, proof-of-concept patient trial and gathered exploratory efficacy data which showed that Arimoclomol was safe and well tolerated. Although Arimoclomol improved some IBM-like pathology in vitro and in vivo in the mutant VCP mouse, we did not see statistically significant evidence of efficacy in this proof of concept patient trial. PMID:27009270

  13. Changes in NGF and NT-3 protein species in the superior cervical ganglion following axotomy of postganglionic axons.

    PubMed

    Walker, Ryan G; Foster, Andrew; Randolph, Chris L; Isaacson, Lori G

    2009-02-19

    Mature sympathetic neurons in the superior cervical ganglion (SCG) are regulated by target-derived neurotrophins such as nerve growth factor (NGF) and neurotrophin-3 (NT-3). High molecular weight NGF species and mature NT-3 are the predominant NGF and NT-3 protein isoforms in the SCG, yet it is unknown whether the presence of these species is dependent on intact connection with the target tissues. In an attempt to determine the role of peripheral targets in regulating the neurotrophin species found in the SCG, we investigated the NGF and NT-3 protein species present in the SCG following axotomy (transection) or injury of the post-ganglionic axons. Following a 7 day axotomy, the 22-24 kDa NGF species and the mature 14 kDa NT-3 species in the SCG were significantly reduced by 99% and 66% respectively, suggesting that intact connection with the target is necessary for the expression of these protein species. As expected, tyrosine hydroxylase (TH) protein in the SCG was significantly reduced by 80% at 7 days following axotomy. In order to distinguish between the effects of injury and loss of target connectivity, the SCG was examined following compression injury to the post-ganglionic nerves. Following injury, no reduction in the 22-24 kDa NGF or 14 kDa mature NT-3 species was observed in the SCG. TH protein was slightly, yet significantly, decreased in the SCG following injury. The findings of this study suggest that the presence of the 22-24 kDa NGF and mature 14 kDa NT-3 species in the SCG is dependent on connection with peripheral targets and may influence, at least in part, TH protein expression in adult sympathetic neurons.

  14. Emerging therapeutic targets in myeloproliferative neoplasms and peripheral T-cell leukemia and lymphomas

    PubMed Central

    Orlova, Anna; Wingelhofer, Bettina; Neubauer, Heidi A.; Maurer, Barbara; Berger-Becvar, Angelika; Keserű, György Miklós; Gunning, Patrick T.; Valent, Peter; Moriggl, Richard

    2018-01-01

    ABSTRACT Introduction: Hematopoietic neoplasms are often driven by gain-of-function mutations of the JAK-STAT pathway together with mutations in chromatin remodeling and DNA damage control pathways. The interconnection between the JAK-STAT pathway, epigenetic regulation or DNA damage control is still poorly understood in cancer cell biology. Areas covered: Here, we focus on a broader description of mutational insights into myeloproliferative neoplasms and peripheral T-cell leukemia and lymphomas, since sequencing efforts have identified similar combinations of driver mutations in these diseases covering different lineages. We summarize how these pathways might be interconnected in normal or cancer cells, which have lost differentiation capacity and drive oncogene transcription. Expert opinion: Due to similarities in driver mutations including epigenetic enzymes, JAK-STAT pathway activation and mutated checkpoint control through TP53, we hypothesize that similar therapeutic approaches could be of benefit in these diseases. We give an overview of how driver mutations in these malignancies contribute to hematopoietic cancer initiation or progression, and how these pathways can be targeted with currently available tools. PMID:29148847

  15. Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins

    DOE PAGES

    Velásquez, André C.; Nomura, Kinya; Cooper, Max D.; ...

    2017-04-19

    The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Here, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs.more » HopM1-specific VLRBs (VLRM1) were expressed in planta in the cytosol, the trans-Golgi network, and the apoplast. Expression of VLRM1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLRM1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLRM1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Later, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta, with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.« less

  16. Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Velásquez, André C.; Nomura, Kinya; Cooper, Max D.

    The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Here, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs.more » HopM1-specific VLRBs (VLRM1) were expressed in planta in the cytosol, the trans-Golgi network, and the apoplast. Expression of VLRM1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLRM1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLRM1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Later, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta, with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.« less

  17. Protein-targeted corona phase molecular recognition

    PubMed Central

    Bisker, Gili; Dong, Juyao; Park, Hoyoung D.; Iverson, Nicole M.; Ahn, Jiyoung; Nelson, Justin T.; Landry, Markita P.; Kruss, Sebastian; Strano, Michael S.

    2016-01-01

    Corona phase molecular recognition (CoPhMoRe) uses a heteropolymer adsorbed onto and templated by a nanoparticle surface to recognize a specific target analyte. This method has not yet been extended to macromolecular analytes, including proteins. Herein we develop a variant of a CoPhMoRe screening procedure of single-walled carbon nanotubes (SWCNT) and use it against a panel of human blood proteins, revealing a specific corona phase that recognizes fibrinogen with high selectivity. In response to fibrinogen binding, SWCNT fluorescence decreases by >80% at saturation. Sequential binding of the three fibrinogen nodules is suggested by selective fluorescence quenching by isolated sub-domains and validated by the quenching kinetics. The fibrinogen recognition also occurs in serum environment, at the clinically relevant fibrinogen concentrations in the human blood. These results open new avenues for synthetic, non-biological antibody analogues that recognize biological macromolecules, and hold great promise for medical and clinical applications. PMID:26742890

  18. Identification of polycystic ovary syndrome potential drug targets based on pathobiological similarity in the protein-protein interaction network

    PubMed Central

    Li, Wan; Wei, Wenqing; Li, Yiran; Xie, Ruiqiang; Guo, Shanshan; Wang, Yahui; Jiang, Jing; Chen, Binbin; Lv, Junjie; Zhang, Nana; Chen, Lina; He, Weiming

    2016-01-01

    Polycystic ovary syndrome (PCOS) is one of the most common endocrinological disorders in reproductive aged women. PCOS and Type 2 Diabetes (T2D) are closely linked in multiple levels and possess high pathobiological similarity. Here, we put forward a new computational approach based on the pathobiological similarity to identify PCOS potential drug target modules (PPDT-Modules) and PCOS potential drug targets in the protein-protein interaction network (PPIN). From the systems level and biological background, 1 PPDT-Module and 22 PCOS potential drug targets were identified, 21 of which were verified by literatures to be associated with the pathogenesis of PCOS. 42 drugs targeting to 13 PCOS potential drug targets were investigated experimentally or clinically for PCOS. Evaluated by independent datasets, the whole PPDT-Module and 22 PCOS potential drug targets could not only reveal the drug response, but also distinguish the statuses between normal and disease. Our identified PPDT-Module and PCOS potential drug targets would shed light on the treatment of PCOS. And our approach would provide valuable insights to research on the pathogenesis and drug response of other diseases. PMID:27191267

  19. Autophagy as an Emerging Common Pathomechanism in Inherited Peripheral Neuropathies

    PubMed Central

    Haidar, Mansour; Timmerman, Vincent

    2017-01-01

    The inherited peripheral neuropathies (IPNs) comprise a growing list of genetically heterogeneous diseases. With mutations in more than 80 genes being reported to cause IPNs, a wide spectrum of functional consequences is expected to follow this genotypic diversity. Hence, the search for a common pathomechanism among the different phenotypes has become the holy grail of functional research into IPNs. During the last decade, studies on several affected genes have shown a direct and/or indirect correlation with autophagy. Autophagy, a cellular homeostatic process, is required for the removal of cell aggregates, long-lived proteins and dead organelles from the cell in double-membraned vesicles destined for the lysosomes. As an evolutionarily highly conserved process, autophagy is essential for the survival and proper functioning of the cell. Recently, neuronal cells have been shown to be particularly vulnerable to disruption of the autophagic pathway. Furthermore, autophagy has been shown to be affected in various common neurodegenerative diseases of both the central and the peripheral nervous system including Alzheimer’s, Parkinson’s, and Huntington’s diseases. In this review we provide an overview of the genes involved in hereditary neuropathies which are linked to autophagy and we propose the disruption of the autophagic flux as an emerging common pathomechanism. We also shed light on the different steps of the autophagy pathway linked to these genes. Finally, we review the concept of autophagy being a therapeutic target in IPNs, and the possibilities and challenges of this pathway-specific targeting. PMID:28553203

  20. Brain Responses to High-Protein Diets12

    PubMed Central

    Journel, Marion; Chaumontet, Catherine; Darcel, Nicolas; Fromentin, Gilles; Tomé, Daniel

    2012-01-01

    Proteins are suspected to have a greater satiating effect than the other 2 macronutrients. After protein consumption, peptide hormones released from the gastrointestinal tract (mainly anorexigenic gut peptides such as cholecystokinin, glucagon peptide 1, and peptide YY) communicate information about the energy status to the brain. These hormones and vagal afferents control food intake by acting on brain regions involved in energy homeostasis such as the brainstem and the hypothalamus. In fact, a high-protein diet leads to greater activation than a normal-protein diet in the nucleus tractus solitarius and in the arcuate nucleus. More specifically, neural mechanisms triggered particularly by leucine consumption involve 2 cellular energy sensors: the mammalian target of rapamycin and AMP-activated protein kinase. In addition, reward and motivation aspects of eating behavior, controlled mainly by neurons present in limbic regions, play an important role in the reduced hedonic response of a high-protein diet. This review examines how metabolic signals emanating from the gastrointestinal tract after protein ingestion target the brain to control feeding, energy expenditure, and hormones. Understanding the functional roles of brain areas involved in the satiating effect of proteins and their interactions will demonstrate how homeostasis and reward are integrated with the signals from peripheral organs after protein consumption. PMID:22585905

  1. Target-specific NMR detection of protein-ligand interactions with antibody-relayed 15N-group selective STD.

    PubMed

    Hetényi, Anasztázia; Hegedűs, Zsófia; Fajka-Boja, Roberta; Monostori, Éva; Kövér, Katalin E; Martinek, Tamás A

    2016-12-01

    Fragment-based drug design has been successfully applied to challenging targets where the detection of the weak protein-ligand interactions is a key element. 1 H saturation transfer difference (STD) NMR spectroscopy is a powerful technique for this work but it requires pure homogeneous proteins as targets. Monoclonal antibody (mAb)-relayed 15 N-GS STD spectroscopy has been developed to resolve the problem of protein mixtures and impure proteins. A 15 N-labelled target-specific mAb is selectively irradiated and the saturation is relayed through the target to the ligand. Tests on the anti-Gal-1 mAb/Gal-1/lactose system showed that the approach is experimentally feasible in a reasonable time frame. This method allows detection and identification of binding molecules directly from a protein mixture in a multicomponent system.

  2. Targeting the Allosteric Site of Oncoprotein BCR-ABL as an Alternative Strategy for Effective Target Protein Degradation.

    PubMed

    Shimokawa, Kenichiro; Shibata, Norihito; Sameshima, Tomoya; Miyamoto, Naoki; Ujikawa, Osamu; Nara, Hiroshi; Ohoka, Nobumichi; Hattori, Takayuki; Cho, Nobuo; Naito, Mikihiko

    2017-10-12

    Protein degradation technology based on hybrid small molecules is an emerging drug modality that has significant potential in drug discovery and as a unique method of post-translational protein knockdown in the field of chemical biology. Here, we report the first example of a novel and potent protein degradation inducer that binds to an allosteric site of the oncogenic BCR-ABL protein. BCR-ABL allosteric ligands were incorporated into the SNIPER (Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers) platform, and a series of in vitro biological assays of binding affinity, target protein modulation, signal transduction, and growth inhibition were carried out. One of the designed compounds, 6 (SNIPER(ABL)-062), showed desirable binding affinities against ABL1, cIAP1/2, and XIAP and consequently caused potent BCR-ABL degradation.

  3. Disease associated prion protein may deposit in the peripheral nervous system in human transmissible spongiform encephalopathies.

    PubMed

    Hainfellner, J A; Budka, H

    1999-11-01

    There is increasing evidence indicating involvement of the peripheral nervous system (PNS) in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Immunocytochemically detectable deposits of TSE-specific abnormal prion protein (PrP(sc)) are considered as a surrogate marker for infectivity. We used anti-PrP immunocytochemistry to trace PrP(sc) deposition in spinal and enteric ganglia, and peripheral nerve in Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), and fatal familial insomnia. Discrete PrP(sc) deposits were detectable only in a few posterior root nerve fibers in an adaxonal location in one of nine CJD and the one GSS patients examined. Follicular dendritic cells of the gut and enteric nervous system were not labeled. Thus, PrP(sc) may spread to the PNS in different forms of human prion disease. In contrast to our observations in experimental scrapie (Groschup et al., Acta Neuropathol, this issue), the deposits were scant. Possible explanations for this discrepancy comprise strain difference, or centripetal (experimental scrapie) versus centrifugal (sporadic and genetic human prion diseases) spread of PrP(sc), resulting in different patterns and amounts of PrP(sc) accumulation in the PNS.

  4. Targeted proteins for diagnostic imaging: does chemistry make a difference?

    PubMed

    Fritzberg, A R; Beaumier, P L

    1992-03-01

    The Oyen et al. study is valuable in that it systematically evaluates several of the factors involved in radiolabeled protein uptake and retention in infectious foci. The role of particular proteins and their receptor specific interactions seems to be inconsequential in agreement with the findings of other. However, the role of the radiolabel was shown to be important and significant differences were delineated from comparisons of the radionuclides and their associated chemistries. The conclusion implicating radionuclide chemistry and associated linkages underscores the need to optimize the attachment and labeling chemical modifications of protein carriers. Evaluation criteria should include serum stability, determination and assessment of the effect of molar substitution ratio, and potential for improving blood clearance without reducing the target-to-non-target ratio. Important areas for future study include characterization of radioactive metabolites and the design and synthesis of new ligands which direct the disposition of metabolites reducing retention in normal organs or accelerating renal excretion. Additionally, intracellular processing of radiolabel, compartmental distribution and strategies for augmenting internalization and retention within the target cell merit detailed exploration. For each radionuclide of interest, 111In, radioiodines, 99mTc and others, improved chemical moieties exist for controlling radiolabel fate. When carrying out mechanistic and evaluative studies, clear-cut conclusions will only be reached when defined and controlled chemistry is used. Having established a "gold standard," simplifications in radiolabeling and other chemical refinements can then be pursued with a quantitative understanding of the trade-offs in targeting agent performance versus other considerations such as cost reduction, simplicity, and convenience.

  5. Identification of Histone Deacetylase (HDAC) as a drug target against MRSA via interolog method of protein-protein interaction prediction.

    PubMed

    Uddin, Reaz; Tariq, Syeda Sumayya; Azam, Syed Sikander; Wadood, Abdul; Moin, Syed Tarique

    2017-08-30

    Patently, Protein-Protein Interactions (PPIs) lie at the core of significant biological functions and make the foundation of host-pathogen relationships. Hence, the current study is aimed to use computational biology techniques to predict host-pathogen Protein-Protein Interactions (HP-PPIs) between MRSA and Humans as potential drug targets ultimately proposing new possible inhibitors against them. As a matter of fact this study is based on the Interolog method which implies that homologous proteins retain their ability to interact. A distant homolog approach based on Interolog method was employed to speculate MRSA protein homologs in Humans using PSI-BLAST. In addition the protein interaction partners of these homologs as listed in Database of Interacting Proteins (DIP) were predicted to interact with MRSA as well. Moreover, a direct approach using BLAST was also applied so as to attain further confidence in the strategy. Consequently, the common HP-PPIs predicted by both approaches are suggested as potential drug targets (22%) whereas, the unique HP-PPIs estimated only through distant homolog approach are presented as novel drug targets (12%). Furthermore, the most repeated entry in our results was found to be MRSA Histone Deacetylase (HDAC) which was then modeled using SWISS-MODEL. Eventually, small molecules from ZINC, selected randomly, were docked against HDAC using Auto Dock and are suggested as potential binders (inhibitors) based on their energetic profiles. Thus the current study provides basis for further in-depth analysis of such data which not only include MRSA but other deadly pathogens as well. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Chemical biology based on target-selective degradation of proteins and carbohydrates using light-activatable organic molecules.

    PubMed

    Toshima, Kazunobu

    2013-05-01

    Proteins and carbohydrates play crucial roles in a wide range of biological processes, including serious diseases. The development of novel and innovative methods for selective control of specific proteins and carbohydrates functions has attracted much attention in the field of chemical biology. In this account article, the development of novel chemical tools, which can degrade target proteins and carbohydrates by irradiation with a specific wavelength of light under mild conditions without any additives, is introduced. This novel class of photochemical agents promise bright prospects for finding not only molecular-targeted bioprobes for understanding of the structure-activity relationships of proteins and carbohydrates but also novel therapeutic drugs targeting proteins and carbohydrates.

  7. Proteomics Analysis of Nucleolar SUMO-1 Target Proteins upon Proteasome Inhibition*

    PubMed Central

    Matafora, Vittoria; D'Amato, Alfonsina; Mori, Silvia; Blasi, Francesco; Bachi, Angela

    2009-01-01

    Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. Recently it has been reported that there is a relationship between sumoylation and ubiquitination. Here we propose that the nucleolus is the key organelle in which SUMO-1 conjugates accumulate in response to proteasome inhibition. We demonstrated that, upon proteasome inhibition, the SUMO-1 nuclear dot localization is redirected to nucleolar structures. To better understand this process we investigated, by quantitative proteomics, the effect of proteasome activity on endogenous nucleolar SUMO-1 targets. 193 potential SUMO-1 substrates were identified, and interestingly in several purified SUMO-1 conjugates ubiquitin chains were found to be present, confirming the coordination of these two modifications. 23 SUMO-1 targets were confirmed by an in vitro sumoylation reaction performed on nuclear substrates. They belong to protein families such as small nuclear ribonucleoproteins, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, histones, RNA-binding proteins, and transcription factor regulators. Among these, histone H1, histone H3, and p160 Myb-binding protein 1A were further characterized as novel SUMO-1 substrates. The analysis of the nature of the SUMO-1 targets identified in this study strongly indicates that sumoylation, acting in coordination with the ubiquitin-proteasome system, regulates the maintenance of nucleolar integrity. PMID:19596686

  8. Conformational stability as a design target to control protein aggregation.

    PubMed

    Costanzo, Joseph A; O'Brien, Christopher J; Tiller, Kathryn; Tamargo, Erin; Robinson, Anne Skaja; Roberts, Christopher J; Fernandez, Erik J

    2014-05-01

    Non-native protein aggregation is a prevalent problem occurring in many biotechnological manufacturing processes and can compromise the biological activity of the target molecule or induce an undesired immune response. Additionally, some non-native aggregation mechanisms lead to amyloid fibril formation, which can be associated with debilitating diseases. For natively folded proteins, partial or complete unfolding is often required to populate aggregation-prone conformational states, and therefore one proposed strategy to mitigate aggregation is to increase the free energy for unfolding (ΔGunf) prior to aggregation. A computational design approach was tested using human γD crystallin (γD-crys) as a model multi-domain protein. Two mutational strategies were tested for their ability to reduce/increase aggregation rates by increasing/decreasing ΔGunf: stabilizing the less stable domain and stabilizing the domain-domain interface. The computational protein design algorithm, RosettaDesign, was implemented to identify point variants. The results showed that although the predicted free energies were only weakly correlated with the experimental ΔGunf values, increased/decreased aggregation rates for γD-crys correlated reasonably well with decreases/increases in experimental ΔGunf, illustrating improved conformational stability as a possible design target to mitigate aggregation. However, the results also illustrate that conformational stability is not the sole design factor controlling aggregation rates of natively folded proteins.

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

    PubMed

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

    2016-09-07

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

  10. Zinc finger protein designed to target 2-long terminal repeat junctions interferes with human immunodeficiency virus integration.

    PubMed

    Sakkhachornphop, Supachai; Barbas, Carlos F; Keawvichit, Rassamee; Wongworapat, Kanlaya; Tayapiwatana, Chatchai

    2012-09-01

    Integration of the human immunodeficiency virus type 1 (HIV-1) genome into the host chromosome is a vital step in the HIV life cycle. The highly conserved cytosine-adenine (CA) dinucleotide sequence immediately upstream of the cleavage site is crucial for integrase (IN) activity. As this viral enzyme has an important role early in the HIV-1 replication cycle, interference with the IN substrate has become an attractive strategy for therapeutic intervention. We demonstrated that a designed zinc finger protein (ZFP) fused to green fluorescent protein (GFP) targets the 2-long terminal repeat (2-LTR) circle junctions of HIV-1 DNA with nanomolar affinity. We report now that 2LTRZFP-GFP stably transduced into 293T cells interfered with the expression of vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral red fluorescent protein (RFP), as shown by the suppression of RFP expression. We also used a third-generation lentiviral vector and pCEP4 expression vector to deliver the 2LTRZFP-GFP transgene into human T-lymphocytic cells, and a stable cell line for long-term expression studies was selected for HIV-1 challenge. HIV-1 integration and replication were inhibited as measured by Alu-gag real-time PCR and p24 antigen assay. In addition, the molecular activity of 2LTRZFP-GFP was evaluated in peripheral blood mononuclear cells. The results were confirmed by Alu-gag real-time PCR for integration interference. We suggest that the expression of 2LTRZFP-GFP limited viral integration on intracellular immunization, and that it has potential for use in HIV gene therapy in the future.

  11. Peripheral Prism Glasses: Effects of Moving and Stationary Backgrounds

    PubMed Central

    Shen, Jieming; Peli, Eli; Bowers, Alex R.

    2015-01-01

    Purpose Unilateral peripheral prisms for homonymous hemianopia (HH) expand the visual field through peripheral binocular visual confusion, a stimulus for binocular rivalry that could lead to reduced predominance (partial local suppression) of the prism image and limit device functionality. Using natural-scene images and motion videos, we evaluated whether detection was reduced in binocular compared to monocular viewing. Methods Detection rates of nine participants with HH or quadranopia and normal binocularity wearing peripheral prisms were determined for static checkerboard perimetry targets briefly presented in the prism expansion area and the seeing hemifield. Perimetry was conducted under monocular and binocular viewing with targets presented over videos of real-world driving scenes and still frame images derived from those videos. Results With unilateral prisms, detection rates in the prism expansion area were significantly lower in binocular than monocular (prism eye) viewing on the motion background (medians 13% and 58%, respectively, p = 0.008), but not the still frame background (63% and 68%, p = 0.123). When the stimulus for binocular rivalry was reduced by fitting prisms bilaterally in 1 HH and 1 normally-sighted subject with simulated HH, prism-area detection rates on the motion background were not significantly different (p > 0.6) in binocular and monocular viewing. Conclusions Conflicting binocular motion appears to be a stimulus for reduced predominance of the prism image in binocular viewing when using unilateral peripheral prisms. However, the effect was only found for relatively small targets. Further testing is needed to determine the extent to which this phenomenon might affect the functionality of unilateral peripheral prisms in more real-world situations. PMID:25785533

  12. Thioredoxin and Thioredoxin Target Proteins: From Molecular Mechanisms to Functional Significance

    PubMed Central

    Lee, Samuel; Kim, Soo Min

    2013-01-01

    Abstract The thioredoxin (Trx) system is one of the central antioxidant systems in mammalian cells, maintaining a reducing environment by catalyzing electron flux from nicotinamide adenine dinucleotide phosphate through Trx reductase to Trx, which reduces its target proteins using highly conserved thiol groups. While the importance of protecting cells from the detrimental effects of reactive oxygen species is clear, decades of research in this field revealed that there is a network of redox-sensitive proteins forming redox-dependent signaling pathways that are crucial for fundamental cellular processes, including metabolism, proliferation, differentiation, migration, and apoptosis. Trx participates in signaling pathways interacting with different proteins to control their dynamic regulation of structure and function. In this review, we focus on Trx target proteins that are involved in redox-dependent signaling pathways. Specifically, Trx-dependent reductive enzymes that participate in classical redox reactions and redox-sensitive signaling molecules are discussed in greater detail. The latter are extensively discussed, as ongoing research unveils more and more details about the complex signaling networks of Trx-sensitive signaling molecules such as apoptosis signal-regulating kinase 1, Trx interacting protein, and phosphatase and tensin homolog, thus highlighting the potential direct and indirect impact of their redox-dependent interaction with Trx. Overall, the findings that are described here illustrate the importance and complexity of Trx-dependent, redox-sensitive signaling in the cell. Our increasing understanding of the components and mechanisms of these signaling pathways could lead to the identification of new potential targets for the treatment of diseases, including cancer and diabetes. Antioxid. Redox Signal. 18, 1165–1207. PMID:22607099

  13. Electrophysiological correlates of target eccentricity in texture segmentation.

    PubMed

    Schaffer, Susann; Schubö, Anna; Meinecke, Cristina

    2011-06-01

    Event-related potentials and behavioural performance as a function of target eccentricity were measured while subjects performed a texture segmentation task. Fit-of-structures, i.e. easiness of target detection was varied: in Experiment 1, a texture with peripheral fit (easier detection of peripheral presented targets) and in Experiment 2, a texture with foveal fit (easier detection of foveal presented targets) was used. In the two experiments, the N2p was sensitive to target eccentricity showing larger amplitudes for foveal targets compared to peripheral targets, and at the foveal position, a reversal of the N2p differential amplitude effect was found. The anterior P2 seemed sensitive to the easiness of target detection. In both experiments the N2pc varied as a function of eccentricity. However, the P3 was neither sensitive to target eccentricity nor to the fit-of-structures. Results show the existence of a P2/N2 complex (Potts and Tucker, 2001) indicating executive functions located in the anterior cortex and perceptual processes located in the posterior cortex. Furthermore, the N2p might indicate the existence of a foveal vs. peripheral subsystem in visual processing. 2011 Elsevier B.V. All rights reserved.

  14. Targeting human Mas-related G protein-coupled receptor X1 to inhibit persistent pain

    PubMed Central

    Li, Zhe; Tseng, Pang-Yen; Tiwari, Vinod; Xu, Qian; He, Shao-Qiu; Wang, Yan; Zheng, Qin; Han, Liang; Wu, Zhiping; Blobaum, Anna L.; Cui, Yiyuan; Tiwari, Vineeta; Sun, Shuohao; Cheng, Yingying; Huang-Lionnet, Julie H. Y.; Geng, Yixun; Xiao, Bo; Peng, Junmin; Hopkins, Corey; Raja, Srinivasa N.; Guan, Yun; Dong, Xinzhong

    2017-01-01

    Human Mas-related G protein-coupled receptor X1 (MRGPRX1) is a promising target for pain inhibition, mainly because of its restricted expression in nociceptors within the peripheral nervous system. However, constrained by species differences across Mrgprs, drug candidates that activate MRGPRX1 do not activate rodent receptors, leaving no responsive animal model to test the effect on pain in vivo. Here, we generated a transgenic mouse line in which we replaced mouse Mrgprs with human MrgprX1. This humanized mouse allowed us to characterize an agonist [bovine adrenal medulla 8–22 (BAM8–22)] and a positive allosteric modulator (PAM), ML382, of MRGPRX1. Cellular studies suggested that ML382 enhances the ability of BAM8–22 to inhibit high-voltage-activated Ca2+ channels and attenuate spinal nociceptive transmission. Importantly, both BAM8–22 and ML382 effectively attenuated evoked, persistent, and spontaneous pain without causing obvious side effects. Notably, ML382 by itself attenuated both evoked pain hypersensitivity and spontaneous pain in MrgprX1 mice after nerve injury without acquiring coadministration of an exogenous agonist. Our findings suggest that humanized MrgprX1 mice provide a promising preclinical model and that activating MRGPRX1 is an effective way to treat persistent pain. PMID:28223516

  15. Natural products used as a chemical library for protein-protein interaction targeted drug discovery.

    PubMed

    Jin, Xuemei; Lee, Kyungro; Kim, Nam Hee; Kim, Hyun Sil; Yook, Jong In; Choi, Jiwon; No, Kyoung Tai

    2018-01-01

    Protein-protein interactions (PPIs), which are essential for cellular processes, have been recognized as attractive therapeutic targets. Therefore, the construction of a PPI-focused chemical library is an inevitable necessity for future drug discovery. Natural products have been used as traditional medicines to treat human diseases for millennia; in addition, their molecular scaffolds have been used in diverse approved drugs and drug candidates. The recent discovery of the ability of natural products to inhibit PPIs led us to use natural products as a chemical library for PPI-targeted drug discovery. In this study, we collected natural products (NPDB) from non-commercial and in-house databases to analyze their similarities to small-molecule PPI inhibitors (iPPIs) and FDA-approved drugs by using eight molecular descriptors. Then, we evaluated the distribution of NPDB and iPPIs in the chemical space, represented by the molecular fingerprint and molecular scaffolds, to identify the promising scaffolds, which could interfere with PPIs. To investigate the ability of natural products to inhibit PPI targets, molecular docking was used. Then, we predicted a set of high-potency natural products by using the iPPI-likeness score based on a docking score-weighted model. These selected natural products showed high binding affinities to the PPI target, namely XIAP, which were validated in an in vitro experiment. In addition, the natural products with novel scaffolds might provide a promising starting point for further medicinal chemistry developments. Overall, our study shows the potency of natural products in targeting PPIs, which might help in the design of a PPI-focused chemical library for future drug discovery. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Identification of specific posttranslational O-mycoloylations mediating protein targeting to the mycomembrane.

    PubMed

    Carel, Clément; Marcoux, Julien; Réat, Valérie; Parra, Julien; Latgé, Guillaume; Laval, Françoise; Demange, Pascal; Burlet-Schiltz, Odile; Milon, Alain; Daffé, Mamadou; Tropis, Maryelle G; Renault, Marie A M

    2017-04-18

    The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum , a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum , we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O -mycoloylation, pyroglutamylation, and N -formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O -acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment.

  17. Identification of specific posttranslational O-mycoloylations mediating protein targeting to the mycomembrane

    PubMed Central

    Carel, Clément; Réat, Valérie; Parra, Julien; Latgé, Guillaume; Laval, Françoise; Burlet-Schiltz, Odile; Milon, Alain; Daffé, Mamadou; Tropis, Maryelle G.; Renault, Marie A. M.

    2017-01-01

    The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum, a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum, we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O-mycoloylation, pyroglutamylation, and N-formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O-acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment. PMID:28373551

  18. Ranking targets in structure-based virtual screening of three-dimensional protein libraries: methods and problems.

    PubMed

    Kellenberger, Esther; Foata, Nicolas; Rognan, Didier

    2008-05-01

    Structure-based virtual screening is a promising tool to identify putative targets for a specific ligand. Instead of docking multiple ligands into a single protein cavity, a single ligand is docked in a collection of binding sites. In inverse screening, hits are in fact targets which have been prioritized within the pool of best ranked proteins. The target rate depends on specificity and promiscuity in protein-ligand interactions and, to a considerable extent, on the effectiveness of the scoring function, which still is the Achilles' heel of molecular docking. In the present retrospective study, virtual screening of the sc-PDB target library by GOLD docking was carried out for four compounds (biotin, 4-hydroxy-tamoxifen, 6-hydroxy-1,6-dihydropurine ribonucleoside, and methotrexate) of known sc-PDB targets and, several ranking protocols based on GOLD fitness score and topological molecular interaction fingerprint (IFP) comparison were evaluated. For the four investigated ligands, the fusion of GOLD fitness and two IFP scores allowed the recovery of most targets, including the rare proteins which are not readily suitable for statistical analysis, while significantly filtering out most false positive entries. The current survey suggests that selecting a small number of targets (<20) for experimental evaluation is achievable with a pure structure-based approach.

  19. Discovery of Peripheral κ-Opioid Receptor Agonists as Novel Analgesics.

    PubMed

    Suzuki, Shinya; Sugawara, Yuji; Inada, Hideaki; Tsuji, Riichiro; Inoue, Atsushi; Tanimura, Ryuji; Shimozono, Rieko; Konno, Mitsuhiro; Ohyama, Tomofumi; Higashi, Eriko; Sakai, Chizuka; Kawai, Koji

    2017-01-01

    κ-Opioid receptor agonists with high selectivity over the μ-opioid receptor and peripheral selectivity are attractive targets in the development of drugs for pain. We have previously attempted to create novel analgesics with peripheral selective κ-opioid receptor agonist on the basis of TRK-820. In this study, we elucidated the biological properties of 17-hydroxy-cyclopropylmethyl and 10α-hydroxy derivatives. These compounds were found to have better κ-opioid receptor selectivity and peripheral selectivity than TRK-820.

  20. Landscape phages and their fusion proteins targeted to breast cancer cells

    PubMed Central

    Fagbohun, Olusegun A.; Bedi, Deepa; Grabchenko, Natalia I.; Deinnocentes, Patricia A.; Bird, Richard C.; Petrenko, Valery A.

    2012-01-01

    Breast cancer is a leading cause of death among women in the USA. The efficacy of existing anticancer therapeutics can be improved by targeting them through conjugation with ligands binding to cellular receptors. Recently, we developed a novel drug targeting strategy based on the use of pre-selected cancer-specific ‘fusion pVIII proteins’ (fpVIII), as targeting ligands. To study the efficiency of this approach in animal models, we developed a panel of breast cancer cell-binding phages as a source of targeted fpVIIIs. Two landscape phage peptide libraries (8-mer f8/8 and 9-mer f8/9) were screened to isolate 132 phage variants that recognize breast carcinoma cells MCF-7 and ZR-75-1 and internalize into the cells. When tested for their interaction with the breast cancer cells in comparison with liver cancer cells HepG2, human mammary cells MCF-10A cells and serum, 16 of the phage probes selectively interacted with the breast cancer cells whereas 32 bound both breast and liver cancer cells. The most prominent cancer-specific phage DMPGTVLP, demonstrating sub-nanomolar Kd in interaction with target cells, was used for affinity chromatography of cellular membrane molecules to reveal its potential binding receptor. The isolated protein was identified by direct sequencing as cellular surface nucleolin. This conclusion was confirmed by inhibition of the phage–cell interaction with nucleolin antibodies. Other prominent phage binders VPTDTDYS, VEEGGYIAA, and DWRGDSMDS demonstrate consensus motifs common to previously identified cancer-specific peptides. Isolated phage proteins exhibit inherent binding specificity towards cancer cells, demonstrating the functional activity of the selected fused peptides. The selected phages, their peptide inserts and intact fusion proteins can serve as promising ligands for the development of targeted nanomedicines and their study in model mice with xenograft of human cells MCF-7 and ZR-75-1. PMID:22490956

  1. Emulsomes Meet S-layer Proteins: An Emerging Targeted Drug Delivery System

    PubMed Central

    Ucisik, Mehmet H.; Sleytr, Uwe B.; Schuster, Bernhard

    2015-01-01

    Here, the use of emulsomes as a drug delivery system is reviewed and compared with other similar lipidic nanoformulations. In particular, we look at surface modification of emulsomes using S-layer proteins, which are self-assembling proteins that cover the surface of many prokaryotic organisms. It has been shown that covering emulsomes with a crystalline S-layer lattice can protect cells from oxidative stress and membrane damage. In the future, the capability to recrystallize S-layer fusion proteins on lipidic nanoformulations may allow the presentation of binding functions or homing protein domains to achieve highly specific targeted delivery of drug-loaded emulsomes. Besides the discussion on several designs and advantages of composite emulsomes, the success of emulsomes for the delivery of drugs to fight against viral and fungal infections, dermal therapy, cancer, and autoimmunity is summarized. Further research might lead to smart, biocompatible emulsomes, which are able to protect and reduce the side effects caused by the drug, but at the same time are equipped with specific targeting molecules to find the desired site of action. PMID:25697368

  2. Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion

    PubMed Central

    Shi, Tujin; Sun, Xuefei; Gao, Yuqian; Fillmore, Thomas L.; Schepmoes, Athena A.; Zhao, Rui; He, Jintang; Moore, Ronald J.; Kagan, Jacob; Rodland, Karin D.; Liu, Tao; Liu, Alvin Y.; Smith, Richard D.; Tang, Keqi; Camp, David G.; Qian, Wei-Jun

    2013-01-01

    We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50–100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion. Limits of quantification (LOQ) at low ng/mL levels with a median coefficient of variation (CV) of ~12% were achieved for proteins spiked into human female serum. PRISM-SRM provided >100-fold improvement in the LOQ when compared to conventional LC-SRM measurements. PRISM-SRM was then applied to measure several low-abundance endogenous serum proteins, including prostate-specific antigen (PSA), in clinical prostate cancer patient sera. PRISM-SRM enabled confident detection of all target endogenous serum proteins except the low pg/mL-level cardiac troponin T. A correlation coefficient >0.99 was observed for PSA between the results from PRISM-SRM and immunoassays. Our results demonstrate that PRISM-SRM can successful quantify low ng/mL proteins in human plasma or serum without depletion. We anticipate broad applications for PRISM-SRM quantification of low-abundance proteins in candidate biomarker verification and systems biology studies. PMID:23763644

  3. Comparison of pencil beam–based homogeneous vs inhomogeneous target dose planning for stereotactic body radiotherapy of peripheral lung tumors through Monte Carlo–based recalculation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ohtakara, Kazuhiro, E-mail: ohtakara@murakami.asahi-u.ac.jp; Hoshi, Hiroaki

    2015-10-01

    This study was conducted to ascertain whether homogeneous target dose planning is suitable for stereotactic body radiotherapy (SBRT) of peripheral lung cancer under appropriate breath-holding. For 20 peripheral lung tumors, paired dynamic conformal arc plans were generated by only adjusting the leaf margin to the planning target volume (PTV) edge for fulfilling the conditions such that the prescription isodose surface (IDS) encompassing exactly 95% of the PTV (PTV D{sub 95}) corresponds to 95% and 80% IDS, normalized to 100% at the PTV isocenter under a pencil beam (PB) algorithm with radiologic path length correction. These plans were recalculated using themore » x-ray voxel Monte Carlo (XVMC) algorithm under otherwise identical conditions, and then compared. Lesions abutting the parietal pleura or not were defined as edge or island tumors, respectively, and the influences of the target volume and its location relative to the chest wall on the target dose were examined. The median (range) leaf margin required for the 95% and 80% plans was 3.9 mm (1.3 to 5.0) and −1.2 mm (−1.8 to 0.1), respectively. Notably, the latter was significantly correlated negatively with PTV. In the 80% plans, the PTV D{sub 95} was slightly higher under XVMC, whereas the PTV D{sub 98} was significantly lower, irrespective of the dose calculation algorithm used. Other PTV and all gross tumor volume doses were significantly higher, while the lung doses outside the PTV were slightly lower. The target doses increased as a function of PTV and were significantly lower for island tumors than for edge tumors. In conclusion, inhomogeneous target dose planning using smaller leaf margin for a larger tumor volume was deemed suitable in ensuring more sufficient target dose while slightly reducing lung dose. In addition, more inhomogeneous target dose planning using <80% IDS (e.g., 70%) for PTV covering would be preferable for island tumors.« less

  4. Molecular engineering of proteins and polymers for targeting and intracellular delivery of therapeutics.

    PubMed

    Stayton, P S; Hoffman, A S; Murthy, N; Lackey, C; Cheung, C; Tan, P; Klumb, L A; Chilkoti, A; Wilbur, F S; Press, O W

    2000-03-01

    There are many protein and DNA based therapeutics under development in the biotechnology and pharmaceutical industries. Key delivery challenges remain before many of these biomolecular therapeutics reach the clinic. Two important barriers are the effective targeting of drugs to specific tissues and cells and the subsequent intracellular delivery to appropriate cellular compartments. In this review, we summarize protein engineering work aimed at improving the stability and refolding efficiency of antibody fragments used in targeting, and at constructing new streptavidin variants which may offer improved performance in pre-targeting delivery strategies. In addition, we review recent work with pH-responsive polymers that mimic the membrane disruptive properties of viruses and toxins. These polymers could serve as alternatives to fusogenic peptides in gene therapy formulations and to enhance the intracellular delivery of protein therapeutics that function in the cytoplasm.

  5. Foveal Processing Under Concurrent Peripheral Load in Profoundly Deaf Adults

    PubMed Central

    2016-01-01

    Development of the visual system typically proceeds in concert with the development of audition. One result is that the visual system of profoundly deaf individuals differs from that of those with typical auditory systems. While past research has suggested deaf people have enhanced attention in the visual periphery, it is still unclear whether or not this enhancement entails deficits in central vision. Profoundly deaf and typically hearing adults were administered a variant of the useful field of view task that independently assessed performance on concurrent central and peripheral tasks. Identification of a foveated target was impaired by a concurrent selective peripheral attention task, more so in profoundly deaf adults than in the typically hearing. Previous findings of enhanced performance on the peripheral task were not replicated. These data are discussed in terms of flexible allocation of spatial attention targeted towards perceived task demands, and support a modified “division of labor” hypothesis whereby attentional resources co-opted to process peripheral space result in reduced resources in the central visual field. PMID:26657078

  6. Concepts of Protein Sorting or Targeting Signals and Membrane Topology in Undergraduate Teaching

    ERIC Educational Resources Information Center

    Tang, Bor Luen; Teng, Felicia Yu Hsuan

    2005-01-01

    The process of protein biogenesis culminates in its correct targeting to specific subcellular locations where it serves a function. Contemporary molecular and cell biology investigations often involve the exogenous expression of epitope- or fluorescent protein-tagged recombinant molecules as well as subsequent analysis of protein-protein…

  7. Antimicrobial Peptides Targeting Gram-Positive Bacteria

    PubMed Central

    Malanovic, Nermina; Lohner, Karl

    2016-01-01

    Antimicrobial peptides (AMPs) have remarkably different structures as well as biological activity profiles, whereupon most of these peptides are supposed to kill bacteria via membrane damage. In order to understand their molecular mechanism and target cell specificity for Gram-positive bacteria, it is essential to consider the architecture of their cell envelopes. Before AMPs can interact with the cytoplasmic membrane of Gram-positive bacteria, they have to traverse the cell wall composed of wall- and lipoteichoic acids and peptidoglycan. While interaction of AMPs with peptidoglycan might rather facilitate penetration, interaction with anionic teichoic acids may act as either a trap for AMPs or a ladder for a route to the cytoplasmic membrane. Interaction with the cytoplasmic membrane frequently leads to lipid segregation affecting membrane domain organization, which affects membrane permeability, inhibits cell division processes or leads to delocalization of essential peripheral membrane proteins. Further, precursors of cell wall components, especially the highly conserved lipid II, are directly targeted by AMPs. Thereby, the peptides do not inhibit peptidoglycan synthesis via binding to proteins like common antibiotics, but form a complex with the precursor molecule, which in addition can promote pore formation and membrane disruption. Thus, the multifaceted mode of actions will make AMPs superior to antibiotics that act only on one specific target. PMID:27657092

  8. Extracting sets of chemical substructures and protein domains governing drug-target interactions.

    PubMed

    Yamanishi, Yoshihiro; Pauwels, Edouard; Saigo, Hiroto; Stoven, Véronique

    2011-05-23

    The identification of rules governing molecular recognition between drug chemical substructures and protein functional sites is a challenging issue at many stages of the drug development process. In this paper we develop a novel method to extract sets of drug chemical substructures and protein domains that govern drug-target interactions on a genome-wide scale. This is made possible using sparse canonical correspondence analysis (SCCA) for analyzing drug substructure profiles and protein domain profiles simultaneously. The method does not depend on the availability of protein 3D structures. From a data set of known drug-target interactions including enzymes, ion channels, G protein-coupled receptors, and nuclear receptors, we extract a set of chemical substructures shared by drugs able to bind to a set of protein domains. These two sets of extracted chemical substructures and protein domains form components that can be further exploited in a drug discovery process. This approach successfully clusters protein domains that may be evolutionary unrelated but that bind a common set of chemical substructures. As shown in several examples, it can also be very helpful for predicting new protein-ligand interactions and addressing the problem of ligand specificity. The proposed method constitutes a contribution to the recent field of chemogenomics that aims to connect the chemical space with the biological space.

  9. Intracellular targeting of CD44+ cells with self-assembling, protein only nanoparticles.

    PubMed

    Pesarrodona, Mireia; Ferrer-Miralles, Neus; Unzueta, Ugutz; Gener, Petra; Tatkiewicz, Witold; Abasolo, Ibane; Ratera, Imma; Veciana, Jaume; Schwartz, Simó; Villaverde, Antonio; Vazquez, Esther

    2014-10-01

    CD44 is a multifunctional cell surface protein involved in proliferation and differentiation, angiogenesis and signaling. The expression of CD44 is up-regulated in several types of human tumors and particularly in cancer stem cells, representing an appealing target for drug delivery in the treatment of cancer. We have explored here several protein ligands of CD44 for the construction of self-assembling modular proteins designed to bind and internalize target cells. Among five tested ligands, two of them (A5G27 and FNI/II/V) drive the formation of protein-only, ring-shaped nanoparticles of about 14 nm that efficiently bind and penetrate CD44(+) cells by an endosomal route. The potential of these newly designed nanoparticles is evaluated regarding the need of biocompatible nanostructured materials for drug delivery in CD44-linked conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Targeted Quantification of Isoforms of a Thylakoid-Bound Protein: MRM Method Development.

    PubMed

    Bru-Martínez, Roque; Martínez-Márquez, Ascensión; Morante-Carriel, Jaime; Sellés-Marchart, Susana; Martínez-Esteso, María José; Pineda-Lucas, José Luis; Luque, Ignacio

    2018-01-01

    Targeted mass spectrometric methods such as selected/multiple reaction monitoring (SRM/MRM) have found intense application in protein detection and quantification which competes with classical immunoaffinity techniques. It provides a universal procedure to develop a fast, highly specific, sensitive, accurate, and cheap methodology for targeted detection and quantification of proteins based on the direct analysis of their surrogate peptides typically generated by tryptic digestion. This methodology can be advantageously applied in the field of plant proteomics and particularly for non-model species since immunoreagents are scarcely available. Here, we describe the issues to take into consideration in order to develop a MRM method to detect and quantify isoforms of the thylakoid-bound protein polyphenol oxidase from the non-model and database underrepresented species Eriobotrya japonica Lindl.

  11. Pac-Man for biotechnology: co-opting degrons for targeted protein degradation to control and alter cell function.

    PubMed

    Yu, Geng; Rosenberg, Julian N; Betenbaugh, Michael J; Oyler, George A

    2015-12-01

    Protein degradation in normal living cells is precisely regulated to match the cells' physiological requirements. The selectivity of protein degradation is determined by an elaborate degron-tagging system. Degron refers to an amino acid sequence that encodes a protein degradation signal, which is oftentimes a poly-ubiquitin chain that can be transferred to other proteins. Current understanding of ubiquitination dependent and independent protein degradation processes has expanded the application of degrons for targeted protein degradation and novel cell engineering strategies. Recent findings suggest that small molecules inducing protein association can be exploited to create degrons that target proteins for degradation. Here, recent applications of degron-based targeted protein degradation in eukaryotic organisms are reviewed. The degron mediated protein degradation represents a rapidly tunable methodology to control protein abundance, which has broad application in therapeutics and cellular function control and monitoring. Copyright © 2015. Published by Elsevier Ltd.

  12. Heat shock proteins as potential targets for protective strategies in neurodegeneration.

    PubMed

    Kampinga, Harm H; Bergink, Steven

    2016-06-01

    Protein aggregates are hallmarks of nearly all age-related neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and several polyglutamine diseases such as Huntington's disease and different forms of spinocerebellar ataxias (SCA; SCA1-3, SCA6, and SCA7). The collapse of cellular protein homoeostasis can be both a cause and a consequence of this protein aggregation. Boosting components of the cellular protein quality control system has been widely investigated as a strategy to counteract protein aggregates or their toxic consequences. Heat shock proteins (HSPs) play a central part in regulating protein quality control and contribute to protein aggregation and disaggregation. Therefore, HSPs are viable targets for the development of drugs aimed at reducing pathogenic protein aggregates that are thought to contribute to the development of so many neurodegenerative disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Peptide drugs to target G protein-coupled receptors.

    PubMed

    Bellmann-Sickert, Kathrin; Beck-Sickinger, Annette G

    2010-09-01

    Major indications for use of peptide-based therapeutics include endocrine functions (especially diabetes mellitus and obesity), infectious diseases, and cancer. Whereas some peptide pharmaceuticals are drugs, acting as agonists or antagonists to directly treat cancer, others (including peptide diagnostics and tumour-targeting pharmaceuticals) use peptides to 'shuttle' a chemotherapeutic agent or a tracer to the tumour and allow sensitive imaging or targeted therapy. Significant progress has been made in the last few years to overcome disadvantages in peptide design such as short half-life, fast proteolytic cleavage, and low oral bioavailability. These advances include peptide PEGylation, lipidisation or multimerisation; the introduction of peptidomimetic elements into the sequences; and innovative uptake strategies such as liposomal, capsule or subcutaneous formulations. This review focuses on peptides targeting G protein-coupled receptors that are promising drug candidates or that have recently entered the pharmaceutical market. Copyright 2010 Elsevier Ltd. All rights reserved.

  14. Dendritic cell targeted chitosan nanoparticles for nasal DNA immunization against SARS CoV nucleocapsid protein.

    PubMed

    Raghuwanshi, Dharmendra; Mishra, Vivek; Das, Dipankar; Kaur, Kamaljit; Suresh, Mavanur R

    2012-04-02

    This work investigates the formulation and in vivo efficacy of dendritic cell (DC) targeted plasmid DNA loaded biotinylated chitosan nanoparticles for nasal immunization against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) as antigen. The induction of antigen-specific mucosal and systemic immune response at the site of virus entry is a major challenge for vaccine design. Here, we designed a strategy for noninvasive receptor mediated gene delivery to nasal resident DCs. The pDNA loaded biotinylated chitosan nanoparticles were prepared using a complex coacervation process and characterized for size, shape, surface charge, plasmid DNA loading and protection against nuclease digestion. The pDNA loaded biotinylated chitosan nanoparticles were targeted with bifunctional fusion protein (bfFp) vector for achieving DC selective targeting. The bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused with anti-DEC-205 single chain antibody (scFv). The core-streptavidin arm of fusion protein binds with biotinylated nanoparticles, while anti-DEC-205 scFv imparts targeting specificity to DC DEC-205 receptor. We demonstrate that intranasal administration of bfFp targeted formulations along with anti-CD40 DC maturation stimuli enhanced magnitude of mucosal IgA as well as systemic IgG against N protein. The strategy led to the detection of augmented levels of N protein specific systemic IgG and nasal IgA antibodies. However, following intranasal delivery of naked pDNA no mucosal and systemic immune responses were detected. A parallel comparison of targeted formulations using intramuscular and intranasal routes showed that the intramuscular route is superior for induction of systemic IgG responses compared with the intranasal route. Our results suggest that targeted pDNA delivery through a noninvasive intranasal route can be a strategy for designing low-dose vaccines.

  15. Data-Driven Approach To Determine Popular Proteins for Targeted Proteomics Translation of Six Organ Systems.

    PubMed

    Lam, Maggie P Y; Venkatraman, Vidya; Xing, Yi; Lau, Edward; Cao, Quan; Ng, Dominic C M; Su, Andrew I; Ge, Junbo; Van Eyk, Jennifer E; Ping, Peipei

    2016-11-04

    Amidst the proteomes of human tissues lie subsets of proteins that are closely involved in conserved pathophysiological processes. Much of biomedical research concerns interrogating disease signature proteins and defining their roles in disease mechanisms. With advances in proteomics technologies, it is now feasible to develop targeted proteomics assays that can accurately quantify protein abundance as well as their post-translational modifications; however, with rapidly accumulating number of studies implicating proteins in diseases, current resources are insufficient to target every protein without judiciously prioritizing the proteins with high significance and impact for assay development. We describe here a data science method to prioritize and expedite assay development on high-impact proteins across research fields by leveraging the biomedical literature record to rank and normalize proteins that are popularly and preferentially published by biomedical researchers. We demonstrate this method by finding priority proteins across six major physiological systems (cardiovascular, cerebral, hepatic, renal, pulmonary, and intestinal). The described method is data-driven and builds upon the collective knowledge of previous publications referenced on PubMed to lend objectivity to target selection. The method and resulting popular protein lists may also be useful for exploring biological processes associated with various physiological systems and research topics, in addition to benefiting ongoing efforts to facilitate the broad translation of proteomics technologies.

  16. Transdermal optogenetic peripheral nerve stimulation

    NASA Astrophysics Data System (ADS)

    Maimon, Benjamin E.; Zorzos, Anthony N.; Bendell, Rhys; Harding, Alexander; Fahmi, Mina; Srinivasan, Shriya; Calvaresi, Peter; Herr, Hugh M.

    2017-06-01

    Objective: A fundamental limitation in both the scientific utility and clinical translation of peripheral nerve optogenetic technologies is the optical inaccessibility of the target nerve due to the significant scattering and absorption of light in biological tissues. To date, illuminating deep nerve targets has required implantable optical sources, including fiber-optic and LED-based systems, both of which have significant drawbacks. Approach: Here we report an alternative approach involving transdermal illumination. Utilizing an intramuscular injection of ultra-high concentration AAV6-hSyn-ChR2-EYFP in rats. Main results: We demonstrate transdermal stimulation of motor nerves at 4.4 mm and 1.9 mm depth with an incident laser power of 160 mW and 10 mW, respectively. Furthermore, we employ this technique to accurately control ankle position by modulating laser power or position on the skin surface. Significance: These results have the potential to enable future scientific optogenetic studies of pathologies implicated in the peripheral nervous system for awake, freely-moving animals, as well as a basis for future clinical studies.

  17. Peripheral leukocyte expression of the potential biomarker proteins Bdnf, Sirt1, and Psen1 is not regulated by promoter methylation in Alzheimer's disease patients.

    PubMed

    Carboni, Lucia; Lattanzio, Francesca; Candeletti, Sanzio; Porcellini, Elisa; Raschi, Elena; Licastro, Federico; Romualdi, Patrizia

    2015-09-25

    The identification of Alzheimer's disease (AD) biomarkers is crucial to support drug discovery. Within putative biomarkers, peripheral Bdnf levels correlate with cognitive decline and AD, although conflicting findings are reported. Sirtuin 1 (Sirt1) serum levels are lower in AD patients and Presenilin 1 (Psen1) is expressed by blood cells. DNA methylation is altered in AD patients, suggesting that epigenetic mechanisms play a role in AD pathophysiology. The objective of this study was to investigate promoter methylation levels of potential biomarkers in AD cases and controls. Peripheral blood DNA methylation levels were analysed by methylation-specific primer real-time PCR. Bdnf promoter methylation levels did not differ between AD patients and controls. Similarly, Sirt1 promoter revealed minimal levels of methylation which did not display significant differences between groups. No significant difference was revealed between AD patients and controls also in Psen1 methylation, showing a large variability of values among subjects. Although peripheral Bdnf expression is associated with differential promoter methylation in psychiatric and neurological disorders, our results suggest that different mechanisms take place in AD. The finding that the control of Sirt1 protein levels in blood is not exerted through the repression of mRNA expression by promoter hypermethylation is in agreement with previous data. In contrast, other studies reported that Psen1 methylation may be increased or decreased in AD patients, suggesting that additional studies are required. In conclusion, this study shows that peripheral levels of the potential AD biomarker proteins Bdnf, Sirt1, and Psen1 are not regulated by different promoter methylation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. The G Protein α Chaperone Ric-8 as a Potential Therapeutic Target

    PubMed Central

    Papasergi, Makaía M.; Patel, Bharti R.

    2015-01-01

    Resistance to inhibitors of cholinesterase (Ric-8)A and Ric-8B are essential genes that encode positive regulators of heterotrimeric G protein α subunits. Controversy persists surrounding the precise way(s) that Ric-8 proteins affect G protein biology and signaling. Ric-8 proteins chaperone nucleotide-free Gα-subunit states during biosynthetic protein folding prior to G protein heterotrimer assembly. In organisms spanning the evolutionary window of Ric-8 expression, experimental perturbation of Ric-8 genes results in reduced functional abundances of G proteins because G protein α subunits are misfolded and degraded rapidly. Ric-8 proteins also act as Gα-subunit guanine nucleotide exchange factors (GEFs) in vitro. However, Ric-8 GEF activity could strictly be an in vitro phenomenon stemming from the ability of Ric-8 to induce partial Gα unfolding, thereby enhancing GDP release. Ric-8 GEF activity clearly differs from the GEF activity of G protein–coupled receptors (GPCRs). G protein βγ is inhibitory to Ric-8 action but obligate for receptors. It remains an open question whether Ric-8 has dual functions in cells and regulates G proteins as both a molecular chaperone and GEF. Clearly, Ric-8 has a profound influence on heterotrimeric G protein function. For this reason, we propose that Ric-8 proteins are as yet untested therapeutic targets in which pharmacological inhibition of the Ric-8/Gα protein–protein interface could serve to attenuate the effects of disease-causing G proteins (constitutively active mutants) and/or GPCR signaling. This minireview will chronicle the understanding of Ric-8 function, provide a comparative discussion of the Ric-8 molecular chaperoning and GEF activities, and support the case for why Ric-8 proteins should be considered potential targets for development of new therapies. PMID:25319541

  19. Musashi RNA-Binding Proteins as Cancer Drivers and Novel Therapeutic Targets.

    PubMed

    Kudinov, Alexander E; Karanicolas, John; Golemis, Erica A; Boumber, Yanis

    2017-05-01

    Aberrant gene expression that drives human cancer can arise from epigenetic dysregulation. Although much attention has focused on altered activity of transcription factors and chromatin-modulating proteins, proteins that act posttranscriptionally can potently affect expression of oncogenic signaling proteins. The RNA-binding proteins (RBP) Musashi-1 (MSI1) and Musashi-2 (MSI2) are emerging as regulators of multiple critical biological processes relevant to cancer initiation, progression, and drug resistance. Following identification of Musashi as a regulator of progenitor cell identity in Drosophila , the human Musashi proteins were initially linked to control of maintenance of hematopoietic stem cells, then stem cell compartments for additional cell types. More recently, the Musashi proteins were found to be overexpressed and prognostic of outcome in numerous cancer types, including colorectal, lung, and pancreatic cancers; glioblastoma; and several leukemias. MSI1 and MSI2 bind and regulate the mRNA stability and translation of proteins operating in essential oncogenic signaling pathways, including NUMB/Notch, PTEN/mTOR, TGFβ/SMAD3, MYC, cMET, and others. On the basis of these activities, MSI proteins maintain cancer stem cell populations and regulate cancer invasion, metastasis, and development of more aggressive cancer phenotypes, including drug resistance. Although RBPs are viewed as difficult therapeutic targets, initial efforts to develop MSI-specific inhibitors are promising, and RNA interference-based approaches to inhibiting these proteins have had promising outcomes in preclinical studies. In the interim, understanding the function of these translational regulators may yield insight into the relationship between mRNA expression and protein expression in tumors, guiding tumor-profiling analysis. This review provides a current overview of Musashi as a cancer driver and novel therapeutic target. Clin Cancer Res; 23(9); 2143-53. ©2017 AACR . ©2017

  20. Targeting the latent cytomegalovirus reservoir with an antiviral fusion toxin protein

    PubMed Central

    Krishna, B. A.; Spiess, K.; Poole, E. L.; Lau, B.; Voigt, S.; Kledal, T. N.; Rosenkilde, M. M.; Sinclair, J. H.

    2017-01-01

    Reactivation of human cytomegalovirus (HCMV) in transplant recipients can cause life-threatening disease. Consequently, for transplant recipients, killing latently infected cells could have far-reaching clinical benefits. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, and one viral gene expressed by latently infected myeloid cells is US28. This viral gene encodes a cell surface G protein-coupled receptor (GPCR) that binds chemokines, triggering its endocytosis. We show that the expression of US28 on the surface of latently infected cells allows monocytes and their progenitor CD34+ cells to be targeted and killed by F49A-FTP, a highly specific fusion toxin protein that binds this viral GPCR. As expected, this specific targeting of latently infected cells by F49A-FTP also robustly reduces virus reactivation in vitro. Consequently, such specific fusion toxin proteins could form the basis of a therapeutic strategy for eliminating latently infected cells before haematopoietic stem cell transplantation. PMID:28148951

  1. Targeted nanodiamonds for identification of subcellular protein assemblies in mammalian cells

    PubMed Central

    Lake, Michael P.; Bouchard, Louis-S.

    2017-01-01

    Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging. PMID:28636640

  2. Differential regulation of protein synthesis by amino acids and insulin in peripheral and visceral tissues of neonatal pigs

    PubMed Central

    Suryawan, Agus; O’Connor, Pamela M. J.; Bush, Jill A.; Nguyen, Hanh V.

    2009-01-01

    The high efficiency of protein deposition during the neonatal period is driven by high rates of protein synthesis, which are maximally stimulated after feeding. In the current study, we examined the individual roles of amino acids and insulin in the regulation of protein synthesis in peripheral and visceral tissues of the neonate by performing pancreatic glucose–amino acid clamps in overnight-fasted 7-day-old pigs. We infused pigs (n = 8–12/group) with insulin at 0, 10, 22, and 110 ng kg−0.66 min−1 to achieve ~0, 2, 6 and 30 μU ml−1 insulin so as to simulate below fasting, fasting, intermediate, and fed insulin levels, respectively. At each insulin dose, amino acids were maintained at the fasting or fed level. In conjunction with the highest insulin dose, amino acids were also allowed to fall below the fasting level. Tissue protein synthesis was measured using a flooding dose of L-[4-3H] phenylalanine. Both insulin and amino acids increased fractional rates of protein synthesis in longissimus dorsi, gastrocnemius, masseter, and diaphragm muscles. Insulin, but not amino acids, increased protein synthesis in the skin. Amino acids, but not insulin, increased protein synthesis in the liver, pancreas, spleen, and lung and tended to increase protein synthesis in the jejunum and kidney. Neither insulin nor amino acids altered protein synthesis in the stomach. The results suggest that the stimulation of protein synthesis by feeding in most tissues of the neonate is regulated by the post-prandial rise in amino acids. However, the feeding-induced stimulation of protein synthesis in skeletal muscles is independently mediated by insulin as well as amino acids. PMID:18683020

  3. Specific targeting of proteins to outer envelope membranes of endosymbiotic organelles, chloroplasts, and mitochondria

    PubMed Central

    Lee, Junho; Kim, Dae Heon; Hwang, Inhwan

    2014-01-01

    Chloroplasts and mitochondria are endosymbiotic organelles thought to be derived from endosymbiotic bacteria. In present-day eukaryotic cells, these two organelles play pivotal roles in photosynthesis and ATP production. In addition to these major activities, numerous reactions, and cellular processes that are crucial for normal cellular functions occur in chloroplasts and mitochondria. To function properly, these organelles constantly communicate with the surrounding cellular compartments. This communication includes the import of proteins, the exchange of metabolites and ions, and interactions with other organelles, all of which heavily depend on membrane proteins localized to the outer envelope membranes. Therefore, correct and efficient targeting of these membrane proteins, which are encoded by the nuclear genome and translated in the cytosol, is critically important for organellar function. In this review, we summarize the current knowledge of the mechanisms of protein targeting to the outer membranes of mitochondria and chloroplasts in two different directions, as well as targeting signals and cytosolic factors. PMID:24808904

  4. Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.

    PubMed

    Liang, Xue-Hai; Sun, Hong; Shen, Wen; Wang, Shiyu; Yao, Joyee; Migawa, Michael T; Bui, Huynh-Hoa; Damle, Sagar S; Riney, Stan; Graham, Mark J; Crooke, Rosanne M; Crooke, Stanley T

    2017-09-19

    A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. A high content in lipid-modified peripheral proteins and integral receptor kinases features in the arabidopsis plasma membrane proteome.

    PubMed

    Marmagne, Anne; Ferro, Myriam; Meinnel, Thierry; Bruley, Christophe; Kuhn, Lauriane; Garin, Jérome; Barbier-Brygoo, Hélène; Ephritikhine, Geneviève

    2007-11-01

    The proteomics of plasma membrane has brought to date only scarce and partial information on the actual protein repertoire. In this work, the plant plasma membrane proteome of Arabidopsis thaliana was investigated. A highly purified plasma membrane fraction was washed by NaCl and Na2CO3 salts, and the insoluble fractions were further analyzed by nano-LC-MS/MS. With 446 proteins identified, we hereby describe the largest plasma membrane proteome diversity reported so far. Half of the proteins were predicted to display transmembrane domains and/or to be anchored to the membrane, validating a posteriori the pertinence of the approach. A fine analysis highlighted two main specific and novel features. First, the main functional category is represented by a majority of as yet unreported signaling proteins, including 11% receptor-like kinases. Second, 16% of the identified proteins are predicted to be lipid-modified, specifically involving double lipid linkage through N-terminal myristoylation, S-palmitoylation, C-terminal prenylation, or glycosylphosphatidylinositol anchors. Thus, our approach led for the first time to the identification of a large number of peripheral proteins as part of the plasma membrane and allowed the functionality of the plasma membrane in the cell context to be reconsidered.

  6. Origins, actions and dynamic expression patterns of the neuropeptide VGF in rat peripheral and central sensory neurones following peripheral nerve injury

    PubMed Central

    Moss, Andrew; Ingram, Rachel; Koch, Stephanie; Theodorou, Andria; Low, Lucie; Baccei, Mark; Hathway, Gareth J; Costigan, Michael; Salton, Stephen R; Fitzgerald, Maria

    2008-01-01

    Background The role of the neurotrophin regulated polypeptide, VGF, has been investigated in a rat spared injury model of neuropathic pain. This peptide has been shown to be associated with synaptic strengthening and learning in the hippocampus and while it is known that VGFmRNA is upregulated in dorsal root ganglia following peripheral nerve injury, the role of this VGF peptide in neuropathic pain has yet to be investigated. Results Prolonged upregulation of VGF mRNA and protein was observed in injured dorsal root ganglion neurons, central terminals and their target dorsal horn neurons. Intrathecal application of TLQP-62, the C-terminal active portion of VGF (5–50 nmol) to naïve rats caused a long-lasting mechanical and cold behavioral allodynia. Direct actions of 50 nM TLQP-62 upon dorsal horn neuron excitability was demonstrated in whole cell patch recordings in spinal cord slices and in receptive field analysis in intact, anesthetized rats where significant actions of VGF were upon spontaneous activity and cold evoked responses. Conclusion VGF expression is therefore highly modulated in nociceptive pathways following peripheral nerve injury and can cause dorsal horn cell excitation and behavioral hypersensitivity in naïve animals. Together the results point to a novel and powerful role for VGF in neuropathic pain. PMID:19077191

  7. Prioritizing and modelling of putative drug target proteins of Candida albicans by systems biology approach.

    PubMed

    Ismail, Tariq; Fatima, Nighat; Muhammad, Syed Aun; Zaidi, Syed Saoud; Rehman, Nisar; Hussain, Izhar; Tariq, Najam Us Sahr; Amirzada, Imran; Mannan, Abdul

    2018-01-01

    Candida albicans (Candida albicans) is one of the major sources of nosocomial infections in humans which may prove fatal in 30% of cases. The hospital acquired infection is very difficult to treat affectively due to the presence of drug resistant pathogenic strains, therefore there is a need to find alternative drug targets to cure this infection. In silico and computational level frame work was used to prioritize and establish antifungal drug targets of Candida albicans. The identification of putative drug targets was based on acquiring 5090 completely annotated genes of Candida albicans from available databases which were categorized into essential and non-essential genes. The result indicated that 9% of proteins were essential and could become potential candidates for intervention which might result in pathogen eradication. We studied cluster of orthologs and the subtractive genomic analysis of these essential proteins against human genome was made as a reference to minimize the side effects. It was seen that 14% of Candida albicans proteins were evolutionary related to the human proteins while 86% are non-human homologs. In the next step of compatible drug target selections, the non-human homologs were sequentially compared to the human microbiome data to minimize the potential effects against gut flora which accumulated to 38% of the essential genome. The sub-cellular localization of these candidate proteins in fungal cellular systems indicated that 80% of them are cytoplasmic, 10% are mitochondrial and the remaining 10% are associated with the cell wall. The role of these non-human and non-gut flora putative target proteins in Candida albicans biological pathways was studied. Due to their integrated and critical role in Candida albicans replication cycle, four proteins were selected for molecular modeling. For drug designing and development, four high quality and reliable protein models with more than 70% sequence identity were constructed. These proteins are

  8. FLOURY ENDOSPERM7 encodes a regulator of starch synthesis and amyloplast development essential for peripheral endosperm development in rice

    PubMed Central

    Zhang, Long; Ren, Yulong; Lu, Bingyue; Yang, Chunyan; Feng, Zhiming; Liu, Zhou; Chen, Jun; Ma, Weiwei; Wang, Ying; Yu, Xiaowen; Wang, Yunlong; Zhang, Wenwei; Wang, Yihua; Liu, Shijia; Wu, Fuqing; Zhang, Xin; Guo, Xiuping; Bao, Yiqun; Jiang, Ling; Wan, Jianmin

    2016-01-01

    In cereal crops, starch synthesis and storage depend mainly on a specialized class of plastids, termed amyloplasts. Despite the importance of starch, the molecular machinery regulating starch synthesis and amyloplast development remains largely unknown. Here, we report the characterization of the rice (Oryza sativa) floury endosperm7 (flo7) mutant, which develops a floury-white endosperm only in the periphery and not in the inner portion. Consistent with the phenotypic alternation in flo7 endosperm, the flo7 mutant had reduced amylose content and seriously disrupted amylopectin structure only in the peripheral endosperm. Notably, flo7 peripheral endosperm cells showed obvious defects in compound starch grain development. Map-based cloning of FLO7 revealed that it encodes a protein of unknown function. FLO7 harbors an N-terminal transit peptide capable of targeting functional FLO7 fused to green fluorescent protein to amyloplast stroma in developing endosperm cells, and a domain of unknown function 1338 (DUF1338) that is highly conserved in green plants. Furthermore, our combined β-glucuronidase activity and RNA in situ hybridization assays showed that the FLO7 gene was expressed ubiquitously but exhibited a specific expression in the endosperm periphery. Moreover, a set of in vivo experiments demonstrated that the missing 32 aa in the flo7 mutant protein are essential for the stable accumulation of FLO7 in the endosperm. Together, our findings identify FLO7 as a unique plant regulator required for starch synthesis and amyloplast development within the peripheral endosperm and provide new insights into the spatial regulation of endosperm development in rice. PMID:26608643

  9. Space-related pharma-motifs for fast search of protein binding motifs and polypharmacological targets

    PubMed Central

    2012-01-01

    Background To discover a compound inhibiting multiple proteins (i.e. polypharmacological targets) is a new paradigm for the complex diseases (e.g. cancers and diabetes). In general, the polypharmacological proteins often share similar local binding environments and motifs. As the exponential growth of the number of protein structures, to find the similar structural binding motifs (pharma-motifs) is an emergency task for drug discovery (e.g. side effects and new uses for old drugs) and protein functions. Results We have developed a Space-Related Pharmamotifs (called SRPmotif) method to recognize the binding motifs by searching against protein structure database. SRPmotif is able to recognize conserved binding environments containing spatially discontinuous pharma-motifs which are often short conserved peptides with specific physico-chemical properties for protein functions. Among 356 pharma-motifs, 56.5% interacting residues are highly conserved. Experimental results indicate that 81.1% and 92.7% polypharmacological targets of each protein-ligand complex are annotated with same biological process (BP) and molecular function (MF) terms, respectively, based on Gene Ontology (GO). Our experimental results show that the identified pharma-motifs often consist of key residues in functional (active) sites and play the key roles for protein functions. The SRPmotif is available at http://gemdock.life.nctu.edu.tw/SRP/. Conclusions SRPmotif is able to identify similar pharma-interfaces and pharma-motifs sharing similar binding environments for polypharmacological targets by rapidly searching against the protein structure database. Pharma-motifs describe the conservations of binding environments for drug discovery and protein functions. Additionally, these pharma-motifs provide the clues for discovering new sequence-based motifs to predict protein functions from protein sequence databases. We believe that SRPmotif is useful for elucidating protein functions and drug discovery

  10. Space-related pharma-motifs for fast search of protein binding motifs and polypharmacological targets.

    PubMed

    Chiu, Yi-Yuan; Lin, Chun-Yu; Lin, Chih-Ta; Hsu, Kai-Cheng; Chang, Li-Zen; Yang, Jinn-Moon

    2012-01-01

    To discover a compound inhibiting multiple proteins (i.e. polypharmacological targets) is a new paradigm for the complex diseases (e.g. cancers and diabetes). In general, the polypharmacological proteins often share similar local binding environments and motifs. As the exponential growth of the number of protein structures, to find the similar structural binding motifs (pharma-motifs) is an emergency task for drug discovery (e.g. side effects and new uses for old drugs) and protein functions. We have developed a Space-Related Pharmamotifs (called SRPmotif) method to recognize the binding motifs by searching against protein structure database. SRPmotif is able to recognize conserved binding environments containing spatially discontinuous pharma-motifs which are often short conserved peptides with specific physico-chemical properties for protein functions. Among 356 pharma-motifs, 56.5% interacting residues are highly conserved. Experimental results indicate that 81.1% and 92.7% polypharmacological targets of each protein-ligand complex are annotated with same biological process (BP) and molecular function (MF) terms, respectively, based on Gene Ontology (GO). Our experimental results show that the identified pharma-motifs often consist of key residues in functional (active) sites and play the key roles for protein functions. The SRPmotif is available at http://gemdock.life.nctu.edu.tw/SRP/. SRPmotif is able to identify similar pharma-interfaces and pharma-motifs sharing similar binding environments for polypharmacological targets by rapidly searching against the protein structure database. Pharma-motifs describe the conservations of binding environments for drug discovery and protein functions. Additionally, these pharma-motifs provide the clues for discovering new sequence-based motifs to predict protein functions from protein sequence databases. We believe that SRPmotif is useful for elucidating protein functions and drug discovery.

  11. New strategy for renal fibrosis: Targeting Smad3 proteins for ubiquitination and degradation.

    PubMed

    Wang, Xin; Feng, Shaozhen; Fan, Jinjin; Li, Xiaoyan; Wen, Qiong; Luo, Ning

    2016-09-15

    Smad3 is a critical signaling protein in renal fibrosis. Proteolysis targeting chimeric molecules (PROTACs) are small molecules designed to degrade target proteins via ubiquitination. They have three components: (1) a recognition motif for E3 ligase; (2) a linker; and (3) a ligand for the target protein. We aimed to design a new PROTAC to prevent renal fibrosis by targeting Smad3 proteins and using hydroxylated pentapeptide of hypoxia-inducible factor-1α as the recognition motif for von Hippel-Lindau (VHL) ubiquitin ligase (E3). Computer-aided drug design was used to find a specific ligand targeting Smad3. Surface plasmon resonance (SPR) was used to verify and optimize screening results. Synthesized PROTAC was validated by two-stage mass spectrometry. The PROTAC's specificity for VHL (E3 ligase) was proved with two human renal carcinoma cell lines, 786-0 (VHL(-)) and ACHN (VHL(+)), and its anti-fibrosis effect was tested in renal fibrosis cell models. Thirteen small molecular compounds (SMCs) were obtained from the Enamine library using GLIDE molecular docking program. SPR results showed that #8 SMC (EN300-72284) combined best with Smad3 (KD=4.547×10(-5)M). Mass spectrometry showed that synthesized PROTAC had the correct peptide molecular weights. Western blot showed Smad3 was degraded by PROTAC with whole-cell lysate of ACHN but not 786-0. Degradation, but not ubiquitination, of Smad3 was inhibited by proteasome inhibitor MG132. The upregulation of fibronectin and Collagen I induced by TGF-β1 in both renal fibroblast and mesangial cells were inhibited by PROTAC. The new PROTAC might prevent renal fibrosis by targeting Smad3 for ubiquitination and degradation. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Overcoming biofluid protein complexity during targeted mass spectrometry detection and quantification of protein biomarkers by MRM cubed (MRM3).

    PubMed

    Jeudy, Jeremy; Salvador, Arnaud; Simon, Romain; Jaffuel, Aurore; Fonbonne, Catherine; Léonard, Jean-François; Gautier, Jean-Charles; Pasquier, Olivier; Lemoine, Jerome

    2014-02-01

    Targeted mass spectrometry in the so-called multiple reaction monitoring mode (MRM) is certainly a promising way for the precise, accurate, and multiplexed measurement of proteins and their genetic or posttranslationally modified isoforms. MRM carried out on a low-resolution triple quadrupole instrument faces a lack of specificity when addressing the quantification of weakly concentrated proteins. In this case, extensive sample fractionation or immunoenrichment alleviates signal contamination by interferences, but in turn decreases assay performance and throughput. Recently, MRM(3) was introduced as an alternative to MRM to improve the limit of quantification of weakly concentrated protein biomarkers. In the present work, we compare MRM and MRM(3) modes for the detection of biomarkers in plasma and urine. Calibration curves drawn with MRM and MRM(3) showed a similar range of linearity (R(2) > 0.99 for both methods) with protein concentrations above 1 μg/mL in plasma and a few nanogram per milliliter in urine. In contrast, optimized MRM(3) methods improve the limits of quantification by a factor of 2 to 4 depending on the targeted peptide. This gain arises from the additional MS(3) fragmentation step, which significantly removes or decreases interfering signals within the targeted transition channels.

  13. Rational modification of protein stability by targeting surface sites leads to complicated results

    PubMed Central

    Xiao, Shifeng; Patsalo, Vadim; Shan, Bing; Bi, Yuan; Green, David F.; Raleigh, Daniel P.

    2013-01-01

    The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions. PMID:23798426

  14. A Universal Stress Protein Involved in Oxidative Stress Is a Phosphorylation Target for Protein Kinase CIPK61

    PubMed Central

    2017-01-01

    Calcineurin B-like interacting protein kinases (CIPKs) decode calcium signals upon interaction with the calcium sensors calcineurin B like proteins into phosphorylation events that result into adaptation to environmental stresses. Few phosphorylation targets of CIPKs are known and therefore the molecular mechanisms underlying their downstream output responses are not fully understood. Tomato (Solanum lycopersicum) Cipk6 regulates immune and susceptible Programmed cell death in immunity transforming Ca2+ signals into reactive oxygen species (ROS) signaling. To investigate SlCipk6-induced molecular mechanisms and identify putative substrates, a yeast two-hybrid approach was carried on and a protein was identified that contained a Universal stress protein (Usp) domain present in bacteria, protozoa and plants, which we named “SlRd2”. SlRd2 was an ATP-binding protein that formed homodimers in planta. SlCipk6 and SlRd2 interacted using coimmunoprecipitation and bimolecular fluorescence complementation (BiFC) assays in Nicotiana benthamiana leaves and the complex localized in the cytosol. SlCipk6 phosphorylated SlRd2 in vitro, thus defining, to our knowledge, a novel target for CIPKs. Heterologous SlRd2 overexpression in yeast conferred resistance to highly toxic LiCl, whereas SlRd2 expression in Escherichia coli UspA mutant restored bacterial viability in response to H2O2 treatment. Finally, transient expression of SlCipk6 in transgenic N. benthamiana SlRd2 overexpressors resulted in reduced ROS accumulation as compared to wild-type plants. Taken together, our results establish that SlRd2, a tomato UspA, is, to our knowledge, a novel interactor and phosphorylation target of a member of the CIPK family, SlCipk6, and functionally regulates SlCipk6-mediated ROS generation. PMID:27899535

  15. A Universal Stress Protein Involved in Oxidative Stress Is a Phosphorylation Target for Protein Kinase CIPK6.

    PubMed

    Gutiérrez-Beltrán, Emilio; Personat, José María; de la Torre, Fernando; Del Pozo, Olga

    2017-01-01

    Calcineurin B-like interacting protein kinases (CIPKs) decode calcium signals upon interaction with the calcium sensors calcineurin B like proteins into phosphorylation events that result into adaptation to environmental stresses. Few phosphorylation targets of CIPKs are known and therefore the molecular mechanisms underlying their downstream output responses are not fully understood. Tomato (Solanum lycopersicum) Cipk6 regulates immune and susceptible Programmed cell death in immunity transforming Ca 2+ signals into reactive oxygen species (ROS) signaling. To investigate SlCipk6-induced molecular mechanisms and identify putative substrates, a yeast two-hybrid approach was carried on and a protein was identified that contained a Universal stress protein (Usp) domain present in bacteria, protozoa and plants, which we named "SlRd2". SlRd2 was an ATP-binding protein that formed homodimers in planta. SlCipk6 and SlRd2 interacted using coimmunoprecipitation and bimolecular fluorescence complementation (BiFC) assays in Nicotiana benthamiana leaves and the complex localized in the cytosol. SlCipk6 phosphorylated SlRd2 in vitro, thus defining, to our knowledge, a novel target for CIPKs. Heterologous SlRd2 overexpression in yeast conferred resistance to highly toxic LiCl, whereas SlRd2 expression in Escherichia coli UspA mutant restored bacterial viability in response to H 2 O 2 treatment. Finally, transient expression of SlCipk6 in transgenic N benthamiana SlRd2 overexpressors resulted in reduced ROS accumulation as compared to wild-type plants. Taken together, our results establish that SlRd2, a tomato UspA, is, to our knowledge, a novel interactor and phosphorylation target of a member of the CIPK family, SlCipk6, and functionally regulates SlCipk6-mediated ROS generation. © 2017 American Society of Plant Biologists. All Rights Reserved.

  16. Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

    PubMed

    Duffy, Fergal J; O'Donovan, Darragh; Devocelle, Marc; Moran, Niamh; O'Connell, David J; Shields, Denis C

    2015-03-23

    Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

  17. Zinc Finger Protein Designed to Target 2-Long Terminal Repeat Junctions Interferes with Human Immunodeficiency Virus Integration

    PubMed Central

    Sakkhachornphop, Supachai; Barbas, Carlos F.; Keawvichit, Rassamee; Wongworapat, Kanlaya

    2012-01-01

    Abstract Integration of the human immunodeficiency virus type 1 (HIV-1) genome into the host chromosome is a vital step in the HIV life cycle. The highly conserved cytosine–adenine (CA) dinucleotide sequence immediately upstream of the cleavage site is crucial for integrase (IN) activity. As this viral enzyme has an important role early in the HIV-1 replication cycle, interference with the IN substrate has become an attractive strategy for therapeutic intervention. We demonstrated that a designed zinc finger protein (ZFP) fused to green fluorescent protein (GFP) targets the 2-long terminal repeat (2-LTR) circle junctions of HIV-1 DNA with nanomolar affinity. We report now that 2LTRZFP-GFP stably transduced into 293T cells interfered with the expression of vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral red fluorescent protein (RFP), as shown by the suppression of RFP expression. We also used a third-generation lentiviral vector and pCEP4 expression vector to deliver the 2LTRZFP-GFP transgene into human T-lymphocytic cells, and a stable cell line for long-term expression studies was selected for HIV-1 challenge. HIV-1 integration and replication were inhibited as measured by Alu-gag real-time PCR and p24 antigen assay. In addition, the molecular activity of 2LTRZFP-GFP was evaluated in peripheral blood mononuclear cells. The results were confirmed by Alu-gag real-time PCR for integration interference. We suggest that the expression of 2LTRZFP-GFP limited viral integration on intracellular immunization, and that it has potential for use in HIV gene therapy in the future. PMID:22429108

  18. Evaluation of the novel algorithm of flexible ligand docking with moveable target-protein atoms.

    PubMed

    Sulimov, Alexey V; Zheltkov, Dmitry A; Oferkin, Igor V; Kutov, Danil C; Katkova, Ekaterina V; Tyrtyshnikov, Eugene E; Sulimov, Vladimir B

    2017-01-01

    We present the novel docking algorithm based on the Tensor Train decomposition and the TT-Cross global optimization. The algorithm is applied to the docking problem with flexible ligand and moveable protein atoms. The energy of the protein-ligand complex is calculated in the frame of the MMFF94 force field in vacuum. The grid of precalculated energy potentials of probe ligand atoms in the field of the target protein atoms is not used. The energy of the protein-ligand complex for any given configuration is computed directly with the MMFF94 force field without any fitting parameters. The conformation space of the system coordinates is formed by translations and rotations of the ligand as a whole, by the ligand torsions and also by Cartesian coordinates of the selected target protein atoms. Mobility of protein and ligand atoms is taken into account in the docking process simultaneously and equally. The algorithm is realized in the novel parallel docking SOL-P program and results of its performance for a set of 30 protein-ligand complexes are presented. Dependence of the docking positioning accuracy is investigated as a function of parameters of the docking algorithm and the number of protein moveable atoms. It is shown that mobility of the protein atoms improves docking positioning accuracy. The SOL-P program is able to perform docking of a flexible ligand into the active site of the target protein with several dozens of protein moveable atoms: the native crystallized ligand pose is correctly found as the global energy minimum in the search space with 157 dimensions using 4700 CPU ∗ h at the Lomonosov supercomputer.

  19. New insights into the targeting of a subset of tail-anchored proteins to the outer mitochondrial membrane

    PubMed Central

    Marty, Naomi J.; Teresinski, Howard J.; Hwang, Yeen Ting; Clendening, Eric A.; Gidda, Satinder K.; Sliwinska, Elwira; Zhang, Daiyuan; Miernyk, Ján A.; Brito, Glauber C.; Andrews, David W.; Dyer, John M.; Mullen, Robert T.

    2014-01-01

    Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins defined by their single C-terminal membrane-spanning domain and their ability to insert post-translationally into specific organelles with an Ncytoplasm-Corganelle interior orientation. The molecular mechanisms by which TA proteins are sorted to the proper organelles are not well-understood. Herein we present results indicating that a dibasic targeting motif (i.e., -R-R/K/H-X{X≠E}) identified previously in the C terminus of the mitochondrial isoform of the TA protein cytochrome b5, also exists in many other A. thaliana outer mitochondrial membrane (OMM)-TA proteins. This motif is conspicuously absent, however, in all but one of the TA protein subunits of the translocon at the outer membrane of mitochondria (TOM), suggesting that these two groups of proteins utilize distinct biogenetic pathways. Consistent with this premise, we show that the TA sequences of the dibasic-containing proteins are both necessary and sufficient for targeting to mitochondria, and are interchangeable, while the TA regions of TOM proteins lacking a dibasic motif are necessary, but not sufficient for localization, and cannot be functionally exchanged. We also present results from a comprehensive mutational analysis of the dibasic motif and surrounding sequences that not only greatly expands the functional definition and context-dependent properties of this targeting signal, but also led to the identification of other novel putative OMM-TA proteins. Collectively, these results provide important insight to the complexity of the targeting pathways involved in the biogenesis of OMM-TA proteins and help define a consensus targeting motif that is utilized by at least a subset of these proteins. PMID:25237314

  20. Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma

    NASA Astrophysics Data System (ADS)

    Neira, José L.; Bintz, Jennifer; Arruebo, María; Rizzuti, Bruno; Bonacci, Thomas; Vega, Sonia; Lanas, Angel; Velázquez-Campoy, Adrián; Iovanna, Juan L.; Abián, Olga

    2017-01-01

    Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the ‘fuzzy’ interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs.

  1. Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma

    PubMed Central

    Neira, José L.; Bintz, Jennifer; Arruebo, María; Rizzuti, Bruno; Bonacci, Thomas; Vega, Sonia; Lanas, Angel; Velázquez-Campoy, Adrián; Iovanna, Juan L.; Abián, Olga

    2017-01-01

    Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the ‘fuzzy’ interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs. PMID:28054562

  2. Functional autoantibodies targeting G protein-coupled receptors in rheumatic diseases.

    PubMed

    Cabral-Marques, Otavio; Riemekasten, Gabriela

    2017-11-01

    G protein-coupled receptors (GPCRs) comprise the largest and most diverse family of integral membrane proteins that participate in different physiological processes such as the regulation of the nervous and immune systems. Besides the endogenous ligands of GPCRs, functional autoantibodies are also able to bind GPCRs to trigger or block intracellular signalling pathways, resulting in agonistic or antagonistic effects, respectively. In this Review, the effects of functional GPCR-targeting autoantibodies on the pathogenesis of autoimmune diseases, including rheumatic diseases, are discussed. Autoantibodies targeting β1 and β2 adrenergic receptors, which are expressed by cardiac and airway smooth muscle cells, respectively, have an important role in the development of asthma and cardiovascular diseases. In addition, high levels of autoantibodies against the muscarinic acetylcholine receptor M3 as well as those targeting endothelin receptor type A and type 1 angiotensin II receptor have several implications in the pathogenesis of rheumatic diseases such as Sjögren syndrome and systemic sclerosis. Expanding the knowledge of the pathophysiological roles of autoantibodies against GPCRs will shed light on the biology of these receptors and open avenues for new therapeutic approaches.

  3. IFPTarget: A Customized Virtual Target Identification Method Based on Protein-Ligand Interaction Fingerprinting Analyses.

    PubMed

    Li, Guo-Bo; Yu, Zhu-Jun; Liu, Sha; Huang, Lu-Yi; Yang, Ling-Ling; Lohans, Christopher T; Yang, Sheng-Yong

    2017-07-24

    Small-molecule target identification is an important and challenging task for chemical biology and drug discovery. Structure-based virtual target identification has been widely used, which infers and prioritizes potential protein targets for the molecule of interest (MOI) principally via a scoring function. However, current "universal" scoring functions may not always accurately identify targets to which the MOI binds from the retrieved target database, in part due to a lack of consideration of the important binding features for an individual target. Here, we present IFPTarget, a customized virtual target identification method, which uses an interaction fingerprinting (IFP) method for target-specific interaction analyses and a comprehensive index (Cvalue) for target ranking. Evaluation results indicate that the IFP method enables substantially improved binding pose prediction, and Cvalue has an excellent performance in target ranking for the test set. When applied to screen against our established target library that contains 11,863 protein structures covering 2842 unique targets, IFPTarget could retrieve known targets within the top-ranked list and identified new potential targets for chemically diverse drugs. IFPTarget prediction led to the identification of the metallo-β-lactamase VIM-2 as a target for quercetin as validated by enzymatic inhibition assays. This study provides a new in silico target identification tool and will aid future efforts to develop new target-customized methods for target identification.

  4. Dual orexin receptor antagonist 12 inhibits expression of proteins in neurons and glia implicated in peripheral and central sensitization.

    PubMed

    Cady, R J; Denson, J E; Sullivan, L Q; Durham, P L

    2014-06-06

    Sensitization and activation of trigeminal nociceptors is implicated in prevalent and debilitating orofacial pain conditions including temporomandibular joint (TMJ) disorders. Orexins are excitatory neuropeptides that function to regulate many physiological processes and are reported to modulate nociception. To determine the role of orexins in an inflammatory model of trigeminal activation, the effects of a dual orexin receptor antagonist (DORA-12) on levels of proteins that promote peripheral and central sensitization and changes in nocifensive responses were investigated. In adult male Sprague-Dawley rats, mRNA for orexin receptor 1 (OX₁R) and receptor 2 (OX₂R) were detected in trigeminal ganglia and spinal trigeminal nucleus (STN). OX₁R immunoreactivity was localized primarily in neuronal cell bodies in the V3 region of the ganglion and in laminas I-II of the STN. Animals injected bilaterally with complete Freund's adjuvant (CFA) in the TMJ capsule exhibited increased expression of P-p38, P-ERK, and lba1 in trigeminal ganglia and P-ERK and lba1 in the STN at 2 days post injection. However, levels of each of these proteins in rats receiving daily oral DORA-12 were inhibited to near basal levels. Similarly, administration of DORA-12 on days 3 and 4 post CFA injection in the TMJ effectively inhibited the prolonged stimulated expression of protein kinase A, NFkB, and Iba1 in the STN on day 5 post injection. While injection of CFA mediated a nocifensive response to mechanical stimulation of the orofacial region at 2h and 3 and 5 days post injection, treatment with DORA-12 suppressed the nocifensive response on day 5. Somewhat surprisingly, nocifensive responses were again observed on day 10 post CFA stimulation in the absence of daily DORA-12 administration. Our results provide evidence that DORA-12 can inhibit CFA-induced stimulation of trigeminal sensory neurons by inhibiting expression of proteins associated with sensitization of peripheral and central

  5. Polymerase Acidic Protein-Basic Protein 1 (PA-PB1) Protein-Protein Interaction as a Target for Next-Generation Anti-influenza Therapeutics.

    PubMed

    Massari, Serena; Goracci, Laura; Desantis, Jenny; Tabarrini, Oriana

    2016-09-08

    The limited therapeutic options against the influenza virus (flu) and increasing challenges in drug resistance make the search for next-generation agents imperative. In this context, heterotrimeric viral PA/PB1/PB2 RNA-dependent RNA polymerase is an attractive target for a challenging but strategic protein-protein interaction (PPI) inhibition approach. Since 2012, the inhibition of the polymerase PA-PB1 subunit interface has become an active field of research following the publication of PA-PB1 crystal structures. In this Perspective, we briefly discuss the validity of flu polymerase as a drug target and its inhibition through a PPI inhibition strategy, including a comprehensive analysis of available PA-PB1 structures. An overview of all of the reported PA-PB1 complex formation inhibitors is provided, and approaches used for identification of the inhibitors, the hit-to-lead studies, and the emerged structure-activity relationship are described. In addition to highlighting the strengths and weaknesses of all of the PA-PB1 heterodimerization inhibitors, we analyze their hypothesized binding modes and alignment with a pharmacophore model that we have developed.

  6. Selective targeting of melanoma by PEG-masked protein-based multifunctional nanoparticles

    PubMed Central

    Vannucci, Luca; Falvo, Elisabetta; Fornara, Manuela; Di Micco, Patrizio; Benada, Oldrich; Krizan, Jiri; Svoboda, Jan; Hulikova-Capkova, Katarina; Morea, Veronica; Boffi, Alberto; Ceci, Pierpaolo

    2012-01-01

    Background Nanoparticle-based systems are promising for the development of imaging and therapeutic agents. The main advantage of nanoparticles over traditional systems lies in the possibility of loading multiple functionalities onto a single molecule, which are useful for therapeutic and/or diagnostic purposes. These functionalities include targeting moieties which are able to recognize receptors overexpressed by specific cells and tissues. However, targeted delivery of nanoparticles requires an accurate system design. We present here a rationally designed, genetically engineered, and chemically modified protein-based nanoplatform for cell/tissue-specific targeting. Methods Our nanoparticle constructs were based on the heavy chain of the human protein ferritin (HFt), a highly symmetrical assembly of 24 subunits enclosing a hollow cavity. HFt-based nanoparticles were produced using both genetic engineering and chemical functionalization methods to impart several functionalities, ie, the α-melanocyte-stimulating hormone peptide as a melanoma-targeting moiety, stabilizing and HFt-masking polyethylene glycol molecules, rhodamine fluorophores, and magnetic resonance imaging agents. The constructs produced were extensively characterized by a number of physicochemical techniques, and assayed for selective melanoma-targeting in vitro and in vivo. Results Our HFt-based nanoparticle constructs functionalized with the α-melanocyte-stimulating hormone peptide moiety and polyethylene glycol molecules were specifically taken up by melanoma cells but not by other cancer cell types in vitro. Moreover, experiments in melanoma-bearing mice indicate that these constructs have an excellent tumor-targeting profile and a long circulation time in vivo. Conclusion By masking human HFt with polyethylene glycol and targeting it with an α-melanocyte-stimulating hormone peptide, we developed an HFt-based melanoma-targeting nanoplatform for application in melanoma diagnosis and treatment

  7. Taste buds as peripheral chemosensory processors

    PubMed Central

    Roper, Stephen D.

    2012-01-01

    Taste buds are peripheral chemosensory organs situated in the oral cavity. Each taste bud consists of a community of 50–100 cells that interact synaptically during gustatory stimulation. At least three distinct cell types are found in mammalian taste buds – Type I cells, Receptor (Type II) cells, and Presynaptic (Type III) cells. Type I cells appear to be glial-like cells. Receptor cells express G protein-coupled taste receptors for sweet, bitter, or umami compounds. Presynaptic cells transduce acid stimuli (sour taste). Cells that sense salt (NaCl) taste have not yet been confidently identified in terms of these cell types. During gustatory stimulation, taste bud cells secrete synaptic, autocrine, and paracrine transmitters. These transmitters include ATP, acetylcholine (ACh), serotonin (5-HT), norepinephrine (NE), and GABA. Glutamate is an efferent transmitter that stimulates Presynaptic cells to release 5-HT. This chapter discusses these transmitters, which cells release them, the postsynaptic targets for the transmitters, and how cell–cell communication shapes taste bud signaling via these transmitters. PMID:23261954

  8. Taste buds as peripheral chemosensory processors.

    PubMed

    Roper, Stephen D

    2013-01-01

    Taste buds are peripheral chemosensory organs situated in the oral cavity. Each taste bud consists of a community of 50-100 cells that interact synaptically during gustatory stimulation. At least three distinct cell types are found in mammalian taste buds - Type I cells, Receptor (Type II) cells, and Presynaptic (Type III) cells. Type I cells appear to be glial-like cells. Receptor cells express G protein-coupled taste receptors for sweet, bitter, or umami compounds. Presynaptic cells transduce acid stimuli (sour taste). Cells that sense salt (NaCl) taste have not yet been confidently identified in terms of these cell types. During gustatory stimulation, taste bud cells secrete synaptic, autocrine, and paracrine transmitters. These transmitters include ATP, acetylcholine (ACh), serotonin (5-HT), norepinephrine (NE), and GABA. Glutamate is an efferent transmitter that stimulates Presynaptic cells to release 5-HT. This chapter discusses these transmitters, which cells release them, the postsynaptic targets for the transmitters, and how cell-cell communication shapes taste bud signaling via these transmitters. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. HIV-1 Proteins, Tat and gp120, Target the Developing Dopamine System

    PubMed Central

    Fitting, Sylvia; Booze, Rosemarie M.; Mactutus, Charles F.

    2015-01-01

    In 2014, 3.2 million children (< 15 years of age) were estimated to be living with HIV and AIDS worldwide, with the 240,000 newly infected children in the past year, i.e., another child infected approximately every two minutes [1]. The primary mode of HIV infection is through mother-to-child transmission (MTCT), occurring either in utero, intrapartum, or during breastfeeding. The effects of HIV-1 on the central nervous system (CNS) are putatively accepted to be mediated, in part, via viral proteins, such as Tat and gp120. The current review focuses on the targets of HIV-1 proteins during the development of the dopamine (DA) system, which appears to be specifically susceptible in HIV-1-infected children. Collectively, the data suggest that the DA system is a clinically relevant target in chronic HIV-1 infection, is one of the major targets in pediatric HIV-1 CNS infection, and may be specifically susceptible during development. The present review discusses the development of the DA system, follows the possible targets of the HIV-1 proteins during the development of the DA system, and suggests potential therapeutic approaches. By coupling our growing understanding of the development of the CNS with the pronounced age-related differences in disease progression, new light may be shed on the neurological and neurocognitive deficits that follow HIV-1 infection. PMID:25613135

  10. AlphaB-crystallin regulates remyelination after peripheral nerve injury

    PubMed Central

    Lim, Erin-Mai F.; Nakanishi, Stan T.; Hoghooghi, Vahid; Eaton, Shane E. A.; Palmer, Alexandra L.; Frederick, Ariana; Stratton, Jo A.; Stykel, Morgan G.; Zochodne, Douglas W.; Biernaskie, Jeffrey; Ousman, Shalina S.

    2017-01-01

    AlphaB-crystallin (αBC) is a small heat shock protein that is constitutively expressed by peripheral nervous system (PNS) axons and Schwann cells. To determine what role this crystallin plays after peripheral nerve damage, we found that loss of αBC impaired remyelination, which correlated with a reduced presence of myelinating Schwann cells and increased numbers of nonmyelinating Schwann cells. The heat shock protein also seems to regulate the cross-talk between Schwann cells and axons, because expected changes in neuregulin levels and ErbB2 receptor expression after PNS injury were disrupted in the absence of αBC. Such dysregulations led to defects in conduction velocity and motor and sensory functions that could be rescued with therapeutic application of the heat shock protein in vivo. Altogether, these findings show that αBC plays an important role in regulating Wallerian degeneration and remyelination after PNS injury. PMID:28137843

  11. Targeted therapies in cancer - challenges and chances offered by newly developed techniques for protein analysis in clinical tissues

    PubMed Central

    Malinowsky, K; Wolff, C; Gündisch, S; Berg, D; Becker, KF

    2011-01-01

    In recent years, new anticancer therapies have accompanied the classical approaches of surgery and radio- and chemotherapy. These new forms of treatment aim to inhibit specific molecular targets namely altered or deregulated proteins, which offer the possibility of individualized therapies. The specificity and efficiency of these new approaches, however, bring about a number of challenges. First of all, it is essential to specifically identify and quantify protein targets in tumor tissues for the reasonable use of such targeted therapies. Additionally, it has become even more obvious in recent years that the presence of a target protein is not always sufficient to predict the outcome of targeted therapies. The deregulation of downstream signaling molecules might also play an important role in the success of such therapeutic approaches. For these reasons, the analysis of tumor-specific protein expression profiles prior to therapy has been suggested as the most effective way to predict possible therapeutic results. To further elucidate signaling networks underlying cancer development and to identify new targets, it is necessary to implement tools that allow the rapid, precise, inexpensive and simultaneous analysis of many network components while requiring only a small amount of clinical material. Reverse phase protein microarray (RPPA) is a promising technology that meets these requirements while enabling the quantitative measurement of proteins. Together with recently developed protocols for the extraction of proteins from formalin-fixed, paraffin-embedded (FFPE) tissues, RPPA may provide the means to quantify therapeutic targets and diagnostic markers in the near future and reliably screen for new protein targets. With the possibility to quantitatively analyze DNA, RNA and protein from a single FFPE tissue sample, the methods are available for integrated patient profiling at all levels of gene expression, thus allowing optimal patient stratification for

  12. Foveal Processing Under Concurrent Peripheral Load in Profoundly Deaf Adults.

    PubMed

    Dye, Matthew W G

    2016-04-01

    Development of the visual system typically proceeds in concert with the development of audition. One result is that the visual system of profoundly deaf individuals differs from that of those with typical auditory systems. While past research has suggested deaf people have enhanced attention in the visual periphery, it is still unclear whether or not this enhancement entails deficits in central vision. Profoundly deaf and typically hearing adults were administered a variant of the useful field of view task that independently assessed performance on concurrent central and peripheral tasks. Identification of a foveated target was impaired by a concurrent selective peripheral attention task, more so in profoundly deaf adults than in the typically hearing. Previous findings of enhanced performance on the peripheral task were not replicated. These data are discussed in terms of flexible allocation of spatial attention targeted towards perceived task demands, and support a modified "division of labor" hypothesis whereby attentional resources co-opted to process peripheral space result in reduced resources in the central visual field. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  13. Peripheral neuropathy

    MedlinePlus

    ... peripheral; Neuritis - peripheral; Nerve disease; Polyneuropathy; Chronic pain - peripheral neuropathy ... Philadelphia, PA: Elsevier; 2016:chap 107. Shy ME. Peripheral neuropathies. In: Goldman L, Schafer AI, eds. Goldman's Cecil ...

  14. Chemotherapy-induced peripheral neuropathy: an update on the current understanding.

    PubMed

    Addington, James; Freimer, Miriam

    2016-01-01

    Chemotherapy-induced peripheral neuropathy is a common side effect of selected chemotherapeutic agents. Previous work has suggested that patients often under report the symptoms of chemotherapy-induced peripheral neuropathy and physicians fail to recognize the presence of such symptoms in a timely fashion. The precise pathophysiology that underlies chemotherapy-induced peripheral neuropathy, in both the acute and the chronic phase, remains complex and appears to be medication specific. Recent work has begun to demonstrate and further clarify potential pathophysiological processes that predispose and, ultimately, lead to the development of chemotherapy-induced peripheral neuropathy. There is increasing evidence that the pathway to neuropathy varies with each agent. With a clearer understanding of how these agents affect the peripheral nervous system, more targeted treatments can be developed in order to optimize treatment and prevent long-term side effects.

  15. Proteomic characterization of Withaferin A-targeted protein networks for the treatment of monoclonal myeloma gammopathies.

    PubMed

    Dom, Martin; Offner, Fritz; Vanden Berghe, Wim; Van Ostade, Xaveer

    2018-05-15

    Withaferin A (WA), a natural steroid lactone from the plant Withania somnifera, is often studied because of its antitumor properties. Although many in vitro and in vivo studies have been performed, the identification of Withaferin A protein targets and its mechanism of antitumor action remain incomplete. We used quantitative chemoproteomics and differential protein expression analysis to characterize the WA antitumor effects on a multiple myeloma cell model. Identified relevant targets were further validated by Ingenuity Pathway Analysis and Western blot and indicate that WA targets protein networks that are specific for monoclonal gammopathy of undetermined significance (MGUS) and other closely related disorders, such as multiple myeloma (MM) and Waldenström macroglobulinemia (WM). By blocking the PSMB10 proteasome subunit, downregulation of ANXA4, potential association with HDAC6 and upregulation of HMOX1, WA puts a massive blockage on both proteotoxic and oxidative stress responses pathways, leaving cancer cells defenseless against WA induced stresses. These results indicate that WA mediated apoptosis is preceded by simultaneous targeting of cellular stress response pathways like proteasome degradation, autophagy and unfolded protein stress response and thus suggests that WA can be used as an effective treatment for MGUS and other closely related disorders. Multifunctional antitumor compounds are of great potential since they reduce the risk of multidrug resistance in chemotherapy. Unfortunately, characterization of all protein targets of a multifunctional compound is lacking. Therefore, we optimized an SILAC quantitative chemoproteomics workflow to identify the potential protein targets of Withaferin A (WA), a natural multifunctional compound with promising antitumor properties. To further understand the antitumor mechanisms of WA, we performed a differential protein expression analysis and combined the altered expression data with chemoproteome WA target data

  16. Protein Drug Targets of Lavandula angustifolia on treatment of Rat Alzheimer's Disease

    PubMed Central

    Zali, Hakimeh; Zamanian-Azodi, Mona; Rezaei Tavirani, Mostafa; Akbar-zadeh Baghban, Alireza

    2015-01-01

    Different treatment strategies of Alzheimer's disease (AD) are being studied for treating or slowing the progression of AD. Many pharmaceutically important regulation systems operate through proteins as drug targets. Here, we investigate the drug target proteins in beta-amyloid (Aβ) injected rat hippocampus treated with Lavandula angustifolia (LA) by proteomics techniques. The reported study showed that lavender extract (LE) improves the spatial performance in AD animal model by diminishing Aβ production in histopathology of hippocampus, so in this study neuroprotective proteins expressed in Aβ injected rats treated with LE were scrutinized. Rats were divided into three groups including normal, Aβ injected, and Aβ injected that was treated with LE. Protein expression profiles of hippocampus tissue were determined by two-dimensional electrophoresis (2DE) method and dysregulated proteins such as Snca, NF-L, Hspa5, Prdx2, Apoa1, and Atp5a1were identified by MALDI-TOF/TOF. KEGG pathway and gene ontology (GO) categories were used by searching DAVID Bioinformatics Resources. All detected protein spots were used to determine predictedinteractions with other proteins in STRING online database. Different isoforms of important protein, Snca that exhibited neuroprotective effects by anti-apoptotic properties were expressed. NF-L involved in the maintenance of neuronal caliber. Hspa5 likewise Prdx2 displays as anti-apoptotic protein that Prdx2 also involved in the neurotrophic effects. Apoa1 has anti-inflammatory activity and Atp5a1, produces ATP from ADP. To sum up, these proteins as potential drug targets were expressed in hippocampus in response to effective components in LA may have therapeutic properties for the treatment of AD and other neurodegenerative diseases. PMID:25561935

  17. Interaction of cellular proteins with BCL-xL targeted to cytoplasmic inclusion bodies in adenovirus infected cells.

    PubMed

    Subramanian, T; Vijayalingam, S; Kuppuswamy, M; Chinnadurai, G

    2015-09-01

    Adenovirus-mediated apoptosis was suppressed when cellular anti-apoptosis proteins (BCL-2 and BCL-xL) were substituted for the viral E1B-19K. For unbiased proteomic analysis of proteins targeted by BCL-xL in adenovirus-infected cells and to visualize the interactions with target proteins, BCL-xL was targeted to cytosolic inclusion bodies utilizing the orthoreovirus µNS protein sequences. The chimeric protein was localized in non-canonical cytosolic factory-like sites and promoted survival of virus-infected cells. The BCL-xL-associated proteins were isolated from the cytosolic inclusion bodies in adenovirus-infected cells and analyzed by LC-MS. These proteins included BAX, BAK, BID, BIK and BIM as well as mitochondrial proteins such as prohibitin 2, ATP synthase and DNA-PKcs. Our studies suggested that in addition to the interaction with various pro-apoptotic proteins, the association with certain mitochondrial proteins such as DNA-PKcs and prohibitins might augment the survival function of BCL-xL in virus infected cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Kininogen in autoimmune uveitis: decrease in peripheral blood stream versus increase in target tissue.

    PubMed

    Zipplies, Johanna K; Hauck, Stefanie M; Schoeffmann, Stephanie; Amann, Barbara; van der Meijden, Christiaan H; Stangassinger, Manfred; Ueffing, Marius; Deeg, Cornelia A

    2010-01-01

    Equine recurrent uveitis (ERU) is an incurable disease affecting the inner eye that leads to blindness, through activated T cells that pass the blood-retinal barrier and destroy the retina. Serum markers are a desirable choice for monitoring development of disease, as serum is easy accessible and the markers could serve to predict the beginning of disease or an imminent relapse. In this study, serum proteomes (depleted of high-abundance serum proteins) of horses with ERU and healthy controls were compared with the 2-D DIGE (two-dimensional gel electrophoresis) technique to identify differentially expressed proteins. The expression pattern of a candidate protein in retina and vitreous was validated by Western blots and immunohistochemistry. Ten differentially expressed proteins could be identified by mass spectrometry (MALDI-TOF/TOF). Five proteins--IgM, IgG4 hc, serotransferrin, alpha-2HS-glycoprotein, and complement factor B--were upregulated in the uveitic state, whereas the five proteins albumin, apolipoprotein A-IV and H, IgG5 hc, and high-molecular-weight kininogen (HK) showed a significantly lower expression in sera of uveitis cases. Of interest, kininogen was significantly upregulated in the target tissues vitreous and retina. HK is a plasma protein with multiple physiological functions, with an important role in inflammation and promoting neovascularization. Most interesting is the as of yet unaddressed association of HK with uveitis. Immunohistochemistry showed coexpression of kininogen and VEGF in inflamed eyes. Since neovascularization plays a major role in the pathogenesis of uveitis, the identification of a proangiogenic factor in the retina presents an important finding and may contribute to elucidating the pathogenesis of uveitis.

  19. Engineering an effective Mn-binding MRI reporter protein by subcellular targeting

    PubMed Central

    Bartelle, Benjamin B.; Mana, Miyeko D.; Suero-Abreu, Giselle A.; Rodriguez, Joe J.; Turnbull, Daniel H.

    2014-01-01

    Purpose Manganese (Mn) is an effective contrast agent and biologically active metal, which has been widely utilized for Mn-enhanced MRI (MEMRI). The purpose of this study was to develop and test a Mn binding protein for use as an genetic reporter for MEMRI. Methods The bacterial Mn-binding protein, MntR was identified as a candidate reporter protein. MntR was engineered for expression in mammalian cells, and targeted to different subcellular organelles, including the Golgi Apparatus where cellular Mn is enriched. Transfected HEK293 cells and B16 melanoma cells were tested in vitro and in vivo, using immunocytochemistry and MR imaging and relaxometry. Results Subcellular targeting of MntR to the cytosol, endoplasmic reticulum and Golgi apparatus was verified with immunocytochemistry. After targeting to the Golgi, MntR expression produced robust R1 changes and T1 contrast in cells, in vitro and in vivo. Co-expression with the divalent metal transporter DMT1, a previously described Mn-based reporter, further enhanced contrast in B16 cells in culture, but in the in vivo B16 tumor model tested was not significantly better than MntR alone. Conclusion This second-generation reporter system both expands the capabilities of genetically-encoded reporters for imaging with MEMRI and provides important insights into the mechanisms of Mn biology which create endogenous MEMRI contrast. PMID:25522343

  20. Dynamin-Related Protein 1 as a therapeutic target in cardiac arrest

    PubMed Central

    Sharp, Willard W.

    2015-01-01

    Despite improvements in cardiopulmonary resuscitation (CPR) quality, defibrillation technologies, and implementation of therapeutic hypothermia, less than 10% of out-of-hospital cardiac arrest (OHCA) victims survive to hospital discharge. New resuscitation therapies have been slow to develop, in part, because the pathophysiologic mechanisms critical for resuscitation are not understood. During cardiac arrest, systemic cessation of blood flow results in whole body ischemia. CPR, and the restoration of spontaneous circulation (ROSC), both result in immediate reperfusion injury of the heart that is characterized by severe contractile dysfunction. Unlike diseases of localized ischemia/reperfusion (IR) injury (myocardial infarction and stroke), global IR injury of organs results in profound organ dysfunction with far shorter ischemic times. The two most commonly injured organs following cardiac arrest resuscitation, the heart and brain, are critically dependent on mitochondrial function. New insights into mitochondrial dynamics and the role of the mitochondrial fission protein Dynamin-related protein 1 (Drp1) in apoptosis have made targeting these mechanisms attractive for IR therapy. In animal models, inhibiting Drp1 following IR injury or cardiac arrest confers protection to both the heart and brain. In this review, the relationship of the major mitochondrial fission protein Drp1 to ischemic changes in the heart and its targeting as a new therapeutic target following cardiac arrest are discussed. PMID:25659608

  1. Activated GTPase movement on an RNA scaffold drives cotranslational protein targeting

    PubMed Central

    Shen, Kuang; Arslan, Sinan; Akopian, David; Ha, Taekjip; Shan, Shu-ou

    2012-01-01

    Roughly one third of the proteome is initially destined for the eukaryotic endoplasmic reticulum or the bacterial plasma membrane1. The proper localization of these proteins is mediated by a universally conserved protein targeting machinery, the signal recognition particle (SRP), which recognizes ribosomes carrying signal sequences2–4 and, via interactions with the SRP receptor5,6, delivers them to the protein translocation machinery on the target membrane7. The SRP is an ancient ribonucleoprotein particle containing an essential, elongated SRP RNA whose precise functions have remained elusive. Here, we used single molecule fluorescence microscopy to demonstrate that the SRP-receptor GTPase complex, after initial assembly at the tetraloop end of SRP RNA, travels over 100 Å to the distal end of this RNA where rapid GTP hydrolysis occurs. This movement is negatively regulated by the translating ribosome and, at a later stage, positively regulated by the SecYEG translocon, providing an attractive mechanism to ensure the productive exchange of the targeting and translocation machineries at the ribosome exit site with exquisite spatial and temporal accuracy. Our results show that large RNAs can act as molecular scaffolds that enable the facile exchange of distinct factors and precise timing of molecular events in a complex cellular process; this concept may be extended to similar phenomena in other ribonucleoprotein complexes. PMID:23235881

  2. Role of Schwann cells in the regeneration of penile and peripheral nerves

    PubMed Central

    Wang, Lin; Sanford, Melissa T; Xin, Zhongcheng; Lin, Guiting; Lue, Tom F

    2015-01-01

    Schwann cells (SCs) are the principal glia of the peripheral nervous system. The end point of SC development is the formation of myelinating and nonmyelinating cells which ensheath large and small diameter axons, respectively. They play an important role in axon regeneration after injury, including cavernous nerve injury that leads to erectile dysfunction (ED). Despite improvement in radical prostatectomy surgical techniques, many patients still suffer from ED postoperatively as surgical trauma causes traction injuries and local inflammatory changes in the neuronal microenvironment of the autonomic fibers innervating the penis resulting in pathophysiological alterations in the end organ. The aim of this review is to summarize contemporary evidence regarding: (1) the origin and development of SCs in the peripheral and penile nerve system; (2) Wallerian degeneration and SC plastic change following peripheral and penile nerve injury; (3) how SCs promote peripheral and penile nerve regeneration by secreting neurotrophic factors; (4) and strategies targeting SCs to accelerate peripheral nerve regeneration. We searched PubMed for articles related to these topics in both animal models and human research and found numerous studies suggesting that SCs could be a novel target for treatment of nerve injury-induced ED. PMID:25999359

  3. Unlocking the secrets to protein–protein interface drug targets using structural mass spectrometry techniques

    PubMed Central

    Dailing, Angela; Luchini, Alessandra; Liotta, Lance

    2016-01-01

    Protein–protein interactions (PPIs) drive all biologic systems at the subcellular and extracellular level. Changes in the specificity and affinity of these interactions can lead to cellular malfunctions and disease. Consequently, the binding interfaces between interacting protein partners are important drug targets for the next generation of therapies that block such interactions. Unfortunately, protein–protein contact points have proven to be very difficult pharmacological targets because they are hidden within complex 3D interfaces. For the vast majority of characterized binary PPIs, the specific amino acid sequence of their close contact regions remains unknown. There has been an important need for an experimental technology that can rapidly reveal the functionally important contact points of native protein complexes in solution. In this review, experimental techniques employing mass spectrometry to explore protein interaction binding sites are discussed. Hydrogen–deuterium exchange, hydroxyl radical footprinting, crosslinking and the newest technology protein painting, are compared and contrasted. PMID:26400464

  4. Quantitative imaging of protein targets in the human brain with PET

    NASA Astrophysics Data System (ADS)

    Gunn, Roger N.; Slifstein, Mark; Searle, Graham E.; Price, Julie C.

    2015-11-01

    PET imaging of proteins in the human brain with high affinity radiolabelled molecules has a history stretching back over 30 years. During this period the portfolio of protein targets that can be imaged has increased significantly through successes in radioligand discovery and development. This portfolio now spans six major categories of proteins; G-protein coupled receptors, membrane transporters, ligand gated ion channels, enzymes, misfolded proteins and tryptophan-rich sensory proteins. In parallel to these achievements in radiochemical sciences there have also been significant advances in the quantitative analysis and interpretation of the imaging data including the development of methods for image registration, image segmentation, tracer compartmental modeling, reference tissue kinetic analysis and partial volume correction. In this review, we analyze the activity of the field around each of the protein targets in order to give a perspective on the historical focus and the possible future trajectory of the field. The important neurobiology and pharmacology is introduced for each of the six protein classes and we present established radioligands for each that have successfully transitioned to quantitative imaging in humans. We present a standard quantitative analysis workflow for these radioligands which takes the dynamic PET data, associated blood and anatomical MRI data as the inputs to a series of image processing and bio-mathematical modeling steps before outputting the outcome measure of interest on either a regional or parametric image basis. The quantitative outcome measures are then used in a range of different imaging studies including tracer discovery and development studies, cross sectional studies, classification studies, intervention studies and longitudinal studies. Finally we consider some of the confounds, challenges and subtleties that arise in practice when trying to quantify and interpret PET neuroimaging data including motion artifacts

  5. Diffusion and retention are major determinants of protein targeting to the inner nuclear membrane

    PubMed Central

    Ungricht, Rosemarie; Klann, Michael; Horvath, Peter

    2015-01-01

    Newly synthesized membrane proteins are constantly sorted from the endoplasmic reticulum (ER) to various membranous compartments. How proteins specifically enrich at the inner nuclear membrane (INM) is not well understood. We have established a visual in vitro assay to measure kinetics and investigate requirements of protein targeting to the INM. Using human LBR, SUN2, and LAP2β as model substrates, we show that INM targeting is energy-dependent but distinct from import of soluble cargo. Accumulation of proteins at the INM relies on both a highly interconnected ER network, which is affected by energy depletion, and an efficient immobilization step at the INM. Nucleoporin depletions suggest that translocation through nuclear pore complexes (NPCs) is rate-limiting and restricted by the central NPC scaffold. Our experimental data combined with mathematical modeling support a diffusion-retention–based mechanism of INM targeting. We experimentally confirmed the sufficiency of diffusion and retention using an artificial reporter lacking natural sorting signals that recapitulates the energy dependence of the process in vivo. PMID:26056139

  6. Imaging cellularity in benign and malignant peripheral nerve sheath tumors: Utility of the "target sign" by diffusion weighted imaging.

    PubMed

    Ahlawat, Shivani; Fayad, Laura M

    2018-05-01

    To determine the utility of "target sign" on diffusion weighted imaging (DWI) and apparent diffusion coefficient (ADC) mapping for peripheral nerve sheath tumor (PNST) characterization. This IRB-approved, HIPAA-compliant study retrospectively reviewed the MR imaging (comprised of T2- FS, DWI (b-values 50, 400, 800 s/mm 2 and ADC mapping), and static contrast-enhanced (CE) T1-W imaging) of 42 patients (mean age: 40 years (range 8-68 years), 48% (20/42) females) with 15 malignant PNSTs (MPNSTs) and 33 benign PNSTs (BPNSTs). MPNSTs were histologically confirmed while BPNSTs were histologically-proven or with stable clinical and imaging appearance for at least 12 months. Two radiologists assessed imaging characteristics (size, signal intensity, heterogeneity, perilesional edema or enhancement) and the presence or absence of "target sign," on each sequence. A "target sign" was defined as a biphasic pattern of peripheral hyperintensity and homogeneous central hypointensity. Descriptive statistics are reported. Cohen's κ statistic or interclass correlation coefficient (ICC) were used to evaluate interobserver agreement between two observers. Univariate and multiple logistic regression analysis were performed to identify MRI features with predictive values. MPNSTs were larger than BPNSTs (6.3 ± 2.5 cm vs 3.5 ± -2.1 cm, p = 0.0002), had perilesional edema (87%(13/15) vs 18%(6/33), p < 0.0001), heterogeneous enhancement (71%(10/14) vs 13%(4/31), p = 0.0001) and perilesional enhancement (79%(11/14) vs 18%(6/31), p = 0.0001), respectively. The "target sign" was present in: 24%(8/33) BPNSTs vs 0/15 MPNST on T2-FS (p = 0.26); 39%(13/33) BPNSTs vs 20%(3/15) MPNST on DWI using b-value = 50 s/mm 2 (p = 0.5); 55%(18/33) BPNSTs vs 6%(1/15) MPNST on DWI using b-value = 400 s/mm 2 (p = 0.002); 48%(16/33) BPNSTs vs 6%(1/15) MPNST on DWI using b-value = 800 s/mm 2 (p = 0.005) and 64%(21/33) benign vs 0/15 MPNST on ADC

  7. Reaching a Moveable Visual Target: Dissociations in Brain Tumour Patients

    ERIC Educational Resources Information Center

    Buiatti, Tania; Skrap, Miran; Shallice, Tim

    2013-01-01

    Damage to the posterior parietal cortex (PPC) can lead to Optic Ataxia (OA), in which patients misreach to peripheral targets. Recent research suggested that the PPC might be involved not only in simple reaching tasks toward peripheral targets, but also in changing the hand movement trajectory in real time if the target moves. The present study…

  8. Mapping a nucleolar targeting sequence of an RNA binding nucleolar protein, Nop25

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fujiwara, Takashi; Suzuki, Shunji; Kanno, Motoko

    2006-06-10

    Nop25 is a putative RNA binding nucleolar protein associated with rRNA transcription. The present study was undertaken to determine the mechanism of Nop25 localization in the nucleolus. Deletion experiments of Nop25 amino acid sequence showed Nop25 to contain a nuclear targeting sequence in the N-terminal and a nucleolar targeting sequence in the C-terminal. By expressing derivative peptides from the C-terminal as GFP-fusion proteins in the cells, a lysine and arginine residue-enriched peptide (KRKHPRRAQDSTKKPPSATRTSKTQRRRR) allowed a GFP-fusion protein to be transported and fully retained in the nucleolus. When the peptide was fused with cMyc epitope and expressed in the cells, amore » cMyc epitope was then detected in the nucleolus. Nop25 did not localize in the nucleolus by deletion of the peptide from Nop25. Furthermore, deletion of a subdomain (KRKHPRRAQ) in the peptide or amino acid substitution of lysine and arginine residues in the subdomain resulted in the loss of Nop25 nucleolar localization. These results suggest that the lysine and arginine residue-enriched peptide is the most prominent nucleolar targeting sequence of Nop25 and that the long stretch of basic residues might play an important role in the nucleolar localization of Nop25. Although Nop25 contained putative SUMOylation, phosphorylation and glycosylation sites, the amino acid substitution in these sites had no effect on the nucleolar localization, thus suggesting that these post-translational modifications did not contribute to the localization of Nop25 in the nucleolus. The treatment of the cells, which expressed a GFP-fusion protein with a nucleolar targeting sequence of Nop25, with RNase A resulted in a complete dislocation of the protein from the nucleolus. These data suggested that the nucleolar targeting sequence might therefore play an important role in the binding of Nop25 to RNA molecules and that the RNA binding of Nop25 might be essential for the nucleolar localization of Nop25.« less

  9. Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shi, Tujin; Sun, Xuefei; Gao, Yuqian

    2013-07-05

    We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50-100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion and the multiplexing potential of this technique. Limits of quantification (LOQs) at low ng/mL levels with a medianmore » CV of ~12% were achieved for proteins spiked into human female serum using as little as 2 µL serum. PRISM-SRM provided up to ~1000-fold improvement in the LOQ when compared to conventional SRM measurements. Multiplexing capability of PRISM-SRM was also evaluated by two sets of serum samples with 6 and 21 target peptides spiked at the low attomole/µL levels. The results from SRM measurements for pooled or post-concatenated samples were comparable to those obtained from individual peptide fractions in terms of signal-to-noise ratios and SRM peak area ratios of light to heavy peptides. PRISM-SRM was applied to measure several ng/mL-level endogenous plasma proteins, including prostate-specific antigen, in clinical patient sera where correlation coefficients > 0.99 were observed between the results from PRISM-SRM and ELISA assays. Our results demonstrate that PRISM-SRM can be successfully used for quantification of low-abundance endogenous proteins in highly complex samples. Moderate throughput (50 samples/week) can be achieved by applying the post-concatenation or fraction multiplexing strategies. We anticipate broad applications for targeted PRISM

  10. Emerging Paradigm of Intracellular Targeting of G Protein-Coupled Receptors.

    PubMed

    Chaturvedi, Madhu; Schilling, Justin; Beautrait, Alexandre; Bouvier, Michel; Benovic, Jeffrey L; Shukla, Arun K

    2018-05-04

    G protein-coupled receptors (GPCRs) recognize a diverse array of extracellular stimuli, and they mediate a broad repertoire of signaling events involved in human physiology. Although the major effort on targeting GPCRs has typically been focused on their extracellular surface, a series of recent developments now unfold the possibility of targeting them from the intracellular side as well. Allosteric modulators binding to the cytoplasmic surface of GPCRs have now been described, and their structural mechanisms are elucidated by high-resolution crystal structures. Furthermore, pepducins, aptamers, and intrabodies targeting the intracellular face of GPCRs have also been successfully utilized to modulate receptor signaling. Moreover, small molecule compounds, aptamers, and synthetic intrabodies targeting β-arrestins have also been discovered to modulate GPCR endocytosis and signaling. Here, we discuss the emerging paradigm of intracellular targeting of GPCRs, and outline the current challenges, potential opportunities, and future outlook in this particular area of GPCR biology. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. People with diabetic peripheral neuropathy display a decreased stepping accuracy during walking: potential implications for risk of tripping.

    PubMed

    Handsaker, J C; Brown, S J; Bowling, F L; Marple-Horvat, D E; Boulton, A J M; Reeves, N D

    2016-05-01

    To examine the stepping accuracy of people with diabetes and diabetic peripheral neuropathy. Fourteen patients with diabetic peripheral neuropathy (DPN), 12 patients with diabetes but no neuropathy (D) and 10 healthy non-diabetic control participants (C). Accuracy of stepping was measured whilst the participants walked along a walkway consisting of 18 stepping targets. Preliminary data on visual gaze characteristics were also captured in a subset of participants (diabetic peripheral neuropathy group: n = 4; diabetes-alone group: n = 4; and control group: n = 4) during the same task. Patients in the diabetic peripheral neuropathy group, and patients in the diabetes-alone group were significantly less accurate at stepping on targets than were control subjects (P < 0.05). Preliminary visual gaze analysis identified that patients diabetic peripheral neuropathy were slower to look between targets, resulting in less time being spent looking at a target before foot-target contact. Impaired motor control is theorized to be a major factor underlying the changes in stepping accuracy, and potentially altered visual gaze behaviour may also play a role. Reduced stepping accuracy may indicate a decreased ability to control the placement of the lower limbs, leading to patients with neuropathy potentially being less able to avoid observed obstacles during walking. © 2015 Diabetes UK.

  12. Macrophage Depletion Ameliorates Peripheral Neuropathy in Aging Mice.

    PubMed

    Yuan, Xidi; Klein, Dennis; Kerscher, Susanne; West, Brian L; Weis, Joachim; Katona, Istvan; Martini, Rudolf

    2018-05-09

    Aging is known as a major risk factor for the structure and function of the nervous system. There is urgent need to overcome such deleterious effects of age-related neurodegeneration. Here we show that peripheral nerves of 24-month-old aging C57BL/6 mice of either sex show similar pathological alterations as nerves from aging human individuals, whereas 12-month-old adult mice lack such alterations. Specifically, nerve fibers showed demyelination, remyelination and axonal lesion. Moreover, in the aging mice, neuromuscular junctions showed features typical for dying-back neuropathies, as revealed by a decline of presynaptic markers, associated with α-bungarotoxin-positive postsynapses. In line with these observations were reduced muscle strengths. These alterations were accompanied by elevated numbers of endoneurial macrophages, partially comprising the features of phagocytosing macrophages. Comparable profiles of macrophages could be identified in peripheral nerve biopsies of aging persons. To determine the pathological impact of macrophages in aging mice, we selectively targeted the cells by applying an orally administered CSF-1R specific kinase (c-FMS) inhibitor. The 6-month-lasting treatment started before development of degenerative changes at 18 months and reduced macrophage numbers in mice by ∼70%, without side effects. Strikingly, nerve structure was ameliorated and muscle strength preserved. We show, for the first time, that age-related degenerative changes in peripheral nerves are driven by macrophages. These findings may pave the way for treating degeneration in the aging peripheral nervous system by targeting macrophages, leading to reduced weakness, improved mobility, and eventually increased quality of life in the elderly. SIGNIFICANCE STATEMENT Aging is a major risk factor for the structure and function of the nervous system. Here we show that peripheral nerves of 24-month-old aging mice show similar degenerative alterations as nerves from aging

  13. A Pathogenic Nematode Targets Recognition Proteins to Avoid Insect Defenses

    PubMed Central

    Toubarro, Duarte; Avila, Mónica Martinez; Montiel, Rafael; Simões, Nelson

    2013-01-01

    Steinernema carpocapsae is a nematode pathogenic in a wide variety of insect species. The great pathogenicity of this nematode has been ascribed to its ability to overcome the host immune response; however, little is known about the mechanisms involved in this process. The analysis of an expressed sequence tags (EST) library in the nematode during the infective phase was performed and a highly abundant contig homologous to serine protease inhibitors was identified. In this work, we show that this contig is part of a 641-bp cDNA that encodes a BPTI-Kunitz family inhibitor (Sc-KU-4), which is up-regulated in the parasite during invasion and installation. Recombinant Sc-KU-4 protein was produced in Escherichia coli and shown to inhibit chymotrypsin and elastase activities in a dose-dependent manner by a competitive mechanism with Ki values of 1.8 nM and 2.6 nM, respectively. Sc-KU-4 also inhibited trypsin and thrombin activities to a lesser extent. Studies of the mode of action of Sc-KU-4 and its effects on insect defenses suggest that although Sc-KU-4 did not inhibit the activation of hemocytes or the formation of clotting fibers, it did inhibit hemocyte aggregation and the entrapment of foreign particles by fibers. Moreover, Sc-KU-4 avoided encapsulation and the deposition of clotting materials, which usually occurs in response to foreign particles. We show by protein-protein interaction that Sc-KU-4 targets recognition proteins of insect immune system such as masquerade-like and serine protease-like homologs. The interaction of Sc-KU-4 with these proteins explains the ability of the nematode to overcome host reactions and its large pathogenic spectrum, once these immune proteins are well conserved in insects. The discovery of this inhibitor targeting insect recognition proteins opens new avenues for the development of S . carpocapsae as a biological control agent and provides a new tool to study host-pathogen interactions. PMID:24098715

  14. Brainstorming: weighted voting prediction of inhibitors for protein targets.

    PubMed

    Plewczynski, Dariusz

    2011-09-01

    The "Brainstorming" approach presented in this paper is a weighted voting method that can improve the quality of predictions generated by several machine learning (ML) methods. First, an ensemble of heterogeneous ML algorithms is trained on available experimental data, then all solutions are gathered and a consensus is built between them. The final prediction is performed using a voting procedure, whereby the vote of each method is weighted according to a quality coefficient calculated using multivariable linear regression (MLR). The MLR optimization procedure is very fast, therefore no additional computational cost is introduced by using this jury approach. Here, brainstorming is applied to selecting actives from large collections of compounds relating to five diverse biological targets of medicinal interest, namely HIV-reverse transcriptase, cyclooxygenase-2, dihydrofolate reductase, estrogen receptor, and thrombin. The MDL Drug Data Report (MDDR) database was used for selecting known inhibitors for these protein targets, and experimental data was then used to train a set of machine learning methods. The benchmark dataset (available at http://bio.icm.edu.pl/∼darman/chemoinfo/benchmark.tar.gz ) can be used for further testing of various clustering and machine learning methods when predicting the biological activity of compounds. Depending on the protein target, the overall recall value is raised by at least 20% in comparison to any single machine learning method (including ensemble methods like random forest) and unweighted simple majority voting procedures.

  15. Comparative proteomic analyses of macular and peripheral retina of cynomolgus monkeys (Macaca fascicularis).

    PubMed

    Okamoto, Haru; Umeda, Shinsuke; Nozawa, Takehiro; Suzuki, Michihiro T; Yoshikawa, Yasuhiro; Matsuura, Etsuko T; Iwata, Takeshi

    2010-01-01

    The central region of the primate retina is called the macula. The fovea is located at the center of the macula, where the photoreceptors are concentrated to create a neural network adapted for high visual acuity. Damage to the fovea, e.g., by macular dystrophies and age-related macular degeneration, can reduce central visual acuity. The molecular mechanisms leading to these diseases are most likely dependent on the proteins in the macula which differ from those in the peripheral retina in expression level. To investigate whether the distribution of proteins in the macula is different from the peripheral retina, proteomic analyses of tissues from these two regions of cynomolgus monkeys were compared. Two-dimensional gel electrophoresis and mass spectrometry identified 26 proteins that were present only in the macular gel spots. The expression levels of five proteins, cone photoreceptor specific arrestin-C, gamma-synuclein, epidermal fatty acid binding protein, tropomyosin 1alpha chain, and heterogeneous nuclear ribonucleoproteins A2/B1, were significantly higher in the macula than in the peripheral retina. Immunostaining of macula sections by antibodies to each identified protein revealed unique localization in the retina, retinal pigment epithelial cells and the choroidal layer. Some of these proteins were located in cells with higher densities in the macula. We suggest that it will be important to study these proteins to determine their contribution to the pathogenesis and progression of macula diseases.

  16. A Systematic Prediction of Drug-Target Interactions Using Molecular Fingerprints and Protein Sequences.

    PubMed

    Huang, Yu-An; You, Zhu-Hong; Chen, Xing

    2018-01-01

    Drug-Target Interactions (DTI) play a crucial role in discovering new drug candidates and finding new proteins to target for drug development. Although the number of detected DTI obtained by high-throughput techniques has been increasing, the number of known DTI is still limited. On the other hand, the experimental methods for detecting the interactions among drugs and proteins are costly and inefficient. Therefore, computational approaches for predicting DTI are drawing increasing attention in recent years. In this paper, we report a novel computational model for predicting the DTI using extremely randomized trees model and protein amino acids information. More specifically, the protein sequence is represented as a Pseudo Substitution Matrix Representation (Pseudo-SMR) descriptor in which the influence of biological evolutionary information is retained. For the representation of drug molecules, a novel fingerprint feature vector is utilized to describe its substructure information. Then the DTI pair is characterized by concatenating the two vector spaces of protein sequence and drug substructure. Finally, the proposed method is explored for predicting the DTI on four benchmark datasets: Enzyme, Ion Channel, GPCRs and Nuclear Receptor. The experimental results demonstrate that this method achieves promising prediction accuracies of 89.85%, 87.87%, 82.99% and 81.67%, respectively. For further evaluation, we compared the performance of Extremely Randomized Trees model with that of the state-of-the-art Support Vector Machine classifier. And we also compared the proposed model with existing computational models, and confirmed 15 potential drug-target interactions by looking for existing databases. The experiment results show that the proposed method is feasible and promising for predicting drug-target interactions for new drug candidate screening based on sizeable features. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. A strategy for targeting recombinant proteins to protein storage vacuoles by fusion to Brassica napus napin in napin-depleted seeds.

    PubMed

    Hegedus, Dwayne D; Baron, Marcus; Labbe, Natalie; Coutu, Cathy; Lydiate, Derek; Lui, Helen; Rozwadowski, Kevin

    2014-03-01

    Seeds are capable of accumulating high levels of seed storage proteins (SSP), as well as heterologous proteins under certain conditions. Arabidopsis thaliana was used to develop a strategy to deplete seeds of an endogenous SSP and then replenish them with the same protein fused to a heterologous protein. In several other studies, competition with endogenous SSP for space and metabolic resources was shown to affect the accumulation of recombinant proteins in seeds. We used RNAi to reduce the expression of the five napin genes and deplete the seeds of this SSP. Targeting a recombinant protein to a vacuole or structure within the seed where it can be protected from cytosolic proteases can also promote its accumulation. To achieve this, a synthetic Brassica napus napin gene (Bn napin) was designed that was both impervious to the A. thaliana napin (At napin) RNAi construct and permitted fusion to a heterologous protein, in this case green fluorescent protein (GFP). GFP was placed in several strategic locations within Bn napin with consideration to maintaining structure, processing sites and possible vacuolar targeting signals. In transgenic A. thaliana plants, GFP was strongly localized to the seed protein storage vacuole in all Bn napin fusion configurations tested, but not when expressed alone. This SSP depletion-replenishment strategy outlined here would be applicable to expression of recombinant proteins in industrial crops that generally have large repertoires of endogenous SSP genes. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

  18. Peripheral nerve injury induces loss of nociceptive neuron-specific Gαi-interacting protein in neuropathic pain rat.

    PubMed

    Liu, Zhen; Wang, Fei; Fischer, Gregory; Hogan, Quinn H; Yu, Hongwei

    2016-01-01

    Gαi-interacting protein (GINIP) is expressed specifically in dorsal root ganglion (DRG) neurons and functions in modulation of peripheral gamma-aminobutyric acid B receptor (GBR). Genetic deletion of GINIP leads to impaired responsiveness to GBR agonist-mediated analgesia in rodent. It is, however, not defined whether nerve injury changes GINIP expression. Immunolabeling with validated antibody revealed GINIP expression in ~40% of total lumbar DRG neurons in normal adult rats. GINIP immunoreactivity was detected in ~80% of IB4-positive (nonpeptidergic) and ~30% of CGRP-positive (peptidergic) neurons. GINIP immunoreactivity in the spinal cord dorsal horn was colabeled with IB4 and partially with CGRP. In addition, GINIP was expressed in DRG neurons immunopositive for GBR1, GBR2, Gαi(s), and Gαo and was also extensively colabeled with multiple nociceptive neuronal markers, including Trpv1, NaV1.7, CaV2.2α1b, CaV3.2α1b, TrkA, and Trek2. Peripheral nerve injury by L5 spinal nerve ligation significantly decreased the proportion of GINIP immunoreactivity-positive neurons from 40 ± 8.4% to 0.8 ± 0.1% (p < 0.01, mean ± SD, four weeks after spinal nerve ligation) and the total GINIP protein to 1.3% ± 0.04% of its basal level (p < 0.01, n = 6 animals in each group, two weeks after spinal nerve ligation) in the ipsilateral L5 DRGs. Our results show that GINIP is predominantly expressed by small nonpeptidergic nociceptive neurons and that nerve injury triggers loss of GINIP expression. Signal transduction roles of GINIP may be diverse as it colabeled with various subgroups of nociceptive neurons. Future studies may investigate details of the signaling mechanism engaged by GINIP, as well as the pathophysiological significance of lost expression of GINIP in neuropathic pain. © The Author(s) 2016.

  19. Membrane-spanning α-helical barrels as tractable protein-design targets.

    PubMed

    Niitsu, Ai; Heal, Jack W; Fauland, Kerstin; Thomson, Andrew R; Woolfson, Derek N

    2017-08-05

    The rational ( de novo ) design of membrane-spanning proteins lags behind that for water-soluble globular proteins. This is due to gaps in our knowledge of membrane-protein structure, and experimental difficulties in studying such proteins compared to water-soluble counterparts. One limiting factor is the small number of experimentally determined three-dimensional structures for transmembrane proteins. By contrast, many tens of thousands of globular protein structures provide a rich source of 'scaffolds' for protein design, and the means to garner sequence-to-structure relationships to guide the design process. The α-helical coiled coil is a protein-structure element found in both globular and membrane proteins, where it cements a variety of helix-helix interactions and helical bundles. Our deep understanding of coiled coils has enabled a large number of successful de novo designs. For one class, the α-helical barrels-that is, symmetric bundles of five or more helices with central accessible channels-there are both water-soluble and membrane-spanning examples. Recent computational designs of water-soluble α-helical barrels with five to seven helices have advanced the design field considerably. Here we identify and classify analogous and more complicated membrane-spanning α-helical barrels from the Protein Data Bank. These provide tantalizing but tractable targets for protein engineering and de novo protein design.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'. © 2017 The Author(s).

  20. Identification and Molecular Characterization of the Chloroplast Targeting Domain of Turnip yellow mosaic virus Replication Proteins

    PubMed Central

    Moriceau, Lucille; Jomat, Lucile; Bressanelli, Stéphane; Alcaide-Loridan, Catherine; Jupin, Isabelle

    2017-01-01

    Turnip yellow mosaic virus (TYMV) is a positive-strand RNA virus infecting plants. The TYMV 140K replication protein is a key organizer of viral replication complex (VRC) assembly, being responsible for recruitment of the viral polymerase and for targeting the VRCs to the chloroplast envelope where viral replication takes place. However, the structural requirements determining the subcellular localization and membrane association of this essential viral protein have not yet been defined. In this study, we investigated determinants for the in vivo chloroplast targeting of the TYMV 140K replication protein. Subcellular localization studies of deletion mutants identified a 41-residue internal sequence as the chloroplast targeting domain (CTD) of TYMV 140K; this sequence is sufficient to target GFP to the chloroplast envelope. The CTD appears to be located in the C-terminal extension of the methyltransferase domain—a region shared by 140K and its mature cleavage product 98K, which behaves as an integral membrane protein during infection. We predicted the CTD to fold into two amphipathic α-helices—a folding that was confirmed in vitro by circular dichroism spectroscopy analyses of a synthetic peptide. The importance for subcellular localization of the integrity of these amphipathic helices, and the function of 140K/98K, was demonstrated by performing amino acid substitutions that affected chloroplast targeting, membrane association and viral replication. These results establish a short internal α-helical peptide as an unusual signal for targeting proteins to the chloroplast envelope membrane, and provide new insights into membrane targeting of viral replication proteins—a universal feature of positive-strand RNA viruses. PMID:29312393

  1. Fearful, but not angry, expressions diffuse attention to peripheral targets in an attentional blink paradigm.

    PubMed

    Taylor, James M; Whalen, Paul J

    2014-06-01

    We previously demonstrated that fearful facial expressions implicitly facilitate memory for contextual events whereas angry facial expressions do not. The current study sought to more directly address the implicit effect of fearful expressions on attention for contextual events within a classic attentional paradigm (i.e., the attentional blink) in which memory is tested on a trial-by-trial basis, thereby providing subjects with a clear, explicit attentional strategy. Neutral faces of a single gender were presented via rapid serial visual presentation (RSVP) while bordered by four gray pound signs. Participants were told to watch for a gender change within the sequence (T1). It is critical to note that the T1 face displayed a neutral, fearful, or angry expression. Subjects were then told to detect a color change (i.e., gray to green; T2) at one of the four peripheral pound sign locations appearing after T1. This T2 color change could appear at one of six temporal positions. Complementing previous attentional blink paradigms, participants were told to respond via button press immediately when a T2 target was detected. We found that, compared with the neutral T1 faces, fearful faces significantly increased target detection ability at four of the six temporal locations (all ps < .05) whereas angry expressions did not. The results of this study demonstrate that fearful facial expressions can uniquely and implicitly enhance environmental monitoring above and beyond explicit attentional effects related to task instructions.

  2. The importance of regulation of blood glucose levels through activation of peripheral 5'-AMP-activated protein kinase on ischemic neuronal damage.

    PubMed

    Harada, Shinichi; Fujita-Hamabe, Wakako; Tokuyama, Shogo

    2010-09-10

    5'-AMP-activated protein kinase (AMPK) is a serine/threonine kinase that plays a key role in energy homeostasis. Recently, it was reported that centrally activated AMPK is involved in the development of ischemic neuronal damage, while the effect of peripherally activated AMPK on ischemic neuronal damage is not known. In addition, we have previously reported that the development of post-ischemic glucose intolerance could be one of the triggers for the aggravation of neuronal damage. In this study, we focused on effect of activation of peripheral or central AMPK on the development of ischemic neuronal damage. Male ddY mice were subjected to 2 h of middle cerebral artery occlusion (MCAO). Neuronal damage was estimated by histological and behavioral analysis after MCAO. In the liver and skeletal muscle, AMPK activity was not affected by MCAO. But, application of intraperitoneal metformin (250 mg/kg), an AMPK activator, significantly suppressed the development of post-ischemic glucose intolerance and ischemic neuronal damage without alteration of central AMPK activity. On the other hand, application of intracerebroventricular metformin (25, 100 microg/mouse) significantly exacerbated the development of neuronal damage observed on day 1 after MCAO, in a dose-dependent manner. These effects were significantly blocked by compound C, a specific AMPK inhibitor. These results suggest that central AMPK was activated by ischemic stress per se, however, peripheral AMPK was not altered. Furthermore, the regulation of post-ischemic glucose intolerance by activation of peripheral AMPK is of assistance for the suppression of cerebral ischemic neuronal damage. 2010 Elsevier B.V. All rights reserved.

  3. Targeting BCL-2-like Proteins to Kill Cancer Cells.

    PubMed

    Cory, Suzanne; Roberts, Andrew W; Colman, Peter M; Adams, Jerry M

    2016-08-01

    Mutations that impair apoptosis contribute to cancer development and reduce the effectiveness of conventional anti-cancer therapies. These insights and understanding of how the B cell lymphoma (BCL)-2 protein family governs apoptosis have galvanized the search for a new class of cancer drugs that target its pro-survival members by mimicking their natural antagonists, the BCL-2 homology (BH)3-only proteins. Successful initial clinical trials of the BH3 mimetic venetoclax/ABT-199, specific for BCL-2, have led to its recent licensing for refractory chronic lymphocytic leukemia and to multiple ongoing trials for other malignancies. Moreover, preclinical studies herald the potential of emerging BH3 mimetics targeting other BCL-2 pro-survival members, particularly myeloid cell leukemia (MCL)-1, for multiple cancer types. Thus, BH3 mimetics seem destined to become powerful new weapons in the arsenal against cancer. This review sketches the discovery of the BCL-2 family and its impact on cancer development and therapy; describes how interactions of family members trigger apoptosis; outlines the development of BH3 mimetic drugs; and discusses their potential to advance cancer therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Steric and not structure-specific factors dictate the endocytic mechanism of glycosylphosphatidylinositol-anchored proteins

    PubMed Central

    Bhagatji, Pinkesh; Leventis, Rania; Comeau, Jonathan; Refaei, Mohammad

    2009-01-01

    Diverse glycosylphosphatidylinositol (GPI)-anchored proteins enter mammalian cells via the clathrin- and dynamin-independent, Arf1-regulated GPI-enriched early endosomal compartment/clathrin-independent carrier endocytic pathway. To characterize the determinants of GPI protein targeting to this pathway, we have used fluorescence microscopic analyses to compare the internalization of artificial lipid-anchored proteins, endogenous membrane proteins, and membrane lipid markers in Chinese hamster ovary cells. Soluble proteins, anchored to cell-inserted saturated or unsaturated phosphatidylethanolamine (PE)-polyethyleneglycols (PEGs), closely resemble the GPI-anchored folate receptor but differ markedly from the transferrin receptor, membrane lipid markers, and even protein-free PE-PEGs, both in their distribution in peripheral endocytic vesicles and in the manner in which their endocytic uptake responds to manipulations of cellular Arf1 or dynamin activity. These findings suggest that the distinctive endocytic targeting of GPI proteins requires neither biospecific recognition of their GPI anchors nor affinity for ordered-lipid microdomains but is determined by a more fundamental property, the steric bulk of the lipid-anchored protein. PMID:19687251

  5. Convergent targeting of a common host protein-network by pathogen effectors from three kingdoms of life

    PubMed Central

    Weßling, Ralf; Epple, Petra; Altmann, Stefan; He, Yijian; Yang, Li; Henz, Stefan R.; McDonald, Nathan; Wiley, Kristin; Bader, Kai Christian; Gläßer, Christine; Mukhtar, M. Shahid; Haigis, Sabine; Ghamsari, Lila; Stephens, Amber E.; Ecker, Joseph R.; Vidal, Marc; Jones, Jonathan D. G.; Mayer, Klaus F. X.; van Themaat, Emiel Ver Loren; Weigel, Detlef; Schulze-Lefert, Paul; Dangl, Jeffery L.; Panstruga, Ralph; Braun, Pascal

    2014-01-01

    SUMMARY While conceptual principles governing plant immunity are becoming clear, its systems-level organization and the evolutionary dynamic of the host-pathogen interface are still obscure. We generated a systematic protein-protein interaction network of virulence effectors from the ascomycete pathogen Golovinomyces orontii and Arabidopsis thaliana host proteins. We combined this dataset with corresponding data for the eubacterial pathogen Pseudomonas syringae and the oomycete pathogen Hyaloperonospora arabidopsidis. The resulting network identifies host proteins onto which intraspecies and interspecies pathogen effectors converge. Phenotyping of 124 Arabidopsis effector-interactor mutants revealed a correlation between intra- and interspecies convergence and several altered immune response phenotypes. The effectors and most heavily targeted host protein co-localized in sub-nuclear foci. Products of adaptively selected Arabidopsis genes are enriched for interactions with effector targets. Our data suggest the existence of a molecular host-pathogen interface that is conserved across Arabidopsis accessions, while evolutionary adaptation occurs in the immediate network neighborhood of effector targets. PMID:25211078

  6. The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

    PubMed

    Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

    2017-07-15

    Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

  7. Immunoblot analysis of cellular expression of Bcl-2 family proteins, Bcl-2, Bax, Bcl-X and Mcl-1, in human peripheral blood and lymphoid tissues.

    PubMed

    Ohta, K; Iwai, K; Kasahara, Y; Taniguchi, N; Krajewski, S; Reed, J C; Miyawaki, T

    1995-11-01

    The ability of Bcl-2 to inhibit apoptotic cell death is well established. Several homologues of the bcl-2 gene, such as bax, bcl-x or mcl-1, have recently been identified. Like Bcl-2, both Bcl-XL and Mcl-1 appear to function as repressors of apoptotic cell death, whereas Bax facilitates it, indicating possible interactions among them in the control of cellular survival. To investigate the in vivo role of expression of bcl-2 gene family products, immunoblot analysis using corresponding specific antisera was performed for peripheral blood cells and some lymphoid tissues in humans. We demonstrated that all Bcl-2 family proteins were expressed at various levels in hematolymphoid cell subpopulations isolated from peripheral blood, tonsil, spleen and thymus. Lymphoid expression of Bcl-2 family proteins tended to increase following activation, but declined with time in culture. Loss of Bcl-2 in cultured lymphoid cells was especially marked. Sole expression of Bax, but not other members of the Bcl-2 family, was observed on neutrophils, seemingly reflecting their shortest life-span among blood leukocytes. The results support the notion that a balance of expression of Bcl-2 family proteins may regulate the life and death of hematolymphoid cells at different stages of cell differentiation and activation.

  8. Proliferation of PD-1+ CD8 T cells in peripheral blood after PD-1-targeted therapy in lung cancer patients.

    PubMed

    Kamphorst, Alice O; Pillai, Rathi N; Yang, Shu; Nasti, Tahseen H; Akondy, Rama S; Wieland, Andreas; Sica, Gabriel L; Yu, Ke; Koenig, Lydia; Patel, Nikita T; Behera, Madhusmita; Wu, Hong; McCausland, Megan; Chen, Zhengjia; Zhang, Chao; Khuri, Fadlo R; Owonikoko, Taofeek K; Ahmed, Rafi; Ramalingam, Suresh S

    2017-05-09

    Exhausted T cells in chronic infections and cancer have sustained expression of the inhibitory receptor programmed cell death 1 (PD-1). Therapies that block the PD-1 pathway have shown promising clinical results in a significant number of advanced-stage cancer patients. Nonetheless, a better understanding of the immunological responses induced by PD-1 blockade in cancer patients is lacking. Identification of predictive biomarkers is a priority in the field, but whether peripheral blood analysis can provide biomarkers to monitor or predict patients' responses to treatment remains to be resolved. In this study, we analyzed longitudinal blood samples from advanced stage non-small cell lung cancer (NSCLC) patients ( n = 29) receiving PD-1-targeted therapies. We detected an increase in Ki-67+ PD-1+ CD8 T cells following therapy in ∼70% of patients, and most responses were induced after the first or second treatment cycle. This T-cell activation was not indiscriminate because we observed only minimal effects on EBV-specific CD8 T cells, suggesting that responding cells may be tumor specific. These proliferating CD8 T cells had an effector-like phenotype (HLA-DR + , CD38 + , Bcl-2 lo ), expressed costimulatory molecules (CD28, CD27, ICOS), and had high levels of PD-1 and coexpression of CTLA-4. We found that 70% of patients with disease progression had either a delayed or absent PD-1+ CD8 T-cell response, whereas 80% of patients with clinical benefit exhibited PD-1+ CD8 T-cell responses within 4 wk of treatment initiation. Our results suggest that peripheral blood analysis may provide valuable insights into NSCLC patients' responses to PD-1-targeted therapies.

  9. GABARAPL1 antibodies: target one protein, get one free!

    PubMed

    Le Grand, Jaclyn Nicole; Chakrama, Fatima Zahra; Seguin-Py, Stéphanie; Fraichard, Annick; Delage-Mourroux, Régis; Jouvenot, Michèle; Risold, Pierre-Yves; Boyer-Guittaut, Michaël

    2011-11-01

    Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA(A) receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.

  10. The RNA binding and transport proteins staufen and fragile X mental retardation protein are expressed by rat primary afferent neurons and localize to peripheral and central axons.

    PubMed

    Price, T J; Flores, C M; Cervero, F; Hargreaves, K M

    2006-09-15

    Neuronal proteins have been traditionally viewed as being derived solely from the soma; however, accumulating evidence indicates that dendritic and axonal sites are capable of a more autonomous role in terms of new protein synthesis. Such extra-somal translation allows for more rapid, on-demand regulation of neuronal structure and function than would otherwise be possible. While mechanisms of dendritic RNA transport have been elucidated, it remains unclear how RNA is trafficked into the axon for this purpose. Primary afferent neurons of the dorsal root (DRG) and trigeminal (TG) ganglia have among the longest axons in the neuraxis and such axonal protein synthesis would be advantageous, given the greater time involved for protein trafficking to occur via axonal transport. Therefore, we hypothesized that these primary sensory neurons might express proteins involved in RNA transport. Rat DRG and TG neurons expressed staufen (stau) 1 and 2 (detected at the mRNA level) and stau2 and fragile x mental retardation protein (FMRP; detected at the protein level). Stau2 mRNA was also detected in human TG neurons. Stau2 and FMRP protein were localized to the sciatic nerve and dorsal roots by immunohistochemistry and to dorsal roots by Western blot. Stau2 and FMRP immunoreactivities colocalized with transient receptor potential channel type 1 immunoreactivity in sensory axons of the sciatic nerve and dorsal root, suggesting that these proteins are being transported into the peripheral and central terminals of nociceptive sensory axons. Based on these findings, we propose that stau2 and FMRP proteins are attractive candidates to subserve RNA transport in sensory neurons, linking somal transcriptional events to axonal translation.

  11. THE RNA BINDING AND TRANSPORT PROTEINS STAUFEN AND FRAGILE X MENTAL RETARDATION PROTEIN ARE EXPRESSED BY RAT PRIMARY AFFERENT NEURONS AND LOCALIZE TO PERIPHERAL AND CENTRAL AXONS

    PubMed Central

    PRICE, T. J.; FLORES, C. M.; CERVERO, F.; HARGREAVES, K. M.

    2007-01-01

    Neuronal proteins have been traditionally viewed as being derived solely from the soma; however, accumulating evidence indicates that dendritic and axonal sites are capable of a more autonomous role in terms of new protein synthesis. Such extra-somal translation allows for more rapid, on-demand regulation of neuronal structure and function than would otherwise be possible. While mechanisms of dendritic RNA transport have been elucidated, it remains unclear how RNA is trafficked into the axon for this purpose. Primary afferent neurons of the dorsal root (DRG) and trigeminal (TG) ganglia have among the longest axons in the neuraxis and such axonal protein synthesis would be advantageous, given the greater time involved for protein trafficking to occur via axonal transport. Therefore, we hypothesized that these primary sensory neurons might express proteins involved in RNA transport. Rat DRG and TG neurons expressed staufen (stau) 1 and 2 (detected at the mRNA level) and stau2 and fragile × mental retardation protein (FMRP; detected at the protein level). Stau2 mRNA was also detected in human TG neurons. Stau2 and FMRP protein were localized to the sciatic nerve and dorsal roots by immunohistochemistry and to dorsal roots by Western blot. Stau2 and FMRP immunoreactivities colocalized with transient receptor potential channel type 1 immunoreactivity in sensory axons of the sciatic nerve and dorsal root, suggesting that these proteins are being transported into the peripheral and central terminals of nociceptive sensory axons. Based on these findings, we propose that stau2 and FMRP proteins are attractive candidates to subserve RNA transport in sensory neurons, linking somal transcriptional events to axonal translation. PMID:16809002

  12. Discovery: an interactive resource for the rational selection and comparison of putative drug target proteins in malaria

    PubMed Central

    Joubert, Fourie; Harrison, Claudia M; Koegelenberg, Riaan J; Odendaal, Christiaan J; de Beer, Tjaart AP

    2009-01-01

    Background Up to half a billion human clinical cases of malaria are reported each year, resulting in about 2.7 million deaths, most of which occur in sub-Saharan Africa. Due to the over-and misuse of anti-malarials, widespread resistance to all the known drugs is increasing at an alarming rate. Rational methods to select new drug target proteins and lead compounds are urgently needed. The Discovery system provides data mining functionality on extensive annotations of five malaria species together with the human and mosquito hosts, enabling the selection of new targets based on multiple protein and ligand properties. Methods A web-based system was developed where researchers are able to mine information on malaria proteins and predicted ligands, as well as perform comparisons to the human and mosquito host characteristics. Protein features used include: domains, motifs, EC numbers, GO terms, orthologs, protein-protein interactions, protein-ligand interactions and host-pathogen interactions among others. Searching by chemical structure is also available. Results An in silico system for the selection of putative drug targets and lead compounds is presented, together with an example study on the bifunctional DHFR-TS from Plasmodium falciparum. Conclusion The Discovery system allows for the identification of putative drug targets and lead compounds in Plasmodium species based on the filtering of protein and chemical properties. PMID:19642978

  13. Specific Increase of Protein Levels by Enhancing Translation Using Antisense Oligonucleotides Targeting Upstream Open Frames.

    PubMed

    Liang, Xue-Hai; Shen, Wen; Crooke, Stanley T

    2017-01-01

    A number of diseases are caused by low levels of key proteins; therefore, increasing the amount of specific proteins in human bodies is of therapeutic interest. Protein expression is downregulated by some structural or sequence elements present in the 5' UTR of mRNAs, such as upstream open reading frames (uORF). Translation initiation from uORF(s) reduces translation from the downstream primary ORF encoding the main protein product in the same mRNA, leading to a less efficient protein expression. Therefore, it is possible to use antisense oligonucleotides (ASOs) to specifically inhibit translation of the uORF by base-pairing with the uAUG region of the mRNA, redirecting translation machinery to initiate from the primary AUG site. Here we review the recent findings that translation of specific mRNAs can be enhanced using ASOs targeting uORF regions. Appropriately designed and optimized ASOs are highly specific, and they act in a sequence- and position-dependent manner, with very minor off-target effects. Protein levels can be increased using this approach in different types of human and mouse cells, and, importantly, also in mice. Since uORFs are present in around half of human mRNAs, the uORF-targeting ASOs may thus have valuable potential as research tools and as therapeutics to increase the levels of proteins for a variety of genes.

  14. Engineered bifunctional proteins and stem cells: next generation of targeted cancer therapeutics.

    PubMed

    Choi, Sung Hugh; Shah, Khalid

    2016-09-01

    Redundant survival signaling pathways and their crosstalk within tumor and/or between tumor and their microenvironment are key impediments to developing effective targeted therapies for cancer. Therefore developing therapeutics that target multiple receptor signaling pathways in tumors and utilizing efficient platforms to deliver such therapeutics are critical to the success of future targeted therapies. During the past two decades, a number of bifunctional multi-targeting antibodies, fusion proteins, and oncolytic viruses have been developed and various stem cell types have been engineered to efficiently deliver them to tumors. In this review, we discuss the design and efficacy of therapeutics targeting multiple pathways in tumors and the therapeutic potential of therapeutic stem cells engineered with bifunctional agents.

  15. Differences in DNA Binding Specificity of Floral Homeotic Protein Complexes Predict Organ-Specific Target Genes.

    PubMed

    Smaczniak, Cezary; Muiño, Jose M; Chen, Dijun; Angenent, Gerco C; Kaufmann, Kerstin

    2017-08-01

    Floral organ identities in plants are specified by the combinatorial action of homeotic master regulatory transcription factors. However, how these factors achieve their regulatory specificities is still largely unclear. Genome-wide in vivo DNA binding data show that homeotic MADS domain proteins recognize partly distinct genomic regions, suggesting that DNA binding specificity contributes to functional differences of homeotic protein complexes. We used in vitro systematic evolution of ligands by exponential enrichment followed by high-throughput DNA sequencing (SELEX-seq) on several floral MADS domain protein homo- and heterodimers to measure their DNA binding specificities. We show that specification of reproductive organs is associated with distinct binding preferences of a complex formed by SEPALLATA3 and AGAMOUS. Binding specificity is further modulated by different binding site spacing preferences. Combination of SELEX-seq and genome-wide DNA binding data allows differentiation between targets in specification of reproductive versus perianth organs in the flower. We validate the importance of DNA binding specificity for organ-specific gene regulation by modulating promoter activity through targeted mutagenesis. Our study shows that intrafamily protein interactions affect DNA binding specificity of floral MADS domain proteins. Differential DNA binding of MADS domain protein complexes plays a role in the specificity of target gene regulation. © 2017 American Society of Plant Biologists. All rights reserved.

  16. Co-translational protein targeting facilitates centrosomal recruitment of PCNT during centrosome maturation in vertebrates

    PubMed Central

    Mahe, Karan; Ou, Tingyoung; Castro, Noemi M; Christensen, Lana N; Cheung, Lee; Jiang, Xueer; Yoon, Daniel; Huang, Bo

    2018-01-01

    As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here, we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near centrosomes, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism. PMID:29708497

  17. Rag Deletion in Peripheral T Cells Blocks TCR Revision

    PubMed Central

    Hale, J. Scott; Ames, Kristina T.; Boursalian, Tamar E.; Fink, Pamela J.

    2010-01-01

    Mature CD4+Vβ5+ T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or T cell receptor (TCR) revision. In Vβ5 transgenic mice, this latter tolerance pathway results in the appearance of CD4+Vβ5−TCRβ+ T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of Vβ-DJβ recombination intermediates in peripheral CD4+ T cells. Because post-thymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We now show that Rag deletion in post-positive selection T cells in Vβ5 transgenic mice blocks TCR revision in vivo, and that mature peripheral T cells sorted to remove cells bearing endogenous TCRβ chains can express newly generated TCRβ molecules in adoptive hosts. These findings unambiguously demonstrate post-thymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4+ T cells. PMID:20435935

  18. Attenuation of TRPV1 by AMG-517 after nerve injury promotes peripheral axonal regeneration in rats.

    PubMed

    Bai, Juan; Liu, Fu; Wu, Li-Fei; Wang, Ya-Fang; Li, Xia-Qing

    2018-01-01

    Aims The main objective was to investigate the effects of the transient receptor potential cation channel subfamily V member 1 (TRPV1) on nerve regeneration following sciatic transection injury by functional blockage of TRPV1 using AMG-517, a specific blocker of TRPV1. Methods AMG-517 was injected into the area surrounding ipsilateral lumbar dorsal root ganglia 30 min after unilateral sciatic nerve transection. The number of sciatic axons and the expression of growth-associated protein-43 (GAP-43) and glial fibrillary acidic protein was examined using semithin sections, Western blot, and immunofluorescence analyses. Results Blockage of TRPV1 with AMG-517 markedly promoted axonal regeneration, especially at two weeks after sciatic injury; the number of axons was similar to the uninjured control group. After sciatic nerve transection, expression of glial fibrillary acidic protein was decreased and GAP-43 was increased at the proximal stump. However, the expression of both glial fibrillary acidic protein and GAP-43 increased significantly in AMG-517-treated groups. Conclusions TRPV1 may be an important therapeutic target to promote peripheral nerve regeneration after injury.

  19. Selection of specific protein binders for pre-defined targets from an optimized library of artificial helicoidal repeat proteins (alphaRep).

    PubMed

    Guellouz, Asma; Valerio-Lepiniec, Marie; Urvoas, Agathe; Chevrel, Anne; Graille, Marc; Fourati-Kammoun, Zaineb; Desmadril, Michel; van Tilbeurgh, Herman; Minard, Philippe

    2013-01-01

    We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a "filtration" procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×10(9) independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties.

  20. Human long intrinsically disordered protein regions are frequent targets of positive selection.

    PubMed

    Afanasyeva, Arina; Bockwoldt, Mathias; Cooney, Christopher R; Heiland, Ines; Gossmann, Toni I

    2018-06-01

    Intrinsically disordered regions occur frequently in proteins and are characterized by a lack of a well-defined three-dimensional structure. Although these regions do not show a higher order of structural organization, they are known to be functionally important. Disordered regions are rapidly evolving, largely attributed to relaxed purifying selection and an increased role of genetic drift. It has also been suggested that positive selection might contribute to their rapid diversification. However, for our own species, it is currently unknown whether positive selection has played a role during the evolution of these protein regions. Here, we address this question by investigating the evolutionary pattern of more than 6600 human proteins with intrinsically disordered regions and their ordered counterparts. Our comparative approach with data from more than 90 mammalian genomes uses a priori knowledge of disordered protein regions, and we show that this increases the power to detect positive selection by an order of magnitude. We can confirm that human intrinsically disordered regions evolve more rapidly, not only within humans but also across the entire mammalian phylogeny. They have, however, experienced substantial evolutionary constraint, hinting at their fundamental functional importance. We find compelling evidence that disordered protein regions are frequent targets of positive selection and estimate that the relative rate of adaptive substitutions differs fourfold between disordered and ordered protein regions in humans. Our results suggest that disordered protein regions are important targets of genetic innovation and that the contribution of positive selection in these regions is more pronounced than in other protein parts. © 2018 Afanasyeva et al.; Published by Cold Spring Harbor Laboratory Press.

  1. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

    NASA Technical Reports Server (NTRS)

    Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M.; Hrabak, Estelle M.; Harmon, Alice C.; Pickard, Barbara G.; Harper, Jeffrey F.; Evans, M. L. (Principal Investigator)

    2003-01-01

    Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

  2. Inhibition of KLF7-Targeting MicroRNA 146b Promotes Sciatic Nerve Regeneration.

    PubMed

    Li, Wen-Yuan; Zhang, Wei-Ting; Cheng, Yong-Xia; Liu, Yan-Cui; Zhai, Feng-Guo; Sun, Ping; Li, Hui-Ting; Deng, Ling-Xiao; Zhu, Xiao-Feng; Wang, Ying

    2018-06-01

    A previous study has indicated that Krüppel-like factor 7 (KLF7), a transcription factor that stimulates Schwann cell (SC) proliferation and axonal regeneration after peripheral nerve injury, is a promising therapeutic transcription factor in nerve injury. We aimed to identify whether inhibition of microRNA-146b (miR-146b) affected SC proliferation, migration, and myelinated axon regeneration following sciatic nerve injury by regulating its direct target KLF7. SCs were transfected with miRNA lentivirus, miRNA inhibitor lentivirus, or KLF7 siRNA lentivirus in vitro. The expression of miR146b and KLF7, as well as SC proliferation and migration, were subsequently evaluated. In vivo, an acellular nerve allograft (ANA) followed by injection of GFP control vector or a lentiviral vector encoding an miR-146b inhibitor was used to assess the repair potential in a model of sciatic nerve gap. miR-146b directly targeted KLF7 by binding to the 3'-UTR, suppressing KLF7. Up-regulation of miR-146b and KLF7 knockdown significantly reduced the proliferation and migration of SCs, whereas silencing miR-146b resulted in increased proliferation and migration. KLF7 protein was localized in SCs in which miR-146b was expressed in vivo. Similarly, 4 weeks after the ANA, anti-miR-146b increased KLF7 and its target gene nerve growth factor cascade, promoting axonal outgrowth. Closer analysis revealed improved nerve conduction and sciatic function index score, and enhanced expression of neurofilaments, P0 (anti-peripheral myelin), and myelinated axon regeneration. Our findings provide new insight into the regulation of KLF7 by miR-146b during peripheral nerve regeneration and suggest a potential therapeutic strategy for peripheral nerve injury.

  3. Distribution of cyclophilin B-binding sites in the subsets of human peripheral blood lymphocytes.

    PubMed

    Denys, A; Allain, F; Foxwell, B; Spik, G

    1997-08-01

    Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway and released in biological fluids. We have recently demonstrated that both free CyPB and CyPB-CsA complex specifically bind to peripheral blood T lymphocytes and are internalized. These results suggest that CyPB might promote the targeting of the drug into sensitive cells. Peripheral blood lymphocytes are subdivided in several populations according to their biological functions and sensitivity to CsA. We have investigated the binding of CyPB to these different subsets using a CyPB derivatized by fluorescein through its single cysteine which retains its binding properties. We have confirmed that only T cells were involved in the interaction with CyPB. The ligand binding was found to be heterogeneously distributed on the different T-cell subsets and surface-bound CyPB was mainly associated with the CD4-positive cells. No significant difference was noted between the CD45RA and CD45RO subsets, demonstrating that CyPB-binding sites were equally distributed between native and memory T cells. CD3 stimulation of T lymphocytes led to a decrease in the CyPB-binding capacity, that may be explained by a down-regulation of the CyPB-receptor expression upon T-cell activation. Finally, we demonstrated that CyPB-receptor-positive cells, isolated on CyPB sulphydryl-coupled affinity matrices, are more sensitive to CyPB-complexed CsA than mixed peripheral blood lymphocytes, suggesting that CyPB potentiates CsA activity through the binding of the complex. Taken together, our results demonstrate that CyPB-binding sites are mainly associated with resting cells of the helper T lymphocyte, and that CyPB might modulate the distribution of CsA through the drug targeting to sensitive cells.

  4. Avidin-Based Targeting and Purification of a Protein IX-Modified, Metabolically Biotinylated Adenoviral Vector

    PubMed Central

    Campos, Samuel K.; Parrott, M. Brandon; Barry, Michael A.

    2014-01-01

    While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (Kd = 10−15 M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (Kd = 10−7 M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors. PMID:15194061

  5. Structural basis of mammalian glycan targeting by Vibrio cholerae cytolysin and biofilm proteins

    PubMed Central

    De, Swastik; Kaus, Katherine; Sinclair, Shada

    2018-01-01

    Vibrio cholerae is an aquatic gram-negative microbe responsible for cholera, a pandemic disease causing life-threatening diarrheal outbreaks in populations with limited access to health care. Like most pathogenic bacteria, V. cholerae secretes virulence factors to assist colonization of human hosts, several of which bind carbohydrate receptors found on cell-surfaces. Understanding how pathogenic virulence proteins specifically target host cells is important for the development of treatment strategies to fight bacterial infections. Vibrio cholerae cytolysin (VCC) is a secreted pore-forming toxin with a carboxy-terminal β-prism domain that targets complex N-glycans found on mammalian cell-surface proteins. To investigate glycan selectivity, we studied the VCC β-prism domain and two additional β-prism domains found within the V. cholerae biofilm matrix protein RbmC. We show that the two RbmC β-prism domains target a similar repertoire of complex N-glycan receptors as VCC and find through binding and modeling studies that a branched pentasaccharide core (GlcNAc2-Man3) represents the likely footprint interacting with these domains. To understand the structural basis of V. cholerae β-prism selectivity, we solved high-resolution crystal structures of fragments of the pentasaccharide core bound to one RbmC β-prism domain and conducted mutagenesis experiments on the VCC toxin. Our results highlight a common strategy for cell-targeting utilized by both toxin and biofilm matrix proteins in Vibrio cholerae and provide a structural framework for understanding the specificity for individual receptors. Our results suggest that a common strategy for disrupting carbohydrate interactions could affect multiple virulence factors produced by V. cholerae, as well as similar β-prism domains found in other vibrio pathogens. PMID:29432487

  6. A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p.

    PubMed

    Ota, Kazuhisa; Kito, Keiji; Okada, Satoshi; Ito, Takashi

    2008-10-01

    Ubiquitination plays various critical roles in eukaryotic cellular regulation and is mediated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1-Cullin-F-box protein complex comprises the largest E3 family, in each member of which a unique F-box protein binds its targets to define substrate specificity. Although genome sequencing uncovers a growing number of F-box proteins, most of them have remained as "orphans" because of the difficulties in identification of their substrates. To address this issue, we tested a quantitative proteomic approach by combining the stable isotope labeling by amino acids in cell culture (SILAC), parallel affinity purification (PAP) that we had developed for efficient enrichment of ubiquitinated proteins, and mass spectrometry (MS). We applied this SILAC-PAP-MS approach to compare ubiquitinated proteins between yeast cells with and without over-expressed Mdm30p, an F-box protein implicated in mitochondrial morphology. Consequently, we identified the mitochondrial outer membrane protein Mdm34p as a target of Mdm30p. Furthermore, we found that mitochondrial defects induced by deletion of MDM30 are not only recapitulated by a mutant Mdm34p defective in interaction with Mdm30p but alleviated by ubiquitination-mimicking forms of Mdm34p. These results indicate that Mdm34p is a physiologically important target of Mdm30p.

  7. Chemical proteomics for target discovery of head-to-tail cyclized mini-proteins

    NASA Astrophysics Data System (ADS)

    Hellinger, Roland; Thell, Kathrin; Vasileva, Mina; Muhammad, Taj; Gunasekera, Sunithi; Kümmel, Daniel; Göransson, Ulf; Becker, Christian W.; Gruber, Christian W.

    2017-10-01

    Target deconvolution is one of the most challenging tasks in drug discovery, but a key step in drug development. In contrast to small molecules, there is a lack of validated and robust methodologies for target elucidation of peptides. In particular, it is difficult to apply these methods to cyclic and cysteine-stabilized peptides since they exhibit reduced amenability to chemical modification and affinity capture; however, such ribosomal synthesized and post-translationally modified peptide natural products are rich sources of promising drug candidates. For example, plant-derived circular peptides called cyclotides have recently attracted much attention due to their immunosuppressive effects and oral activity in the treatment of multiple sclerosis in mice, but their molecular target has hitherto not been reported. In this study a chemical proteomics approach using photo-affinity crosslinking was developed to determine a target of the circular peptide [T20K]kalata B1. Using this prototypic nature-derived peptide enabled the identification of a possible modulation of 14-3-3 proteins. This biochemical interaction was validated via competition pull down assays as well as a cellular reporter assay indicating an effect on 14-3-3-dependent transcriptional activity. As proof of concept, the presented approach may be applicable for target elucidation of various cyclic peptides and mini-proteins, in particular cyclotides, which represent a promising class of molecules in drug discovery and development.

  8. Drug Distribution into Peripheral Nerve.

    PubMed

    Liu, Houfu; Chen, Yan; Huang, Liang; Sun, Xueying; Fu, Tingting; Wu, Shengqian; Zhu, Xiaoyan; Zhen, Wei; Liu, Jihong; Lu, Gang; Cai, Wei; Yang, Ting; Zhang, Wandong; Yu, Xiaohong; Wan, Zehong; Wang, Jianfei; Summerfield, Scott G; Dong, Kelly; Terstappen, Georg C

    2018-05-01

    Little is known about the impact of the blood-nerve barrier (BNB) on drug distribution into peripheral nerves. In this study, we examined the peripheral nerve penetration in rats of 11 small-molecule drugs possessing diverse physicochemical and transport properties and ProTx-II, a tarantula venom peptide with molecular mass of 3826 Daltons. Each drug was administered as constant rate intravenous infusion for 6 hours (small molecules) or 24 hours (ProTx-II). Blood and tissues including brain, spinal cord, sciatic nerve, and dorsal root ganglion (DRG) were collected for drug concentration measurements. Unbound fractions of a set of compounds were determined by equilibrium dialysis method in rat blood, brains, spinal cords, sciatic nerves, and DRG. We also investigated the influence of N -[4-[2-(6,7-dimethoxy-3,4-dihydro-1 H -isoquinolin-2-yl)ethyl]phenyl]-5-methoxy-9-oxo-10 H -acridine-4-carboxamide (GF120918), a P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) inhibitor, on the peripheral nerve and central nervous system (CNS) tissue penetration of imatinib. We found that: 1) the unbound fraction in brain tissue homogenate highly correlates with that in the spinal cord, sciatic nerve, and DRG for a set of compounds and thus provides a good surrogate for spinal cord and peripheral nerve tissues, 2) small-molecule drugs investigated can penetrate the DRG and sciatic nerve, 3) P-gp and BCRP have a limited impact on the distribution of small-molecule drugs into peripheral nerves, and 4) DRG is permeable to ProTx-II, but its distribution into sciatic nerve and CNS tissues is restricted. These results demonstrate that small-molecule drugs investigated can penetrate peripheral nerve tissues, and P-gp/BCRP may not be a limiting factor at the BNB. Biologics as large as ProTx-II can access the DRG but not sciatic nerve and CNS tissues. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  9. Deep-Dive Targeted Quantification for Ultrasensitive Analysis of Proteins in Nondepleted Human Blood Plasma/Serum and Tissues

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nie, Song; Shi, Tujin; Fillmore, Thomas L.

    Mass spectrometry-based targeted proteomics (e.g., selected reaction monitoring, SRM) is emerging as an attractive alternative to immunoassays for protein quantification. Recently we have made significant progress in SRM sensitivity for enabling quantification of low ng/mL to sub-ng/mL level proteins in nondepleted human blood plasma/serum without affinity enrichment. However, precise quantification of extremely low abundant but biologically important proteins (e.g., ≤100 pg/mL in blood plasma/serum) using targeted proteomics approaches still remains challenging. To address this need, we have developed an antibody-independent Deep-Dive SRM (DD-SRM) approach that capitalizes on multidimensional high-resolution reversed-phase liquid chromatography (LC) separation for target peptide enrichment combined withmore » precise selection of target peptide fractions of interest, significantly improving SRM sensitivity by ~5 orders of magnitude when compared to conventional LC-SRM. Application of DD-SRM to human serum and tissue has been demonstrated to enable precise quantification of endogenous proteins at ~10 pg/mL level in nondepleted serum and at <10 copies per cell level in tissue. Thus, DD-SRM holds great promise for precisely measuring extremely low abundance proteins or protein modifications, especially when high-quality antibody is not available.« less

  10. Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

    PubMed

    Ho, J C S; Nadeem, A; Rydström, A; Puthia, M; Svanborg, C

    2016-02-18

    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.

  11. Obesity treatment: novel peripheral targets

    PubMed Central

    Field, Benjamin C T; Chaudhri, Owais B; Bloom, Stephen R

    2009-01-01

    Our knowledge of the complex mechanisms underlying energy homeostasis has expanded enormously in recent years. Food intake and body weight are tightly regulated by the hypothalamus, brainstem and reward circuits, on the basis both of cognitive inputs and of diverse humoral and neuronal signals of nutritional status. Several gut hormones, including cholecystokinin, glucagon-like peptide-1, peptide YY, oxyntomodulin, amylin, pancreatic polypeptide and ghrelin, have been shown to play an important role in regulating short-term food intake. These hormones therefore represent potential targets in the development of novel anti-obesity drugs. This review focuses on the role of gut hormones in short- and long-term regulation of food intake, and on the current state of development of gut hormone-based obesity therapies. PMID:20002077

  12. A Protein Chimera Strategy Supports Production of a Model "Difficult-to-Express" Recombinant Target.

    PubMed

    Hussain, Hirra; Fisher, David I; Roth, Robert G; Abbott, W Mark; Carballo-Amador, Manuel Alejandro; Warwicker, Jim; Dickson, Alan J

    2018-06-22

    Due in part to the needs of the biopharmaceutical industry, there has been an increased drive to generate high quality recombinant proteins in large amounts. However, achieving high yields can be a challenge as the novelty and increased complexity of new targets often makes them 'difficult-to-express'. This study aimed to define the molecular features that restrict the production of a model 'difficult-to-express' recombinant protein, Tissue Inhibitor Metalloproteinase-3 (TIMP-3). Building from experimental data, computational approaches were used to rationalise the re-design of this recombinant target to generate a chimera with enhanced secretion. The results highlight the importance of early identification of unfavourable sequence attributes, enabling the generation of engineered protein forms that bypass 'secretory' bottlenecks and result in efficient recombinant protein production. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  13. How protein targeting to primary plastids via the endomembrane system could have evolved? A new hypothesis based on phylogenetic studies.

    PubMed

    Gagat, Przemysław; Bodył, Andrzej; Mackiewicz, Paweł

    2013-07-11

    It is commonly assumed that a heterotrophic ancestor of the supergroup Archaeplastida/Plantae engulfed a cyanobacterium that was transformed into a primary plastid; however, it is still unclear how nuclear-encoded proteins initially were imported into the new organelle. Most proteins targeted to primary plastids carry a transit peptide and are transported post-translationally using Toc and Tic translocons. There are, however, several proteins with N-terminal signal peptides that are directed to higher plant plastids in vesicles derived from the endomembrane system (ES). The existence of these proteins inspired a hypothesis that all nuclear-encoded, plastid-targeted proteins initially carried signal peptides and were targeted to the ancestral primary plastid via the host ES. We present the first phylogenetic analyses of Arabidopsis thaliana α-carbonic anhydrase (CAH1), Oryza sativa nucleotide pyrophosphatase/phosphodiesterase (NPP1), and two O. sativa α-amylases (αAmy3, αAmy7), proteins that are directed to higher plant primary plastids via the ES. We also investigated protein disulfide isomerase (RB60) from the green alga Chlamydomonas reinhardtii because of its peculiar dual post- and co-translational targeting to both the plastid and ES. Our analyses show that these proteins all are of eukaryotic rather than cyanobacterial origin, and that their non-plastid homologs are equipped with signal peptides responsible for co-translational import into the host ES. Our results indicate that vesicular trafficking of proteins to primary plastids evolved long after the cyanobacterial endosymbiosis (possibly only in higher plants) to permit their glycosylation and/or transport to more than one cellular compartment. The proteins we analyzed are not relics of ES-mediated protein targeting to the ancestral primary plastid. Available data indicate that Toc- and Tic-based translocation dominated protein import into primary plastids from the beginning. Only a handful of host

  14. The association of DNA-dependent protein kinase activity of peripheral blood lymphocytes with prognosis of cancer

    PubMed Central

    Someya, M; Sakata, K-i; Matsumoto, Y; Kamdar, R P; Kai, M; Toyota, M; Hareyama, M

    2011-01-01

    Background: Repair of various types of DNA damages is critical for genomic stability. DNA-dependent protein kinase (DNA-PK) has an important role in DNA double-strand break repair. We examined whether there may be a correlation between DNA-PK activity in peripheral blood lymphocytes (PBLs) and survival percentages in various cancer patients. We also investigated the changes of DNA-PK activity in PBLs after radiotherapy. Methods: A total of 167 of untreated cancer patients participated in this study. Peripheral blood was collected, separated, and centrifuged. DNA-PK activity was measured by DNA-pull-down assay. Chromosomal aberrations were examined by cytogenetic methods. Results: DNA-PK activity of PBLs in advanced cancer patients was significantly lower than that in early stage. The patients with lower DNA-PK activity in PBLs tended to have the lower disease-specific survivals and distant metastasis-free survivals than those with higher DNA-PK activity in advanced stages. There was also a tendency of inverse correlation between DNA-PK activity and excess fragments. The DNA-PK activity of PBLs in most patients decreased in response to radiation as the equivalent whole-body dose increased. Conclusion: Cancer patients in advanced stage, with lower DNA-PK activity of PBLs might have higher distant metastasis and exhibit poorer prognosis. Therefore, DNA-PK activity in PBLs could be used as a marker to predict the chromosomal instability and poorer prognosis. PMID:21559021

  15. Correlation between C-Reactive Protein in Peripheral Vein and Coronary Sinus in Stable and Unstable Angina

    PubMed Central

    Leite, Weverton Ferreira; Ramires, José Antonio Franchini; Moreira, Luiz Felipe Pinho; Strunz, Célia Maria Cassaro; Mangione, José Armando

    2015-01-01

    Background High sensitivity C-reactive protein (hs-CRP) is commonly used in clinical practice to assess cardiovascular risk. However, a correlation has not yet been established between the absolute levels of peripheral and central hs-CRP. Objective To assess the correlation between serum hs-CRP levels (mg/L) in a peripheral vein in the left forearm (LFPV) with those in the coronary sinus (CS) of patients with coronary artery disease (CAD) and a diagnosis of stable angina (SA) or unstable angina (UA). Methods This observational, descriptive, and cross-sectional study was conducted at the Instituto do Coração, Hospital das Clinicas, Faculdade de Medicina, Universidade de São Paulo, and at the Hospital Beneficência Portuguesa de Sao Paulo, where CAD patients referred to the hospital for coronary angiography were evaluated. Results Forty patients with CAD (20 with SA and 20 with UA) were included in the study. Blood samples from LFPV and CS were collected before coronary angiography. Furthermore, analysis of the correlation between serum levels of hs-CRP in LFPV versus CS showed a strong linear correlation for both SA (r = 0.993, p < 0.001) and UA (r = 0.976, p < 0.001) and for the entire sample (r = 0.985, p < 0.001). Conclusion Our data suggest a strong linear correlation between hs-CRP levels in LFPV versus CS in patients with SA and UA. PMID:25494014

  16. Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep)

    PubMed Central

    Chevrel, Anne; Graille, Marc; Fourati-Kammoun, Zaineb; Desmadril, Michel; van Tilbeurgh, Herman; Minard, Philippe

    2013-01-01

    We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a “filtration” procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×109 independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties. PMID:24014183

  17. Heterodimerization of the Entamoeba histolytica EhCPADH virulence complex through molecular dynamics and protein-protein docking.

    PubMed

    Montaño, Sarita; Orozco, Esther; Correa-Basurto, José; Bello, Martiniano; Chávez-Munguía, Bibiana; Betanzos, Abigail

    2017-02-01

    EhCPADH is a protein complex involved in the virulence of Entamoeba histolytica, the protozoan responsible for human amebiasis. It is formed by the EhCP112 cysteine protease and the EhADH adhesin. To explore the molecular basis of the complex formation, three-dimensional models were built for both proteins and molecular dynamics simulations (MDS) and docking calculations were performed. Results predicted that the pEhCP112 proenzyme and the mEhCP112 mature enzyme were globular and peripheral membrane proteins. Interestingly, in pEhCP112, the propeptide appeared hiding the catalytic site (C167, H329, N348); while in mEhCP112, this site was exposed and its residues were found structurally closer than in pEhCP112. EhADH emerged as an extended peripheral membrane protein with high fluctuation in Bro1 and V shape domains. 500 ns-long MDS and protein-protein docking predictions evidenced different heterodimeric complexes with the lowest free energy. pEhCP112 interacted with EhADH by the propeptide and C-terminal regions and mEhCP112 by the C-terminal through hydrogen bonds. In contrast, EhADH bound to mEhCP112 by 442-479 residues, adjacent to the target cell-adherence region (480-600 residues), and by the Bro1 domain (9-349 residues). Calculations of the effective binding free energy and per residue free energy decomposition showed that EhADH binds to mEhCP112 with a higher binding energy than to pEhCP112, mainly through van der Waals interactions and the nonpolar part of solvation energy. The EhADH and EhCP112 structural relationship was validated in trophozoites by immunofluorescence, TEM, and immunoprecipitation assays. Experimental findings fair agreed with in silico results.

  18. Peripheral elastic and inelastic scattering of 17,18O on light targets at 12 MeV/nucleon

    NASA Astrophysics Data System (ADS)

    Carstoiu, F.; Al-Abdullah, T.; Gagliardi, C. A.; Trache, L.

    2015-02-01

    The elastic and inelastic scattering of 17,18O with light targets has been undertaken at 12 MeV/nucleon in order to determine the optical potentials needed for the transfer reaction 13C (17O ,18O )12C . Optical potentials in both incoming and outgoing channels have been determined in a single experiment. This transfer reaction was used to infer the direct capture rate to the 17F ( p ,γ)18Ne which is essential to estimate the production of 18F at stellar energies in ONe novae. We demonstrate the stability of the ANC method and OMP results using good quality elastic and inelastic scattering data with stable beams. The peripherality of our reaction is inferred from a semiclassical decomposition of the total scattering amplitude into barrier and internal barrier components. Comparison between elastic scattering of 17O , 18O and 16O projectiles is made.

  19. Mutant HSPB1 causes loss of translational repression by binding to PCBP1, an RNA binding protein with a possible role in neurodegenerative disease.

    PubMed

    Geuens, Thomas; De Winter, Vicky; Rajan, Nicholas; Achsel, Tilmann; Mateiu, Ligia; Almeida-Souza, Leonardo; Asselbergh, Bob; Bouhy, Delphine; Auer-Grumbach, Michaela; Bagni, Claudia; Timmerman, Vincent

    2017-01-11

    The small heat shock protein HSPB1 (Hsp27) is an ubiquitously expressed molecular chaperone able to regulate various cellular functions like actin dynamics, oxidative stress regulation and anti-apoptosis. So far disease causing mutations in HSPB1 have been associated with neurodegenerative diseases such as distal hereditary motor neuropathy, Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis. Most mutations in HSPB1 target its highly conserved α-crystallin domain, while other mutations affect the C- or N-terminal regions or its promotor. Mutations inside the α-crystallin domain have been shown to enhance the chaperone activity of HSPB1 and increase the binding to client proteins. However, the HSPB1-P182L mutation, located outside and downstream of the α-crystallin domain, behaves differently. This specific HSPB1 mutation results in a severe neuropathy phenotype affecting exclusively the motor neurons of the peripheral nervous system. We identified that the HSPB1-P182L mutant protein has a specifically increased interaction with the RNA binding protein poly(C)binding protein 1 (PCBP1) and results in a reduction of its translational repressive activity. RNA immunoprecipitation followed by RNA sequencing on mouse brain lead to the identification of PCBP1 mRNA targets. These targets contain larger 3'- and 5'-UTRs than average and are enriched in an RNA motif consisting of the CTCCTCCTCCTCC consensus sequence. Interestingly, next to the clear presence of neuronal transcripts among the identified PCBP1 targets we identified known genes associated with hereditary peripheral neuropathies and hereditary spastic paraplegias. We therefore conclude that HSPB1 can mediate translational repression through interaction with an RNA binding protein further supporting its role in neurodegenerative disease.

  20. Molecular characterization of myelin protein zero in Xenopus laevis peripheral nerve

    NASA Astrophysics Data System (ADS)

    Xie, Bo; Luo, Xiaoyang; Zhao, Cheng; Priest, Christina Marie; Chan, Shiu-Yung; O'Connor, Peter B.; Kirschner, Daniel A.; Costello, Catherine E.

    2007-12-01

    Myelin protein zero (P0), a glycosylated single-pass transmembrane protein, is essential in the formation and maintenance of peripheral nervous system (PNS) compact myelin. P0 in Xenopus (xP0) exists primarily as a dimeric form that remains stable after various physical and chemical treatments. In exploring the nature of the interactions underlying the dimer stability, we found that xP0 dimer dissociated into monomer during continuous elution gel electrophoresis and conventional SDS-PAGE, indicating that the dimer is stabilized by non-covalent interactions. Furthermore, as some of the gel-purified monomer re-associated into dimer on SDS-PAGE gels, there is likely a dynamic equilibrium between xP0 dimer and monomer in vivo. Because the carbohydrate and fatty acyl moieties may be crucial for the adhesion role of P0, we used sensitive mass spectrometry approaches to elucidate the detailed N-glycosylation and S-acylation profiles of xP0. Asn92 was determined to be the single, fully-occupied glycosylation site of xP0, and a total of 12 glycans was detected that exhibited new structural features compared with those observed from P0 in other species: (1) the neutral glycans were composed mainly of high mannose and hybrid types; (2) 5 of 12 were acidic glycans, among which three were sialylated and the other two were sulfated; (3) none of the glycans had core fucosylation; and (4) no glucuronic acid, hence no HNK-1 epitope, was detected. The drastically different carbohydrate structures observed here support the concept of the species-specific variation in N-glycosylation of P0. Cys152 was found to be acylated with stearoyl (C18:0), whereas palmitoyl (C16:0) is the corresponding predominant fatty acyl group on P0 from higher vertebrates. We propose that the unique glycosylation and acylation patterns of Xenopus P0 may underlie its unusual dimerization behavior. Our results should shed light on the understanding of the phylogenetic development of P0's adhesion role in PNS

  1. Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics.

    PubMed

    Hossler, Patrick; McDermott, Sean; Racicot, Christopher; Chumsae, Christopher; Raharimampionona, Haly; Zhou, Yu; Ouellette, David; Matuck, Joseph; Correia, Ivan; Fann, John; Li, Jianmin

    2014-01-01

    Protein glycosylation is an important post-translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N-glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody-dependent cell-mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability. © 2014 American Institute of Chemical Engineers.

  2. A fusion-protein approach enabling mammalian cell production of tumor targeting protein domains for therapeutic development.

    PubMed

    Hu, Jia; Chen, Xiang; Zhang, Xuhua; Yuan, Xiaopeng; Yang, Mingjuan; Dai, Hui; Yang, Wei; Zhou, Qinghua; Wen, Weihong; Wang, Qirui; Qin, Weijun; Zhao, Aizhi

    2018-05-01

    A single chain Fv fragment (scFv) is a fusion of the variable regions of heavy (V H ) and light (V L ) chains of immunoglobulins. They are important elements of chimeric antigen receptors for cancer therapy. We sought to produce a panel of 16 extracellular protein domains of tumor markers for use in scFv yeast library screenings. A series of vectors comprising various combinations of expression elements was made, but expression was unpredictable and more than half of the protein domains could not be produced using any of the constructs. Here we describe a novel fusion expression system based on mouse TEM7 (tumor endothelial marker 7), which could facilitate protein expression. With this approach we could produce all but one of the tumor marker domains that could not otherwise be expressed. In addition, we demonstrated that the tumor associated antigen hFZD10 produced as a fusion protein with mTEM7 could be used to enrich scFv antibodies from a yeast display library. Collectively our study demonstrates the potential of specific fusion proteins based on mTEM7 in enabling mammalian cell production of tumor targeting protein domains for therapeutic development. © 2018 The Protein Society.

  3. Method for Targeted Therapeutic Delivery of Proteins into Cells | NCI Technology Transfer Center | TTC

    Cancer.gov

    The Protein Expression Laboratory at the National Cancer Institute in Frederick, MD is seeking statements of capability or interest from parties interested in collaborative research to further develop a platform technology for the targeted intra-cellular delivery of proteins using virus-like particles (VLPs).

  4. Sir-two-homolog 2 (Sirt2) modulates peripheral myelination through polarity protein Par-3/atypical protein kinase C (aPKC) signaling.

    PubMed

    Beirowski, Bogdan; Gustin, Jason; Armour, Sean M; Yamamoto, Hiroyasu; Viader, Andreu; North, Brian J; Michán, Shaday; Baloh, Robert H; Golden, Judy P; Schmidt, Robert E; Sinclair, David A; Auwerx, Johan; Milbrandt, Jeffrey

    2011-10-25

    The formation of myelin by Schwann cells (SCs) occurs via a series of orchestrated molecular events. We previously used global expression profiling to examine peripheral nerve myelination and identified the NAD(+)-dependent deacetylase Sir-two-homolog 2 (Sirt2) as a protein likely to be involved in myelination. Here, we show that Sirt2 expression in SCs is correlated with that of structural myelin components during both developmental myelination and remyelination after nerve injury. Transgenic mice lacking or overexpressing Sirt2 specifically in SCs show delays in myelin formation. In SCs, we found that Sirt2 deacetylates Par-3, a master regulator of cell polarity. The deacetylation of Par-3 by Sirt2 decreases the activity of the polarity complex signaling component aPKC, thereby regulating myelin formation. These results demonstrate that Sirt2 controls an essential polarity pathway in SCs during myelin assembly and provide insights into the association between intracellular metabolism and SC plasticity.

  5. Melatonin membrane receptors in peripheral tissues: Distribution and functions

    PubMed Central

    Slominski, Radomir M.; Reiter, Russel J.; Schlabritz-Loutsevitch, Natalia; Ostrom, Rennolds S.; Slominski, Andrzej T.

    2012-01-01

    Many of melatonin’s actions are mediated through interaction with the G-protein coupled membrane bound melatonin receptors type 1 and type 2 (MT1 and MT2, respectively) or, indirectly with nuclear orphan receptors from the RORα/RZR family. Melatonin also binds to the quinone reductase II enzyme, previously defined the MT3 receptor. Melatonin receptors are widely distributed in the body; herein we summarize their expression and actions in non-neural tissues. Several controversies still exist regarding, for example, whether melatonin binds the RORα/RZR family. Studies of the peripheral distribution of melatonin receptors are important since they are attractive targets for immunomodulation, regulation of endocrine, reproductive and cardiovascular functions, modulation of skin pigmentation, hair growth, cancerogenesis, and aging. Melatonin receptor agonists and antagonists have an exciting future since they could define multiple mechanisms by which melatonin modulates the complexity of such a wide variety of physiological and pathological processes. PMID:22245784

  6. Deletion of Sarm1 gene is neuroprotective in two models of peripheral neuropathy.

    PubMed

    Turkiew, Elliot; Falconer, Debbie; Reed, Nicole; Höke, Ahmet

    2017-09-01

    Distal axon degeneration seen in many peripheral neuropathies is likely to share common molecular mechanisms with Wallerian degeneration. Although several studies in mouse models of peripheral neuropathy showed prevention of axon degeneration in the slow Wallerian degeneration (Wlds) mouse, the role of a recently identified player in Wallerian degeneration, Sarm1, has not been explored extensively. In this study, we show that mice lacking the Sarm1 gene are resistant to distal axonal degeneration in a model of chemotherapy induced peripheral neuropathy caused by paclitaxel and a model of high fat diet induced putative metabolic neuropathy. This study extends the role of Sarm1 to axon degeneration seen in peripheral neuropathies and identifies it as a likely target for therapeutic development. © 2017 Peripheral Nerve Society.

  7. Ligand cluster-based protein network and ePlatton, a multi-target ligand finder.

    PubMed

    Du, Yu; Shi, Tieliu

    2016-01-01

    Small molecules are information carriers that make cells aware of external changes and couple internal metabolic and signalling pathway systems with each other. In some specific physiological status, natural or artificial molecules are used to interact with selective biological targets to activate or inhibit their functions to achieve expected biological and physiological output. Millions of years of evolution have optimized biological processes and pathways and now the endocrine and immune system cannot work properly without some key small molecules. In the past thousands of years, the human race has managed to find many medicines against diseases by trail-and-error experience. In the recent decades, with the deepening understanding of life and the progress of molecular biology, researchers spare no effort to design molecules targeting one or two key enzymes and receptors related to corresponding diseases. But recent studies in pharmacogenomics have shown that polypharmacology may be necessary for the effects of drugs, which challenge the paradigm, 'one drug, one target, one disease'. Nowadays, cheminformatics and structural biology can help us reasonably take advantage of the polypharmacology to design next-generation promiscuous drugs and drug combination therapies. 234,591 protein-ligand interactions were extracted from ChEMBL. By the 2D structure similarity, 13,769 ligand emerged from 156,151 distinct ligands which were recognized by 1477 proteins. Ligand cluster- and sequence-based protein networks (LCBN, SBN) were constructed, compared and analysed. For assisting compound designing, exploring polypharmacology and finding possible drug combination, we integrated the pathway, disease, drug adverse reaction and the relationship of targets and ligand clusters into the web platform, ePlatton, which is available at http://www.megabionet.org/eplatton. Although there were some disagreements between the LCBN and SBN, communities in both networks were largely the same

  8. Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers.

    PubMed

    Fuchs, Hendrik; Weng, Alexander; Gilabert-Oriol, Roger

    2016-07-01

    The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments.

  9. Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers

    PubMed Central

    Fuchs, Hendrik; Weng, Alexander; Gilabert-Oriol, Roger

    2016-01-01

    The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments. PMID:27376327

  10. Neuronal calcium sensor proteins are direct targets of the insulinotropic agent repaglinide.

    PubMed Central

    Okada, Miki; Takezawa, Daisuke; Tachibanaki, Shuji; Kawamura, Satoru; Tokumitsu, Hiroshi; Kobayashi, Ryoji

    2003-01-01

    The NCS (neuronal calcium sensor) proteins, including neurocalcins, recoverins and visinin-like proteins are members of a family of Ca2+-sensitive regulators, each with three Ca2+-binding EF-hand motifs. In plants, lily CCaMK [chimaeric Ca2+/CaM (calmodulin)-dependent protein kinase] and its PpCaMK ( Physcomitrella patens CCaMK) homologue are characterized by a visinin-like domain with three EF-hands. In the present study, in an effort to discover NCS antagonists, we screened a total of 43 compounds using Ca2+-dependent drug affinity chromatography and found that the insulinotropic agent repaglinide targets the NCS protein family. Repaglinide was found to bind to NCS proteins, but not to CaM or S100 proteins, in a Ca2+-dependent manner. Furthermore, the drug antagonized the inhibitory action of recoverin in a rhodopsin kinase assay with IC50 values of 400 microM. Moreover, repaglinide tightly bound to the visinin-like domain of CCaMK and PpCaMK in a Ca2+-dependent manner and antagonized the regulatory function of the domain with IC50 values of 55 and 4 microM for CCaMK and PpCaMK respectively. Although both repaglinide and a potent insulin secretagogue, namely glibenclamide, blocked K(ATP) channels with similar potency, glibenclamide had no antagonizing effect on the Ca2+-stimulated CCaMK and PpCaMK autophosphorylation, mediated by their visinin-like domain. In addition, a typical CaM antagonist, trifluoperazine, had no effect on the CCaMK and PpCaMK autophosphorylation. Repaglinide appears to be the first antagonist of NCS proteins and visinin-like domain-bearing enzymes. It may serve as a useful tool for evaluating the physiological functions of the NCS protein family. In addition, since repaglinide selectively targets NCS proteins among the EF-hand Ca2+-binding proteins, it is a potential lead compound for the development of more potent NCS antagonists. PMID:12844348

  11. Synaptic activity induces input-specific rearrangements in a targeted synaptic protein interaction network.

    PubMed

    Lautz, Jonathan D; Brown, Emily A; VanSchoiack, Alison A Williams; Smith, Stephen E P

    2018-05-27

    Cells utilize dynamic, network level rearrangements in highly interconnected protein interaction networks to transmit and integrate information from distinct signaling inputs. Despite the importance of protein interaction network dynamics, the organizational logic underlying information flow through these networks is not well understood. Previously, we developed the quantitative multiplex co-immunoprecipitation platform, which allows for the simultaneous and quantitative measurement of the amount of co-association between large numbers of proteins in shared complexes. Here, we adapt quantitative multiplex co-immunoprecipitation to define the activity dependent dynamics of an 18-member protein interaction network in order to better understand the underlying principles governing glutamatergic signal transduction. We first establish that immunoprecipitation detected by flow cytometry can detect activity dependent changes in two known protein-protein interactions (Homer1-mGluR5 and PSD-95-SynGAP). We next demonstrate that neuronal stimulation elicits a coordinated change in our targeted protein interaction network, characterized by the initial dissociation of Homer1 and SynGAP-containing complexes followed by increased associations among glutamate receptors and PSD-95. Finally, we show that stimulation of distinct glutamate receptor types results in different modular sets of protein interaction network rearrangements, and that cells activate both modules in order to integrate complex inputs. This analysis demonstrates that cells respond to distinct types of glutamatergic input by modulating different combinations of protein co-associations among a targeted network of proteins. Our data support a model of synaptic plasticity in which synaptic stimulation elicits dissociation of preexisting multiprotein complexes, opening binding slots in scaffold proteins and allowing for the recruitment of additional glutamatergic receptors. This article is protected by copyright. All

  12. An optimized transit peptide for effective targeting of diverse foreign proteins into chloroplasts in rice.

    PubMed

    Shen, Bo-Ran; Zhu, Cheng-Hua; Yao, Zhen; Cui, Li-Li; Zhang, Jian-Jun; Yang, Cheng-Wei; He, Zheng-Hui; Peng, Xin-Xiang

    2017-04-11

    Various chloroplast transit peptides (CTP) have been used to successfully target some foreign proteins into chloroplasts, but for other proteins these same CTPs have reduced localization efficiencies or fail completely. The underlying cause of the failures remains an open question, and more effective CTPs are needed. In this study, we initially observed that two E.coli enzymes, EcTSR and EcGCL, failed to be targeted into rice chloroplasts by the commonly-used rice rbcS transit peptide (rCTP) and were subsequently degraded. Further analyses revealed that the N-terminal unfolded region of cargo proteins is critical for their localization capability, and that a length of about 20 amino acids is required to attain the maximum localization efficiency. We considered that the unfolded region may alleviate the steric hindrance produced by the cargo protein, by functioning as a spacer to which cytosolic translocators can bind. Based on this inference, an optimized CTP, named RC2, was constructed. Analyses showed that RC2 can more effectively target diverse proteins, including EcTSR and EcGCL, into rice chloroplasts. Collectively, our results provide further insight into the mechanism of CTP-mediated chloroplastic localization, and more importantly, RC2 can be widely applied in future chloroplastic metabolic engineering, particularly for crop plants.

  13. Rapid computational identification of the targets of protein kinase inhibitors.

    PubMed

    Rockey, William M; Elcock, Adrian H

    2005-06-16

    We describe a method for rapidly computing the relative affinities of an inhibitor for all individual members of a family of homologous receptors. The approach, implemented in a new program, SCR, models inhibitor-receptor interactions in full atomic detail with an empirical energy function and includes an explicit account of flexibility in homology-modeled receptors through sampling of libraries of side chain rotamers. SCR's general utility was demonstrated by application to seven different protein kinase inhibitors: for each inhibitor, relative binding affinities with panels of approximately 20 protein kinases were computed and compared with experimental data. For five of the inhibitors (SB203580, purvalanol B, imatinib, H89, and hymenialdisine), SCR provided excellent reproduction of the experimental trends and, importantly, was capable of identifying the targets of inhibitors even when they belonged to different kinase families. The method's performance in a predictive setting was demonstrated by performing separate training and testing applications, and its key assumptions were tested by comparison with a number of alternative approaches employing the ligand-docking program AutoDock (Morris et al. J. Comput. Chem. 1998, 19, 1639-1662). These comparison tests included using AutoDock in nondocking and docking modes and performing energy minimizations of inhibitor-kinase complexes with the molecular mechanics code GROMACS (Berendsen et al. Comput. Phys. Commun. 1995, 91, 43-56). It was found that a surprisingly important aspect of SCR's approach is its assumption that the inhibitor be modeled in the same orientation for each kinase: although this assumption is in some respects unrealistic, calculations that used apparently more realistic approaches produced clearly inferior results. Finally, as a large-scale application of the method, SB203580, purvalanol B, and imatinib were screened against an almost full complement of 493 human protein kinases using SCR in

  14. Targeting pH regulating proteins for cancer therapy-Progress and limitations.

    PubMed

    Parks, Scott K; Pouysségur, Jacques

    2017-04-01

    Tumour acidity induced by metabolic alterations and incomplete vascularisation sets cancer cells apart from normal cellular physiology. This distinguishing tumour characteristic has been an area of intense study, as cellular pH (pH i ) disturbances disrupt protein function and therefore multiple cellular processes. Tumour cells effectively utilise pH i regulating machinery present in normal cells with enhancements provided by additional oncogenic or hypoxia induced protein modifications. This overall improvement of pH regulation enables maintenance of an alkaline pH i in the continued presence of external acidification (pH e ). Considerable experimentation has revealed targets that successfully disrupt tumour pH i regulation in efforts to develop novel means to weaken or kill tumour cells. However, redundancy in these pH-regulating proteins, which include Na + /H + exchangers (NHEs), carbonic anhydrases (CAs), Na + /HCO 3 - co-transporters (NBCs) and monocarboxylate transporters (MCTs) has prevented effective disruption of tumour pH i when individual protein targeting is performed. Here we synthesise recent advances in understanding both normoxic and hypoxic pH regulating mechanisms in tumour cells with an ultimate focus on the disruption of tumour growth, survival and metastasis. Interactions between tumour acidity and other cell types are also proving to be important in understanding therapeutic applications such as immune therapy. Promising therapeutic developments regarding pH manipulation along with current limitations are highlighted to provide a framework for future research directives. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mulari, Mika T.K.; Centre for Military Medicine, Research Department, Lahti; Nars, Martin

    2008-05-01

    Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSDmore » to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-{beta}-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption.« less

  16. Petri net-based prediction of therapeutic targets that recover abnormally phosphorylated proteins in muscle atrophy.

    PubMed

    Jung, Jinmyung; Kwon, Mijin; Bae, Sunghwa; Yim, Soorin; Lee, Doheon

    2018-03-05

    Muscle atrophy, an involuntary loss of muscle mass, is involved in various diseases and sometimes leads to mortality. However, therapeutics for muscle atrophy thus far have had limited effects. Here, we present a new approach for therapeutic target prediction using Petri net simulation of the status of phosphorylation, with a reasonable assumption that the recovery of abnormally phosphorylated proteins can be a treatment for muscle atrophy. The Petri net model was employed to simulate phosphorylation status in three states, i.e. reference, atrophic and each gene-inhibited state based on the myocyte-specific phosphorylation network. Here, we newly devised a phosphorylation specific Petri net that involves two types of transitions (phosphorylation or de-phosphorylation) and two types of places (activation with or without phosphorylation). Before predicting therapeutic targets, the simulation results in reference and atrophic states were validated by Western blotting experiments detecting five marker proteins, i.e. RELA, SMAD2, SMAD3, FOXO1 and FOXO3. Finally, we determined 37 potential therapeutic targets whose inhibition recovers the phosphorylation status from an atrophic state as indicated by the five validated marker proteins. In the evaluation, we confirmed that the 37 potential targets were enriched for muscle atrophy-related terms such as actin and muscle contraction processes, and they were also significantly overlapping with the genes associated with muscle atrophy reported in the Comparative Toxicogenomics Database (p-value < 0.05). Furthermore, we noticed that they included several proteins that could not be characterized by the shortest path analysis. The three potential targets, i.e. BMPR1B, ROCK, and LEPR, were manually validated with the literature. In this study, we suggest a new approach to predict potential therapeutic targets of muscle atrophy with an analysis of phosphorylation status simulated by Petri net. We generated a list of the potential

  17. A Program for Iron Economy during Deficiency Targets Specific Fe Proteins.

    PubMed

    Hantzis, Laura J; Kroh, Gretchen E; Jahn, Courtney E; Cantrell, Michael; Peers, Graham; Pilon, Marinus; Ravet, Karl

    2018-01-01

    Iron (Fe) is an essential element for plants, utilized in nearly every cellular process. Because the adjustment of uptake under Fe limitation cannot satisfy all demands, plants need to acclimate their physiology and biochemistry, especially in their chloroplasts, which have a high demand for Fe. To investigate if a program exists for the utilization of Fe under deficiency, we analyzed how hydroponically grown Arabidopsis ( Arabidopsis thaliana ) adjusts its physiology and Fe protein composition in vegetative photosynthetic tissue during Fe deficiency. Fe deficiency first affected photosynthetic electron transport with concomitant reductions in carbon assimilation and biomass production when effects on respiration were not yet significant. Photosynthetic electron transport function and protein levels of Fe-dependent enzymes were fully recovered upon Fe resupply, indicating that the Fe depletion stress did not cause irreversible secondary damage. At the protein level, ferredoxin, the cytochrome- b 6 f complex, and Fe-containing enzymes of the plastid sulfur assimilation pathway were major targets of Fe deficiency, whereas other Fe-dependent functions were relatively less affected. In coordination, SufA and SufB, two proteins of the plastid Fe-sulfur cofactor assembly pathway, were also diminished early by Fe depletion. Iron depletion reduced mRNA levels for the majority of the affected proteins, indicating that loss of enzyme was not just due to lack of Fe cofactors. SufB and ferredoxin were early targets of transcript down-regulation. The data reveal a hierarchy for Fe utilization in photosynthetic tissue and indicate that a program is in place to acclimate to impending Fe deficiency. © 2018 American Society of Plant Biologists. All Rights Reserved.

  18. Topologically Diverse Human Membrane Proteins Partition to Liquid-Disordered Domains in Phase-Separated Lipid Vesicles.

    PubMed

    Schlebach, Jonathan P; Barrett, Paul J; Day, Charles A; Kim, Ji Hun; Kenworthy, Anne K; Sanders, Charles R

    2016-02-23

    The integration of membrane proteins into "lipid raft" membrane domains influences many biochemical processes. The intrinsic structural properties of membrane proteins are thought to mediate their partitioning between membrane domains. However, whether membrane topology influences the targeting of proteins to rafts remains unclear. To address this question, we examined the domain preference of three putative raft-associated membrane proteins with widely different topologies: human caveolin-3, C99 (the 99 residue C-terminal domain of the amyloid precursor protein), and peripheral myelin protein 22. We find that each of these proteins are excluded from the ordered domains of giant unilamellar vesicles containing coexisting liquid-ordered and liquid-disordered phases. Thus, the intrinsic structural properties of these three topologically distinct disease-linked proteins are insufficient to confer affinity for synthetic raft-like domains.

  19. Small-Molecule Targeting of BET Proteins in Cancer.

    PubMed

    French, C A

    2016-01-01

    BET proteins have recently become recognized for their role in a broad range of cancers and are defined by the presence of two acetyl-histone reading bromodomains and an ET domain. This family of proteins includes BRD2, BRD3, BRD4, and BRDT. BRD4 is the most-studied BET protein in cancer, and normally serves as an epigenetic reader that links active chromatin marks to transcriptional elongation through activation of RNA polymerase II. The role of BRD3 and BRD4 first became known in cancer as mutant oncoproteins fused to the p300-recruiting NUT protein in a rare aggressive subtype of squamous cell cancer known as NUT midline carcinoma (NMC). BET inhibitors are acetyl-histone mimetics that specifically bind BET bromodomains, competitively inhibiting its engagement with chromatin. The antineoplastic effects of BET inhibitors were first demonstrated in NMC and have since been shown to be effective at inhibiting the growth of many different cancers, particularly acute leukemia. BET inhibitors have also been instrumental as tool compounds that have demonstrated the key role of BRD4 in driving NMC and non-NMC cancer growth. Many clinical trials enrolling patients with hematologic and solid tumors are ongoing, with encouraging preliminary findings. BET proteins BRD2, BRD3, and BRD4 are expressed in nearly all cells of the body, so there are concerns of toxicity with BET inhibitors, as well as the development of resistance. Toxicity and resistance may be overcome by combining BET inhibitors with other targeted inhibitors, or through the use of novel BET inhibitor derivatives. © 2016 Elsevier Inc. All rights reserved.

  20. Addressing the Immunogenicity of the Cargo and of the Targeting Antibodies with a Focus on Deimmunized Bacterial Toxins and on Antibody-Targeted Human Effector Proteins

    PubMed Central

    Grinberg, Yehudit; Benhar, Itai

    2017-01-01

    Third-generation immunotoxins are composed of a human, or humanized, targeting moiety, usually a monoclonal antibody or an antibody fragment, and a non-human effector molecule. Due to the non-human origin of the cytotoxic domain, these molecules stimulate potent anti-drug immune responses, which limit treatment options. Efforts are made to deimmunize such immunotoxins or to combine treatment with immunosuppression. An alternative approach is using the so-called “human cytotoxic fusion proteins”, in which antibodies are used to target human effector proteins. Here, we present three relevant approaches for reducing the immunogenicity of antibody-targeted protein therapeutics: (1) reducing the immunogenicity of the bacterial toxin, (2) fusing human cytokines to antibodies to generate immunocytokines and (3) addressing the immunogenicity of the targeting antibodies. PMID:28574434

  1. Monoclonal Gammopathy Associated Peripheral Neuropathy: Diagnosis and Management

    PubMed Central

    Chaudhry, Hafsa M.; Mauermann, Michelle L.; Rajkumar, S. Vincent

    2017-01-01

    Monoclonal gammopathies consist of a spectrum of clonal plasma cell disorders that includes monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM) and Waldenstrom Macroglobulinemia (WM). In this review, we outline the epidemiology, etiology, classification, diagnosis, and treatment of monoclonal gammopathy associated peripheral neuropathy. Monoclonal gammopathy of undetermined significance (MGUS) is relatively common in the general population, with a prevalence of 3–4% among those over the age of 50. Therefore, the presence of M protein in a patient with neuropathy does not automatically indicate a causal relationship. Monoclonal gammopathy associated peripheral neuropathy is often a difficult diagnosis with limited treatment options. Studies addressing the optimal approach to diagnosis and management of this entity are limited. In addition to a review of the literature, we present a diagnostic approach to patients with monoclonal gammopathy associated peripheral neuropathy and discuss available data and options for treatment. PMID:28473042

  2. Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins.

    PubMed

    Martinko, Alexander J; Truillet, Charles; Julien, Olivier; Diaz, Juan E; Horlbeck, Max A; Whiteley, Gordon; Blonder, Josip; Weissman, Jonathan S; Bandyopadhyay, Sourav; Evans, Michael J; Wells, James A

    2018-01-23

    While there have been tremendous efforts to target oncogenic RAS signaling from inside the cell, little effort has focused on the cell-surface. Here, we used quantitative surface proteomics to reveal a signature of proteins that are upregulated on cells transformed with KRAS G12V , and driven by MAPK pathway signaling. We next generated a toolkit of recombinant antibodies to seven of these RAS-induced proteins. We found that five of these proteins are broadly distributed on cancer cell lines harboring RAS mutations. In parallel, a cell-surface CRISPRi screen identified integrin and Wnt signaling proteins as critical to RAS-transformed cells. We show that antibodies targeting CDCP1, a protein common to our proteomics and CRISPRi datasets, can be leveraged to deliver cytotoxic and immunotherapeutic payloads to RAS-transformed cancer cells and report for RAS signaling status in vivo. Taken together, this work presents a technological platform for attacking RAS from outside the cell. © 2018, Martinko et al.

  3. Biotinylated probes of artemisinin with labeling affinity toward Trypanosoma brucei brucei target proteins.

    PubMed

    Konziase, Benetode

    2015-08-01

    We studied the target proteins of artemisinin in Trypanosoma brucei brucei using the affinity-labeling method. We designed and synthesized four biotinylated probes of artemisinin for use as molecular tools. Their in vitro trypanocidal activities (data not shown) proved that they mimicked the biological action of artemisinin. We assessed the chemical stability for all of the probes in the parasite culture medium and lysate using reversed-phase high-performance liquid chromatography (HPLC). After 3-h incubations, the probes remained undecomposed in a range of 40 to 65% in the parasite culture medium, whereas approximately 80% of the probes remained stable in the parasite lysate. Using liquid chromatography mass spectrometry (LC-MS), we demonstrated that, with respect to all of the probes, uptakes into the parasite ranging from 81 to 96% occurred after 30-min incubations. In a competitive binding assay between artemisinin and the four biotinylated probes, we searched for the trypanosomal target protein of artemisinin. Consequently, we observed that only the diazirine-free probe 5 could provide the desired result with high affinity-labeling efficiency. Using the horseradish peroxidase-tagged streptavidin-biotin method, we showed that artemisinin could specifically bind to candidate target proteins of approximately 60, 40, and 39 kDa. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Protein Targeting: ER Leads the Way to the Inner Nuclear Envelope.

    PubMed

    Blackstone, Craig

    2017-12-04

    Efficient targeting of newly synthesized membrane proteins from the endoplasmic reticulum to the inner nuclear membrane depends on nucleotide hydrolysis. A new study shows that this dependence reflects critical actions of the atlastin family of GTPases in maintaining the morphology of the endoplasmic reticulum network. Published by Elsevier Ltd.

  5. Foveal analysis and peripheral selection during active visual sampling

    PubMed Central

    Ludwig, Casimir J. H.; Davies, J. Rhys; Eckstein, Miguel P.

    2014-01-01

    Human vision is an active process in which information is sampled during brief periods of stable fixation in between gaze shifts. Foveal analysis serves to identify the currently fixated object and has to be coordinated with a peripheral selection process of the next fixation location. Models of visual search and scene perception typically focus on the latter, without considering foveal processing requirements. We developed a dual-task noise classification technique that enables identification of the information uptake for foveal analysis and peripheral selection within a single fixation. Human observers had to use foveal vision to extract visual feature information (orientation) from different locations for a psychophysical comparison. The selection of to-be-fixated locations was guided by a different feature (luminance contrast). We inserted noise in both visual features and identified the uptake of information by looking at correlations between the noise at different points in time and behavior. Our data show that foveal analysis and peripheral selection proceeded completely in parallel. Peripheral processing stopped some time before the onset of an eye movement, but foveal analysis continued during this period. Variations in the difficulty of foveal processing did not influence the uptake of peripheral information and the efficacy of peripheral selection, suggesting that foveal analysis and peripheral selection operated independently. These results provide important theoretical constraints on how to model target selection in conjunction with foveal object identification: in parallel and independently. PMID:24385588

  6. Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

    PubMed

    Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

    1995-01-01

    Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

  7. How protein targeting to primary plastids via the endomembrane system could have evolved? A new hypothesis based on phylogenetic studies

    PubMed Central

    2013-01-01

    Background It is commonly assumed that a heterotrophic ancestor of the supergroup Archaeplastida/Plantae engulfed a cyanobacterium that was transformed into a primary plastid; however, it is still unclear how nuclear-encoded proteins initially were imported into the new organelle. Most proteins targeted to primary plastids carry a transit peptide and are transported post-translationally using Toc and Tic translocons. There are, however, several proteins with N-terminal signal peptides that are directed to higher plant plastids in vesicles derived from the endomembrane system (ES). The existence of these proteins inspired a hypothesis that all nuclear-encoded, plastid-targeted proteins initially carried signal peptides and were targeted to the ancestral primary plastid via the host ES. Results We present the first phylogenetic analyses of Arabidopsis thaliana α-carbonic anhydrase (CAH1), Oryza sativa nucleotide pyrophosphatase/phosphodiesterase (NPP1), and two O. sativa α-amylases (αAmy3, αAmy7), proteins that are directed to higher plant primary plastids via the ES. We also investigated protein disulfide isomerase (RB60) from the green alga Chlamydomonas reinhardtii because of its peculiar dual post- and co-translational targeting to both the plastid and ES. Our analyses show that these proteins all are of eukaryotic rather than cyanobacterial origin, and that their non-plastid homologs are equipped with signal peptides responsible for co-translational import into the host ES. Our results indicate that vesicular trafficking of proteins to primary plastids evolved long after the cyanobacterial endosymbiosis (possibly only in higher plants) to permit their glycosylation and/or transport to more than one cellular compartment. Conclusions The proteins we analyzed are not relics of ES-mediated protein targeting to the ancestral primary plastid. Available data indicate that Toc- and Tic-based translocation dominated protein import into primary plastids from the

  8. Discovery-2: an interactive resource for the rational selection and comparison of putative drug target proteins in malaria

    PubMed Central

    2013-01-01

    Background Drug resistance to anti-malarial compounds remains a serious problem, with resistance to newer pharmaceuticals developing at an alarming rate. The development of new anti-malarials remains a priority, and the rational selection of putative targets is a key element of this process. Discovery-2 is an update of the original Discovery in silico resource for the rational selection of putative drug target proteins, enabling researchers to obtain information for a protein which may be useful for the selection of putative drug targets, and to perform advanced filtering of proteins encoded by the malaria genome based on a series of molecular properties. Methods An updated in silico resource has been developed where researchers are able to mine information on malaria proteins and predicted ligands, as well as perform comparisons to the human and mosquito host characteristics. Protein properties used include: domains, motifs, EC numbers, GO terms, orthologs, protein-protein interactions, protein-ligand interactions. Newly added features include drugability measures from ChEMBL, automated literature relations and links to clinical trial information. Searching by chemical structure is also available. Results The updated functionality of the Discovery-2 resource is presented, together with a detailed case study of the Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase (PfSAHH) protein. A short example of a chemical search with pyrimethamine is also illustrated. Conclusion The updated Discovery-2 resource allows researchers to obtain detailed properties of proteins from the malaria genome, which may be of interest in the target selection process, and to perform advanced filtering and selection of proteins based on a relevant range of molecular characteristics. PMID:23537208

  9. Targeting accuracy of single-isocenter intensity-modulated radiosurgery for multiple lesions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Calvo-Ortega, J.F., E-mail: jfcdrr@yahoo.es; Pozo, M.; Moragues, S.

    To investigate the targeting accuracy of intensity-modulated SRS (IMRS) plans designed to simultaneously treat multiple brain metastases with a single isocenter. A home-made acrylic phantom able to support a film (EBT3) in its coronal plane was used. The phantom was CT scanned and three coplanar small targets (a central and two peripheral) were outlined in the Eclipse system. Peripheral targets were 6 cm apart from the central one. A reference IMRS plan was designed to simultaneously treat the three targets, but only a single isocenter located at the center of the central target was used. After positioning the phantom onmore » the linac using the room lasers, a CBCT scan was acquired and the reference plan were mapped on it, by placing the planned isocenter at the intersection of the landmarks used in the film showing the linac isocenter. The mapped plan was then recalculated and delivered. The film dose distribution was derived using a cloud computing application ( (www.radiochromic.com)) that uses a triple-channel dosimetry algorithm. Comparison of dose distributions using the gamma index (5%/1 mm) were performed over a 5 × 5 cm{sup 2} region centered over each target. 2D shifts required to get the best gamma passing rates on the peripheral target regions were compared with the reported ones for the central target. The experiment was repeated ten times in different sessions. Average 2D shifts required to achieve optimal gamma passing rates (99%, 97%, 99%) were 0.7 mm (SD: 0.3 mm), 0.8 mm (SD: 0.4 mm) and 0.8 mm (SD: 0.3 mm), for the central and the two peripheral targets, respectively. No statistical differences (p > 0.05) were found for targeting accuracy between the central and the two peripheral targets. The study revealed a targeting accuracy within 1 mm for off-isocenter targets within 6 cm of the linac isocenter, when a single-isocenter IMRS plan is designed.« less

  10. Targeting accuracy of single-isocenter intensity-modulated radiosurgery for multiple lesions.

    PubMed

    Calvo-Ortega, J F; Pozo, M; Moragues, S; Casals, J

    2017-01-01

    To investigate the targeting accuracy of intensity-modulated SRS (IMRS) plans designed to simultaneously treat multiple brain metastases with a single isocenter. A home-made acrylic phantom able to support a film (EBT3) in its coronal plane was used. The phantom was CT scanned and three coplanar small targets (a central and two peripheral) were outlined in the Eclipse system. Peripheral targets were 6 cm apart from the central one. A reference IMRS plan was designed to simultaneously treat the three targets, but only a single isocenter located at the center of the central target was used. After positioning the phantom on the linac using the room lasers, a CBCT scan was acquired and the reference plan were mapped on it, by placing the planned isocenter at the intersection of the landmarks used in the film showing the linac isocenter. The mapped plan was then recalculated and delivered. The film dose distribution was derived using a cloud computing application (www.radiochromic.com) that uses a triple-channel dosimetry algorithm. Comparison of dose distributions using the gamma index (5%/1 mm) were performed over a 5 × 5 cm 2 region centered over each target. 2D shifts required to get the best gamma passing rates on the peripheral target regions were compared with the reported ones for the central target. The experiment was repeated ten times in different sessions. Average 2D shifts required to achieve optimal gamma passing rates (99%, 97%, 99%) were 0.7 mm (SD: 0.3 mm), 0.8 mm (SD: 0.4 mm) and 0.8 mm (SD: 0.3 mm), for the central and the two peripheral targets, respectively. No statistical differences (p > 0.05) were found for targeting accuracy between the central and the two peripheral targets. The study revealed a targeting accuracy within 1 mm for off-isocenter targets within 6 cm of the linac isocenter, when a single-isocenter IMRS plan is designed. Copyright © 2017 American Association of Medical Dosimetrists. Published by Elsevier Inc. All rights reserved.

  11. Peripheral nerve injury induces loss of nociceptive neuron-specific Gαi-interacting protein in neuropathic pain rat

    PubMed Central

    Liu, Zhen; Wang, Fei; Fischer, Gregory; Hogan, Quinn H.

    2016-01-01

    Background Gαi-interacting protein (GINIP) is expressed specifically in dorsal root ganglion (DRG) neurons and functions in modulation of peripheral gamma-aminobutyric acid B receptor (GBR). Genetic deletion of GINIP leads to impaired responsiveness to GBR agonist-mediated analgesia in rodent. It is, however, not defined whether nerve injury changes GINIP expression. Results Immunolabeling with validated antibody revealed GINIP expression in ∼40% of total lumbar DRG neurons in normal adult rats. GINIP immunoreactivity was detected in ∼80% of IB4-positive (nonpeptidergic) and ∼30% of CGRP-positive (peptidergic) neurons. GINIP immunoreactivity in the spinal cord dorsal horn was colabeled with IB4 and partially with CGRP. In addition, GINIP was expressed in DRG neurons immunopositive for GBR1, GBR2, Gαi(s), and Gαo and was also extensively colabeled with multiple nociceptive neuronal markers, including Trpv1, NaV1.7, CaV2.2α1b, CaV3.2α1b, TrkA, and Trek2. Peripheral nerve injury by L5 spinal nerve ligation significantly decreased the proportion of GINIP immunoreactivity-positive neurons from 40 ± 8.4% to 0.8 ± 0.1% (p < 0.01, mean ± SD, four weeks after spinal nerve ligation) and the total GINIP protein to 1.3% ± 0.04% of its basal level (p < 0.01, n = 6 animals in each group, two weeks after spinal nerve ligation) in the ipsilateral L5 DRGs. Conclusion Our results show that GINIP is predominantly expressed by small nonpeptidergic nociceptive neurons and that nerve injury triggers loss of GINIP expression. Signal transduction roles of GINIP may be diverse as it colabeled with various subgroups of nociceptive neurons. Future studies may investigate details of the signaling mechanism engaged by GINIP, as well as the pathophysiological significance of lost expression of GINIP in neuropathic pain. PMID:27145804

  12. Engineered Proteins Program Mammalian Cells to Target Inflammatory Disease Sites.

    PubMed

    Qudrat, Anam; Mosabbir, Abdullah Al; Truong, Kevin

    2017-06-22

    Disease sites in atherosclerosis and cancer feature cell masses (e.g., plaques/tumors), a low pH extracellular microenvironment, and various pro-inflammatory cytokines such as tumor necrosis factor α (TNFα). The ability to engineer a cell to seek TNFα sources allows for targeted therapeutic delivery. To accomplish this, here we introduced a system of proteins: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca 2+ -activated RhoA (named CaRQ), vesicular stomatitis virus glycoprotein G (VSVG), and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca 2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs that allow migration toward the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, after ganciclovir treatment cells undergo death via the thymidine kinase suicide mechanism. Hence, we assembled a system of proteins that forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low-pH microenvironment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Deficiency of a membrane skeletal protein, 4.1G, results in myelin abnormalities in the peripheral nervous system.

    PubMed

    Saitoh, Yurika; Ohno, Nobuhiko; Yamauchi, Junji; Sakamoto, Takeharu; Terada, Nobuo

    2017-12-01

    We previously demonstrated that a membrane skeletal molecular complex, 4.1G-membrane palmitoylated protein 6 (MPP6)-cell adhesion molecule 4, is incorporated in Schwann cells in the peripheral nervous system (PNS). In this study, we evaluated motor activity and myelin ultrastructures in 4.1G-deficient (-/-) mice. When suspended by the tail, aged 4.1G -/- mice displayed spastic leg extension, especially after overwork. Motor-conduction velocity in 4.1G -/- mice was slower than that in wild-type mice. Using electron microscopy, 4.1G -/- mice exhibited myelin abnormalities: myelin was thicker in internodes, and attachment of myelin tips was distorted in some paranodes. In addition, we found a novel function of 4.1G for sorting a scaffold protein, Lin7, due to disappearance of the immunolocalization and reduction of the production of Lin7c and Lin7a in 4.1G -/- sciatic nerves, as well as the interaction of MPP6 and Lin7 with immunoprecipitation. Thus, we herein propose 4.1G functions as a signal for proper formation of myelin in PNS.

  14. Influence of Translation Initiation on Organellar Protein Targeting in Arabidopsis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sally A. Mackenzie

    2011-04-18

    A primary focus of the Mackenzie laboratory is the elucidation of processes and machinery for mitochondrial genome maintenance and transmission in higher plants. We have found that numerous organellar DNA maintenance components in plants appear to be dual targeted to mitochondria and plastids. Of particular interest was the observation that some twin (tandemly arrayed) dual targeting presequences appeared to utilize non-AUG alternative translation initiation, allowing for multiple translation starts at a single gene. Two aspects of this phenomenon were of particular interest: (1) Alternative translation initiation might provide a mechanism to regulate protein targeting temporally and spatially, a possibility thatmore » had not been demonstrated previously, and (2) alternative translation initiation might occur in genes involved in nuclear-controlled mitochondrial genome recombination, thought to be exclusively mitochondrial in their function. During the course of this research, we pursued three aims, with an emphasis on two specific genes of interest: POLgamma2, an organellar DNA polymerase, and MSH1, a MutS homolog thought to participate in mitochondrial, but not plastid, genome recombination surveillance. Our aims were to (1) Identify additional genes within Arabidopsis and other genomes that employ non-AUG alternative translation initiation, (2) Locate sequences upstream to the annotated AUG that confer alternative non-AUG translation initiation activity, and (3) Identify cis and trans factors that influence start site selection in genes with non-AUG starts. Toward these ends, we have shown that non-AUG initiation occurs in a number of genes, likely influencing targeting behavior of the protein. We have also shown that start site selection is strongly influenced by Kozak consensus sequence environment, indicating that alternative translation initiation in plants occurs by relaxation of ribosome scanning.« less

  15. Obesity: Current and potential pharmacotherapeutics and targets.

    PubMed

    Narayanaswami, Vidya; Dwoskin, Linda P

    2017-02-01

    Obesity is a global epidemic that contributes to a number of health complications including cardiovascular disease, type 2 diabetes, cancer and neuropsychiatric disorders. Pharmacotherapeutic strategies to treat obesity are urgently needed. Research over the past two decades has increased substantially our knowledge of central and peripheral mechanisms underlying homeostatic energy balance. Homeostatic mechanisms involve multiple components including neuronal circuits, some originating in hypothalamus and brain stem, as well as peripherally-derived satiety, hunger and adiposity signals that modulate neural activity and regulate eating behavior. Dysregulation of one or more of these homeostatic components results in obesity. Coincident with obesity, reward mechanisms that regulate hedonic aspects of food intake override the homeostatic regulation of eating. In addition to functional interactions between homeostatic and reward systems in the regulation of food intake, homeostatic signals have the ability to alter vulnerability to drug abuse. Regarding the treatment of obesity, pharmacological monotherapies primarily focus on a single protein target. FDA-approved monotherapy options include phentermine (Adipex-P®), orlistat (Xenical®), lorcaserin (Belviq®) and liraglutide (Saxenda®). However, monotherapies have limited efficacy, in part due to the recruitment of alternate and counter-regulatory pathways. Consequently, a multi-target approach may provide greater benefit. Recently, two combination products have been approved by the FDA to treat obesity, including phentermine/topiramate (Qsymia®) and naltrexone/bupropion (Contrave®). The current review provides an overview of homeostatic and reward mechanisms that regulate energy balance, potential therapeutic targets for obesity and current treatment options, including some candidate therapeutics in clinical development. Finally, challenges in anti-obesity drug development are discussed. Copyright © 2016 Elsevier

  16. Obesity: Current and Potential Pharmacotherapeutics and Targets

    PubMed Central

    Narayanaswami, Vidya; Dwoskin, Linda P.

    2016-01-01

    Obesity is a global epidemic that contributes to a number of health complications including cardiovascular disease, type 2 diabetes, cancer and neuropsychiatric disorders. Pharmacotherapeutic strategies to treat obesity are urgently needed. Research over the past two decades has increased substantially our knowledge of central and peripheral mechanisms underlying homeostatic energy balance. Homeostatic mechanisms involve multiple components including neuronal circuits, some originating in hypothalamus and brain stem, as well as peripherally-derived satiety, hunger and adiposity signals that modulate neural activity and regulate eating behavior. Dysregulation of one or more of these homeostatic components results in obesity. Coincident with obesity, reward mechanisms that regulate hedonic aspects of food intake override the homeostatic regulation of eating. In addition to functional interactions between homeostatic and reward systems in the regulation of food intake, homeostatic signals have the ability to alter vulnerability to drug abuse. Regarding the treatment of obesity, pharmacological monotherapies primarily focus on a single protein target. FDA-approved monotherapy options include phentermine (Adipex-P®), orlistat (Xenical®), lorcaserin (Belviq®) and liraglutide (Saxenda®). However, monotherapies have limited efficacy, in part due to the recruitment of alternate and counter-regulatory pathways. Consequently, a multi-target approach may provide greater benefit. Recently, two combination products have been approved by the FDA to treat obesity, including phentermine/topiramate (Qsymia®) and naltrexone/bupropion (Contrave®). The current review provides an overview of homeostatic and reward mechanisms that regulate energy balance, potential therapeutic targets for obesity and current treatment options, including some candidate therapeutics in clinical development. Finally, challenges in anti-obesity drug development are discussed. PMID:27773782

  17. Micro-Environmental Signature of The Interactions between Druggable Target Protein, Dipeptidyl Peptidase-IV, and Anti-Diabetic Drugs.

    PubMed

    Chakraborty, Chiranjib; Mallick, Bidyut; Sharma, Ashish Ranjan; Sharma, Garima; Jagga, Supriya; Doss, C George Priya; Nam, Ju-Suk; Lee, Sang-Soo

    2017-01-01

    Druggability of a target protein depends on the interacting micro-environment between the target protein and drugs. Therefore, a precise knowledge of the interacting micro-environment between the target protein and drugs is requisite for drug discovery process. To understand such micro-environment, we performed in silico interaction analysis between a human target protein, Dipeptidyl Peptidase-IV (DPP-4), and three anti-diabetic drugs (saxagliptin, linagliptin and vildagliptin). During the theoretical and bioinformatics analysis of micro-environmental properties, we performed drug-likeness study, protein active site predictions, docking analysis and residual interactions with the protein-drug interface. Micro-environmental landscape properties were evaluated through various parameters such as binding energy, intermolecular energy, electrostatic energy, van der Waals'+H-bond+desolvo energy (E VHD ) and ligand efficiency (LE) using different in silico methods. For this study, we have used several servers and software, such as Molsoft prediction server, CASTp server, AutoDock software and LIGPLOT server. Through micro-environmental study, highest log P value was observed for linagliptin (1.07). Lowest binding energy was also observed for linagliptin with DPP-4 in the binding plot. We also identified the number of H-bonds and residues involved in the hydrophobic interactions between the DPP-4 and the anti-diabetic drugs. During interaction, two H-bonds and nine residues, two H-bonds and eleven residues as well as four H-bonds and nine residues were found between the saxagliptin, linagliptin as well as vildagliptin cases and DPP-4, respectively. Our in silico data obtained for drug-target interactions and micro-environmental signature demonstrates linagliptin as the most stable interacting drug among the tested anti-diabetic medicines.

  18. New tools for evaluating protein tyrosine sulphation: Tyrosyl Protein Sulphotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors.

    PubMed

    Byrne, Dominic P; Li, Yong; Ngamlert, Pawin; Ramakrishnan, Krithika; Eyers, Claire E; Wells, Carrow; Drewry, David H; Zuercher, William J; Berry, Neil G; Fernig, David G; Eyers, Patrick A

    2018-06-22

    Protein tyrosine sulphation is a post-translational modification best known for regulating extracellular protein-protein interactions. Tyrosine sulphation is catalysed by two Golgi-resident enzymes termed Tyrosyl Protein Sulpho Transferases (TPSTs) 1 and 2, which transfer sulphate from the co-factor PAPS (3'-phosphoadenosine 5'-phosphosulphate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulphation assays has hampered the development of chemical biology approaches for the identification of small molecule inhibitors of tyrosine sulphation. In this paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulphation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and TPST2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set (PKIS), we identified oxindole-based inhibitors of the Ser/Thr kinase RAF as low micromolar inhibitors of TPST1 and TPST2.  Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulphotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulphation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors. ©2018 The Author(s).

  19. Malignant peripheral nerve sheath tumor of the uterine cervix expressing both S-100 protein and HMB-45.

    PubMed

    Kim, Na Rae; Chung, Dong-Hae; Park, Chan Yong; Ha, Seung Yeon

    2009-12-01

    A 50-year-old woman presented with a large cervical polypoid mass. Grossly, the mass occupied a substantial proportion of the cervical canal, measuring 6 cm. Histologically, the mass showed a spindle cell malignancy arranged in large fascicles that penetrated deeply into the fibromuscular wall of the cervix. The spindle cells were immunoreactive for both S-100 protein and HMB-45 antigen, but were negative for Melan-A. Electron microscopy showed that cytoplasmic processes of the spindle to oval tumor cells contained microtubules and were lined by basal lamina and abundant intercellular collagen spacing with no melanosomes in any stage. As far as we are aware, this is the ninth reported case of cervical malignant peripheral nerve sheath tumor (MPNST), and the second reported case of MPNST expressing HMB-45 antigen.

  20. The use of shock waves in peripheral nerve regeneration: new perspectives?

    PubMed

    Hausner, Thomas; Nógrádi, Antal

    2013-01-01

    Low-energy extracorporeal shock wave treatment (ESWT) is a relatively new therapeutic tool that is widely used for the treatment of epicondylitis and plantar fasciitis and to foster bone and wound healing. Shock waves, sonic pulses with high energy impact, are thought to induce biochemical changes within the targeted tissues through mechanotransduction. The biological effects of ESWT are manifested in improved vascularization, the local release of growth factors, and local anti-inflammatory effects, but the target cells too are influenced. ESWT appears to have differential effects on peripheral nerves and has been proved to promote axonal regeneration after axotomy. This review discusses the effects of ESWT on intact and injured peripheral nerves and suggests a multiple mechanism of action. © 2013 Elsevier Inc. All rights reserved.

  1. Co-evolution of SNF spliceosomal proteins with their RNA targets in trans-splicing nematodes.

    PubMed

    Strange, Rex Meade; Russelburg, L Peyton; Delaney, Kimberly J

    2016-08-01

    Although the mechanism of pre-mRNA splicing has been well characterized, the evolution of spliceosomal proteins is poorly understood. The U1A/U2B″/SNF family (hereafter referred to as the SNF family) of RNA binding spliceosomal proteins participates in both the U1 and U2 small interacting nuclear ribonucleoproteins (snRNPs). The highly constrained nature of this system has inhibited an analysis of co-evolutionary trends between the proteins and their RNA binding targets. Here we report accelerated sequence evolution in the SNF protein family in Phylum Nematoda, which has allowed an analysis of protein:RNA co-evolution. In a comparison of SNF genes from ecdysozoan species, we found a correlation between trans-splicing species (nematodes) and increased phylogenetic branch lengths of the SNF protein family, with respect to their sister clade Arthropoda. In particular, we found that nematodes (~70-80 % of pre-mRNAs are trans-spliced) have experienced higher rates of SNF sequence evolution than arthropods (predominantly cis-spliced) at both the nucleotide and amino acid levels. Interestingly, this increased evolutionary rate correlates with the reliance on trans-splicing by nematodes, which would alter the role of the SNF family of spliceosomal proteins. We mapped amino acid substitutions to functionally important regions of the SNF protein, specifically to sites that are predicted to disrupt protein:RNA and protein:protein interactions. Finally, we investigated SNF's RNA targets: the U1 and U2 snRNAs. Both are more divergent in nematodes than arthropods, suggesting the RNAs have co-evolved with SNF in order to maintain the necessarily high affinity interaction that has been characterized in other species.

  2. Regulators of G-protein signaling and their Gα substrates: promises and challenges in their use as drug discovery targets.

    PubMed

    Kimple, Adam J; Bosch, Dustin E; Giguère, Patrick M; Siderovski, David P

    2011-09-01

    Because G-protein coupled receptors (GPCRs) continue to represent excellent targets for the discovery and development of small-molecule therapeutics, it is posited that additional protein components of the signal transduction pathways emanating from activated GPCRs themselves are attractive as drug discovery targets. This review considers the drug discovery potential of two such components: members of the "regulators of G-protein signaling" (RGS protein) superfamily, as well as their substrates, the heterotrimeric G-protein α subunits. Highlighted are recent advances, stemming from mouse knockout studies and the use of "RGS-insensitivity" and fast-hydrolysis mutations to Gα, in our understanding of how RGS proteins selectively act in (patho)physiologic conditions controlled by GPCR signaling and how they act on the nucleotide cycling of heterotrimeric G-proteins in shaping the kinetics and sensitivity of GPCR signaling. Progress is documented regarding recent activities along the path to devising screening assays and chemical probes for the RGS protein target, not only in pursuits of inhibitors of RGS domain-mediated acceleration of Gα GTP hydrolysis but also to embrace the potential of finding allosteric activators of this RGS protein action. The review concludes in considering the Gα subunit itself as a drug target, as brought to focus by recent reports of activating mutations to GNAQ and GNA11 in ocular (uveal) melanoma. We consider the likelihood of several strategies for antagonizing the function of these oncogene alleles and their gene products, including the use of RGS proteins with Gα(q) selectivity.

  3. Serologic reactivity to purified recombinant and native 29-kilodalton peripheral membrane protein of pathogenic Entamoeba histolytica.

    PubMed Central

    Flores, B M; Reed, S L; Ravdin, J I; Torian, B E

    1993-01-01

    The 29-kDa peripheral membrane protein of Entamoeba histolytica has recently been demonstrated to have epitopes on pathogenic clinical isolates which were not detected by monoclonal antibodies on nonpathogenic isolates. To analyze the serological response to this protein, we tested 93 serum specimens (from 33 patients with amebic liver abscess, 7 patients with colitis, 2 patients with ameboma, 18 individuals harboring a nonpathogenic zymodeme strain, 10 healthy Mexican migrant workers, and 23 healthy controls) by enzyme-linked immunosorbent assay (ELISA) using immunoaffinity-purified native or recombinant protein. When tested by ELISA with the native antigen, 79% (26 of 33) of the serum specimens from patients with amebic liver abscess, 4 of 9 serum specimens from symptomatic patients with colitis or ameboma, and serum from one migrant worker were positive. None of the 18 subjects harboring a nonpathogenic strain or 23 control individuals were seropositive to the native antigen (sensitivity, 71%; specificity, 98%). Of 30 serum specimens from patients with amebic liver abscess tested with recombinant antigen, 27 were seropositive (90%). In addition, six patients with colitis or ameboma and two individuals who harbored a nonpathogenic strain were seropositive to the recombinant antigen. One healthy Mexican migrant worker tested positive by both ELISAs (sensitivity, 87%; specificity, 94%). Immunoblotting of 51 serum specimens to sodium dodecyl sulfate-denatured native 29-kDa protein was less sensitive (65%) than ELISA in detecting serum antibodies to the antigen. These results suggest a similar antibody response to native and recombinant antigens (r = 0.86) and support the potential utility of a quantitative assay with defined recombinant antigen for the serodiagnosis of invasive amebiasis in nonendemic areas in conjunction with other diagnostic tools. Images PMID:8314979

  4. Identifying mRNA sequence elements for target recognition by human Argonaute proteins

    PubMed Central

    Li, Jingjing; Kim, TaeHyung; Nutiu, Razvan; Ray, Debashish; Hughes, Timothy R.; Zhang, Zhaolei

    2014-01-01

    It is commonly known that mammalian microRNAs (miRNAs) guide the RNA-induced silencing complex (RISC) to target mRNAs through the seed-pairing rule. However, recent experiments that coimmunoprecipitate the Argonaute proteins (AGOs), the central catalytic component of RISC, have consistently revealed extensive AGO-associated mRNAs that lack seed complementarity with miRNAs. We herein test the hypothesis that AGO has its own binding preference within target mRNAs, independent of guide miRNAs. By systematically analyzing the data from in vivo cross-linking experiments with human AGOs, we have identified a structurally accessible and evolutionarily conserved region (∼10 nucleotides in length) that alone can accurately predict AGO–mRNA associations, independent of the presence of miRNA binding sites. Within this region, we further identified an enriched motif that was replicable on independent AGO-immunoprecipitation data sets. We used RNAcompete to enumerate the RNA-binding preference of human AGO2 to all possible 7-mer RNA sequences and validated the AGO motif in vitro. These findings reveal a novel function of AGOs as sequence-specific RNA-binding proteins, which may aid miRNAs in recognizing their targets with high specificity. PMID:24663241

  5. Human Spermatozoa Contain Multiple Targets for Protein S-Nitrosylation: An Alternative Mechanism of the Modulation of Sperm Function by Nitric Oxide?

    PubMed Central

    Lefièvre, Linda; Chen, Yongjian; Conner, Sarah J; Scott, Joanna L; Publicover, Steve J; Ford, W Christopher L; Barratt, Christopher LR

    2009-01-01

    Nitric oxide (NO) enhances human sperm motility and capacitation associated with increased protein phosphorylation. NO activates soluble guanylyl cyclase, but can also modify protein function covalently via S-nitrosylation of cysteine. Remarkably, this mechanism remains unexplored in sperm although they depend on post-translational protein modification to achieve changes in function required for fertilisation. Our objective was to identify targets for S-nitrosylation in human sperm. Spermatozoa were incubated with NO donors and S-nitrosylated proteins were identified using the biotin switch assay and a proteomic approach using tandem mass spectrometry. 240 S-nitrosylated proteins were detected in sperm incubated with S-nitrosoglutathione. Minimal levels were observed in glutathione or untreated samples. Proteins identified consistently based on multiple peptides included established targets for S-nitrosylation in other cells e.g. tubulin,, glutathione-S-transferase and heat shock proteins but also novel targets including A-kinase anchoring protein (AKAP) types 3 and 4, voltage-dependent anion-selective channel protein 3 and semenogelin 1 and 2. In situ localisation revealed S-nitrosylated targets on the post-acrosomal region of the head and throughout the flagellum. Potential targets for S-nitrosylation in human sperm include physiologically significant proteins not previously reported in other cells. Their identification will provide novel insight into the mechanism of action of NO in spermatozoa. PMID:17683036

  6. Effects of ocular transverse chromatic aberration on peripheral word identification.

    PubMed

    Yang, Shun-Nan; Tai, Yu-chi; Laukkanen, Hannu; Sheedy, James E

    2011-11-01

    Transverse chromatic aberration (TCA) smears the retinal image of peripheral stimuli. We previously found that TCA significantly reduces the ability to recognize letters presented in the near fovea by degrading image quality and exacerbating crowding effect from adjacent letters. The present study examined whether TCA has a significant effect on near foveal and peripheral word identification, and whether within-word orthographic facilitation interacts with TCA effect to affect word identification. Subjects were briefly presented a 6- to 7-letter word of high or low frequency in each trial. Target words were generated with weak or strong horizontal color fringe to attenuate the TCA in the right periphery and exacerbate it in the left. The center of the target word was 1°, 2°, 4°, and 6° to the left or right of a fixation point. Subject's eye position was monitored with an eye-tracker to ensure proper fixation before target presentation. They were required to report the identity of the target word as soon and accurately as possible. Results show significant effect of color fringe on the latency and accuracy of word recognition, indicating existing TCA effect. Observed TCA effect was more salient in the right periphery, and was affected by word frequency more there. Individuals' subjective preference of color-fringed text was correlated to the TCA effect in the near periphery. Our results suggest that TCA significantly affects peripheral word identification, especially when it is located in the right periphery. Contextual facilitation such as word frequency interacts with TCA to influence the accuracy and latency of word recognition. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Proliferation of PD-1+ CD8 T cells in peripheral blood after PD-1–targeted therapy in lung cancer patients

    PubMed Central

    Kamphorst, Alice O.; Pillai, Rathi N.; Yang, Shu; Nasti, Tahseen H.; Sica, Gabriel L.; Yu, Ke; Koenig, Lydia; Patel, Nikita T.; Behera, Madhusmita; Wu, Hong; McCausland, Megan; Chen, Zhengjia; Zhang, Chao; Khuri, Fadlo R.; Owonikoko, Taofeek K.; Ahmed, Rafi; Ramalingam, Suresh S.

    2017-01-01

    Exhausted T cells in chronic infections and cancer have sustained expression of the inhibitory receptor programmed cell death 1 (PD-1). Therapies that block the PD-1 pathway have shown promising clinical results in a significant number of advanced-stage cancer patients. Nonetheless, a better understanding of the immunological responses induced by PD-1 blockade in cancer patients is lacking. Identification of predictive biomarkers is a priority in the field, but whether peripheral blood analysis can provide biomarkers to monitor or predict patients’ responses to treatment remains to be resolved. In this study, we analyzed longitudinal blood samples from advanced stage non–small cell lung cancer (NSCLC) patients (n = 29) receiving PD-1–targeted therapies. We detected an increase in Ki-67+ PD-1+ CD8 T cells following therapy in ∼70% of patients, and most responses were induced after the first or second treatment cycle. This T-cell activation was not indiscriminate because we observed only minimal effects on EBV-specific CD8 T cells, suggesting that responding cells may be tumor specific. These proliferating CD8 T cells had an effector-like phenotype (HLA-DR+, CD38+, Bcl-2lo), expressed costimulatory molecules (CD28, CD27, ICOS), and had high levels of PD-1 and coexpression of CTLA-4. We found that 70% of patients with disease progression had either a delayed or absent PD-1+ CD8 T-cell response, whereas 80% of patients with clinical benefit exhibited PD-1+ CD8 T-cell responses within 4 wk of treatment initiation. Our results suggest that peripheral blood analysis may provide valuable insights into NSCLC patients’ responses to PD-1–targeted therapies. PMID:28446615

  8. Peripheral Refraction, Peripheral Eye Length, and Retinal Shape in Myopia.

    PubMed

    Verkicharla, Pavan K; Suheimat, Marwan; Schmid, Katrina L; Atchison, David A

    2016-09-01

    To investigate how peripheral refraction and peripheral eye length are related to retinal shape. Relative peripheral refraction (RPR) and relative peripheral eye length (RPEL) were determined in 36 young adults (M +0.75D to -5.25D) along horizontal and vertical visual field meridians out to ±35° and ±30°, respectively. Retinal shape was determined in terms of vertex radius of curvature Rv, asphericity Q, and equivalent radius of curvature REq using a partial coherence interferometry method involving peripheral eye lengths and model eye raytracing. Second-order polynomial fits were applied to RPR and RPEL as functions of visual field position. Linear regressions were determined for the fits' second order coefficients and for retinal shape estimates as functions of central spherical refraction. Linear regressions investigated relationships of RPR and RPEL with retinal shape estimates. Peripheral refraction, peripheral eye lengths, and retinal shapes were significantly affected by meridian and refraction. More positive (hyperopic) relative peripheral refraction, more negative RPELs, and steeper retinas were found along the horizontal than along the vertical meridian and in myopes than in emmetropes. RPR and RPEL, as represented by their second-order fit coefficients, correlated significantly with retinal shape represented by REq. Effects of meridian and refraction on RPR and RPEL patterns are consistent with effects on retinal shape. Patterns derived from one of these predict the others: more positive (hyperopic) RPR predicts more negative RPEL and steeper retinas, more negative RPEL predicts more positive relative peripheral refraction and steeper retinas, and steeper retinas derived from peripheral eye lengths predict more positive RPR.

  9. In Vivo Imaging of the Central and Peripheral Effects of Sleep Deprivation and Suprachiasmatic Nuclei Lesion on PERIOD-2 Protein in Mice

    PubMed Central

    Curie, Thomas; Maret, Stephanie; Emmenegger, Yann; Franken, Paul

    2015-01-01

    Study Objectives: That sleep deprivation increases the brain expression of various clock genes has been well documented. Based on these and other findings we hypothesized that clock genes not only underlie circadian rhythm generation but are also implicated in sleep homeostasis. However, long time lags have been reported between the changes in the clock gene messenger RNA levels and their encoded proteins. It is therefore crucial to establish whether also protein levels increase within the time frame known to activate a homeostatic sleep response. We report on the central and peripheral effects of sleep deprivation on PERIOD-2 (PER2) protein both in intact and suprachiasmatic nuclei-lesioned mice. Design: In vivo and in situ PER2 imaging during baseline, sleep deprivation, and recovery. Settings: Mouse sleep-recording facility. Participants: Per2::Luciferase knock-in mice. Interventions: N/A. Measurements and Results: Six-hour sleep deprivation increased PER2 not only in the brain but also in liver and kidney. Remarkably, the effects in the liver outlasted those observed in the brain. Within the brain the increase in PER2 concerned the cerebral cortex mainly, while leaving suprachiasmatic nuclei (SCN) levels unaffected. Against expectation, sleep deprivation did not increase PER2 in the brain of arrhythmic SCN-lesioned mice because of higher PER2 levels in baseline. In contrast, liver PER2 levels did increase in these mice similar to the sham and partially lesioned controls. Conclusions: Our results stress the importance of considering both sleep-wake dependent and circadian processes when quantifying clock-gene levels. Because sleep deprivation alters PERIOD-2 in the brain as well as in the periphery, it is tempting to speculate that clock genes constitute a common pathway mediating the shared and well-known adverse effects of both chronic sleep loss and disrupted circadian rhythmicity on metabolic health. Citation: Curie T, Maret S, Emmenegger Y, Franken P. In

  10. Fragmentation of relativistic nuclei in peripheral interactions in nuclear track emulsion

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Artemenkov, D. A., E-mail: artemenkov@lhe.jinr.ru; Bradnova, V.; Chernyavsky, M. M.

    2008-09-15

    The technique of nuclear track emulsions is used to explore the fragmentation of light relativistic nuclei down to the most peripheral interactions: nuclear 'white' stars. A complete pattern of the relativistic dissociation of a 8B nucleus with target fragment accompaniment is presented. Relativistic dissociation {sup 9}Be {yields} 2{alpha} is explored using significant statistics, and a relative contribution of {sup 8}Be decays from 0+ and 2+ states is established. Target fragment accompaniments are shown for relativistic fragmentation {sup 14}N {yields} 3He +H and {sup 22}Ne {yields} 5He. The leading role of the electromagnetic dissociation on heavy nuclei with respect to breakupsmore » on target protons is demonstrated in all these cases. It is possible to conclude that the peripheral dissociation of relativistic nuclei in nuclear track emulsion is a unique tool to study many-body systems composed of the lightest nuclei and nucleons in the energy scale relevant for nuclear astrophysics.« less

  11. Challenging the state of the art in protein structure prediction: Highlights of experimental target structures for the 10th Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10.

    PubMed

    Kryshtafovych, Andriy; Moult, John; Bales, Patrick; Bazan, J Fernando; Biasini, Marco; Burgin, Alex; Chen, Chen; Cochran, Frank V; Craig, Timothy K; Das, Rhiju; Fass, Deborah; Garcia-Doval, Carmela; Herzberg, Osnat; Lorimer, Donald; Luecke, Hartmut; Ma, Xiaolei; Nelson, Daniel C; van Raaij, Mark J; Rohwer, Forest; Segall, Anca; Seguritan, Victor; Zeth, Kornelius; Schwede, Torsten

    2014-02-01

    For the last two decades, CASP has assessed the state of the art in techniques for protein structure prediction and identified areas which required further development. CASP would not have been possible without the prediction targets provided by the experimental structural biology community. In the latest experiment, CASP10, more than 100 structures were suggested as prediction targets, some of which appeared to be extraordinarily difficult for modeling. In this article, authors of some of the most challenging targets discuss which specific scientific question motivated the experimental structure determination of the target protein, which structural features were especially interesting from a structural or functional perspective, and to what extent these features were correctly reproduced in the predictions submitted to CASP10. Specifically, the following targets will be presented: the acid-gated urea channel, a difficult to predict transmembrane protein from the important human pathogen Helicobacter pylori; the structure of human interleukin (IL)-34, a recently discovered helical cytokine; the structure of a functionally uncharacterized enzyme OrfY from Thermoproteus tenax formed by a gene duplication and a novel fold; an ORFan domain of mimivirus sulfhydryl oxidase R596; the fiber protein gene product 17 from bacteriophage T7; the bacteriophage CBA-120 tailspike protein; a virus coat protein from metagenomic samples of the marine environment; and finally, an unprecedented class of structure prediction targets based on engineered disulfide-rich small proteins. Copyright © 2013 The Authors. Wiley Periodicals, Inc.

  12. Challenging the state-of-the-art in protein structure prediction: Highlights of experimental target structures for the 10th Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10

    PubMed Central

    Kryshtafovych, Andriy; Moult, John; Bales, Patrick; Bazan, J. Fernando; Biasini, Marco; Burgin, Alex; Chen, Chen; Cochran, Frank V.; Craig, Timothy K.; Das, Rhiju; Fass, Deborah; Garcia-Doval, Carmela; Herzberg, Osnat; Lorimer, Donald; Luecke, Hartmut; Ma, Xiaolei; Nelson, Daniel C.; van Raaij, Mark J.; Rohwer, Forest; Segall, Anca; Seguritan, Victor; Zeth, Kornelius; Schwede, Torsten

    2014-01-01

    For the last two decades, CASP has assessed the state of the art in techniques for protein structure prediction and identified areas which required further development. CASP would not have been possible without the prediction targets provided by the experimental structural biology community. In the latest experiment, CASP10, over 100 structures were suggested as prediction targets, some of which appeared to be extraordinarily difficult for modeling. In this paper, authors of some of the most challenging targets discuss which specific scientific question motivated the experimental structure determination of the target protein, which structural features were especially interesting from a structural or functional perspective, and to what extent these features were correctly reproduced in the predictions submitted to CASP10. Specifically, the following targets will be presented: the acid-gated urea channel, a difficult to predict trans-membrane protein from the important human pathogen Helicobacter pylori; the structure of human interleukin IL-34, a recently discovered helical cytokine; the structure of a functionally uncharacterized enzyme OrfY from Thermoproteus tenax formed by a gene duplication and a novel fold; an ORFan domain of mimivirus sulfhydryl oxidase R596; the fibre protein gp17 from bacteriophage T7; the Bacteriophage CBA-120 tailspike protein; a virus coat protein from metagenomic samples of the marine environment; and finally an unprecedented class of structure prediction targets based on engineered disulfide-rich small proteins. PMID:24318984

  13. Targeting Heat Shock Proteins in Cancer: A Promising Therapeutic Approach

    PubMed Central

    Burns, Timothy F.

    2017-01-01

    Heat shock proteins (HSPs) are a large family of chaperones that are involved in protein folding and maturation of a variety of “client” proteins protecting them from degradation, oxidative stress, hypoxia, and thermal stress. Hence, they are significant regulators of cellular proliferation, differentiation and strongly implicated in the molecular orchestration of cancer development and progression as many of their clients are well established oncoproteins in multiple tumor types. Interestingly, tumor cells are more HSP chaperonage-dependent than normal cells for proliferation and survival because the oncoproteins in cancer cells are often misfolded and require augmented chaperonage activity for correction. This led to the development of several inhibitors of HSP90 and other HSPs that have shown promise both preclinically and clinically in the treatment of cancer. In this article, we comprehensively review the roles of some of the important HSPs in cancer, and how targeting them could be efficacious, especially when traditional cancer therapies fail. PMID:28914774

  14. Targeting Heat Shock Proteins in Cancer: A Promising Therapeutic Approach.

    PubMed

    Chatterjee, Suman; Burns, Timothy F

    2017-09-15

    Heat shock proteins (HSPs) are a large family of chaperones that are involved in protein folding and maturation of a variety of "client" proteins protecting them from degradation, oxidative stress, hypoxia, and thermal stress. Hence, they are significant regulators of cellular proliferation, differentiation and strongly implicated in the molecular orchestration of cancer development and progression as many of their clients are well established oncoproteins in multiple tumor types. Interestingly, tumor cells are more HSP chaperonage-dependent than normal cells for proliferation and survival because the oncoproteins in cancer cells are often misfolded and require augmented chaperonage activity for correction. This led to the development of several inhibitors of HSP90 and other HSPs that have shown promise both preclinically and clinically in the treatment of cancer. In this article, we comprehensively review the roles of some of the important HSPs in cancer, and how targeting them could be efficacious, especially when traditional cancer therapies fail.

  15. Large-scale identification of target proteins of a glycosyltransferase isozyme by Lectin-IGOT-LC/MS, an LC/MS-based glycoproteomic approach

    PubMed Central

    Sugahara, Daisuke; Kaji, Hiroyuki; Sugihara, Kazushi; Asano, Masahide; Narimatsu, Hisashi

    2012-01-01

    Model organisms containing deletion or mutation in a glycosyltransferase-gene exhibit various physiological abnormalities, suggesting that specific glycan motifs on certain proteins play important roles in vivo. Identification of the target proteins of glycosyltransferase isozymes is the key to understand the roles of glycans. Here, we demonstrated the proteome-scale identification of the target proteins specific for a glycosyltransferase isozyme, β1,4-galactosyltransferase-I (β4GalT-I). Although β4GalT-I is the most characterized glycosyltransferase, its distinctive contribution to β1,4-galactosylation has been hardly described so far. We identified a large number of candidates for the target proteins specific to β4GalT-I by comparative analysis of β4GalT-I-deleted and wild-type mice using the LC/MS-based technique with the isotope-coded glycosylation site-specific tagging (IGOT) of lectin-captured N-glycopeptides. Our approach to identify the target proteins in a proteome-scale offers common features and trends in the target proteins, which facilitate understanding of the mechanism that controls assembly of a particular glycan motif on specific proteins. PMID:23002422

  16. False-positive rate determination of protein target discovery using a covalent modification- and mass spectrometry-based proteomics platform.

    PubMed

    Strickland, Erin C; Geer, M Ariel; Hong, Jiyong; Fitzgerald, Michael C

    2014-01-01

    Detection and quantitation of protein-ligand binding interactions is important in many areas of biological research. Stability of proteins from rates of oxidation (SPROX) is an energetics-based technique for identifying the proteins targets of ligands in complex biological mixtures. Knowing the false-positive rate of protein target discovery in proteome-wide SPROX experiments is important for the correct interpretation of results. Reported here are the results of a control SPROX experiment in which chemical denaturation data is obtained on the proteins in two samples that originated from the same yeast lysate, as would be done in a typical SPROX experiment except that one sample would be spiked with the test ligand. False-positive rates of 1.2-2.2% and <0.8% are calculated for SPROX experiments using Q-TOF and Orbitrap mass spectrometer systems, respectively. Our results indicate that the false-positive rate is largely determined by random errors associated with the mass spectral analysis of the isobaric mass tag (e.g., iTRAQ®) reporter ions used for peptide quantitation. Our results also suggest that technical replicates can be used to effectively eliminate such false positives that result from this random error, as is demonstrated in a SPROX experiment to identify yeast protein targets of the drug, manassantin A. The impact of ion purity in the tandem mass spectral analyses and of background oxidation on the false-positive rate of protein target discovery using SPROX is also discussed.

  17. Loss of intracellular lipid binding proteins differentially impacts saturated fatty acid uptake and nuclear targeting in mouse hepatocytes

    PubMed Central

    Storey, Stephen M.; McIntosh, Avery L.; Huang, Huan; Martin, Gregory G.; Landrock, Kerstin K.; Landrock, Danilo; Payne, H. Ross; Kier, Ann B.

    2012-01-01

    The liver expresses high levels of two proteins with high affinity for long-chain fatty acids (LCFAs): liver fatty acid binding protein (L-FABP) and sterol carrier protein-2 (SCP-2). Real-time confocal microscopy of cultured primary hepatocytes from gene-ablated (L-FABP, SCP-2/SCP-x, and L-FABP/SCP-2/SCP-x null) mice showed that the loss of L-FABP reduced cellular uptake of 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazo)-aminostearic acid (a fluorescent-saturated LCFA analog) by ∼50%. Importantly, nuclear targeting of the LCFA was enhanced when L-FABP was upregulated (SCP-2/SCP-x null) but was significantly reduced when L-FABP was ablated (L-FABP null), thus impacting LCFA nuclear targeting. These effects were not associated with a net decrease in expression of key membrane proteins involved in LCFA or glucose transport. Since hepatic LCFA uptake and metabolism are closely linked to glucose uptake, the effect of glucose on L-FABP-mediated LCFA uptake and nuclear targeting was examined. Increasing concentrations of glucose decreased cellular LCFA uptake and even more extensively decreased LCFA nuclear targeting. Loss of L-FABP exacerbated the decrease in LCFA nuclear targeting, while loss of SCP-2 reduced the glucose effect, resulting in enhanced LCFA nuclear targeting compared with control. Simply, ablation of L-FABP decreases LCFA uptake and even more extensively decreases its nuclear targeting. PMID:22859366

  18. Kinetic recognition of the retinoblastoma tumor suppressor by a specific protein target.

    PubMed

    Chemes, Lucía B; Sánchez, Ignacio E; de Prat-Gay, Gonzalo

    2011-09-16

    The retinoblastoma tumor suppressor (Rb) plays a key role in cell cycle control and is linked to various types of human cancer. Rb binds to the LxCxE motif, present in a number of cellular and viral proteins such as AdE1A, SV40 large T-antigen and human papillomavirus (HPV) E7, all instrumental in revealing fundamental mechanisms of tumor suppression, cell cycle control and gene expression. A detailed kinetic study of RbAB binding to the HPV E7 oncoprotein shows that an LxCxE-containing E7 fragment binds through a fast two-state reaction strongly favored by electrostatic interactions. Conversely, full-length E7 binds through a multistep process involving a pre-equilibrium between E7 conformers, a fast electrostatically driven association step guided by the LxCxE motif and a slow conformational rearrangement. This kinetic complexity arises from the conformational plasticity and intrinsically disordered nature of E7 and from multiple interaction surfaces present in both proteins. Affinity differences between E7N domains from high- and low-risk types are explained by their dissociation rates. In fact, since Rb is at the center of a large protein interaction network, fast and tight recognition provides an advantage for disruption by the viral proteins, where the balance of physiological and pathological interactions is dictated by kinetic ligand competition. The localization of the LxCxE motif within an intrinsically disordered domain provides the fast, diffusion-controlled interaction that allows viral proteins to outcompete physiological targets. We describe the interaction mechanism of Rb with a protein ligand, at the same time an LxCxE-containing model target, and a paradigmatic intrinsically disordered viral oncoprotein. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Adverse drug reaction prediction using scores produced by large-scale drug-protein target docking on high-performance computing machines.

    PubMed

    LaBute, Montiago X; Zhang, Xiaohua; Lenderman, Jason; Bennion, Brian J; Wong, Sergio E; Lightstone, Felice C

    2014-01-01

    Late-stage or post-market identification of adverse drug reactions (ADRs) is a significant public health issue and a source of major economic liability for drug development. Thus, reliable in silico screening of drug candidates for possible ADRs would be advantageous. In this work, we introduce a computational approach that predicts ADRs by combining the results of molecular docking and leverages known ADR information from DrugBank and SIDER. We employed a recently parallelized version of AutoDock Vina (VinaLC) to dock 906 small molecule drugs to a virtual panel of 409 DrugBank protein targets. L1-regularized logistic regression models were trained on the resulting docking scores of a 560 compound subset from the initial 906 compounds to predict 85 side effects, grouped into 10 ADR phenotype groups. Only 21% (87 out of 409) of the drug-protein binding features involve known targets of the drug subset, providing a significant probe of off-target effects. As a control, associations of this drug subset with the 555 annotated targets of these compounds, as reported in DrugBank, were used as features to train a separate group of models. The Vina off-target models and the DrugBank on-target models yielded comparable median area-under-the-receiver-operating-characteristic-curves (AUCs) during 10-fold cross-validation (0.60-0.69 and 0.61-0.74, respectively). Evidence was found in the PubMed literature to support several putative ADR-protein associations identified by our analysis. Among them, several associations between neoplasm-related ADRs and known tumor suppressor and tumor invasiveness marker proteins were found. A dual role for interstitial collagenase in both neoplasms and aneurysm formation was also identified. These associations all involve off-target proteins and could not have been found using available drug/on-target interaction data. This study illustrates a path forward to comprehensive ADR virtual screening that can potentially scale with increasing number

  20. Contrast-enhanced peripheral MRA: technique and contrast agents.

    PubMed

    Nielsen, Yousef W; Thomsen, Henrik S

    2012-09-01

    In the last decade contrast-enhanced magnetic resonance angiography (CE-MRA) has gained wide acceptance as a valuable tool in the diagnostic work-up of patients with peripheral arterial disease. This review presents current concepts in peripheral CE-MRA with emphasis on MRI technique and contrast agents. Peripheral CE-MRA is defined as an MR angiogram of the arteries from the aortic bifurcation to the feet. Advantages of CE-MRA include minimal invasiveness and lack of ionizing radiation. The basic technique employed for peripheral CE-MRA is the bolus-chase method. With this method a paramagnetic MRI contrast agent is injected intravenously and T1-weighted images are acquired in the subsequent arterial first-pass phase. In order to achieve high quality MR angiograms without interfering venous contamination or artifacts, a number of factors need to be taken into account. This includes magnetic field strength of the MRI system, receiver coil configuration, use of parallel imaging, contrast bolus timing technique, and k-space filling strategies. Furthermore, it is possible to optimize peripheral CE-MRA using venous compression techniques, hybrid scan protocols, time-resolved imaging, and steady-state MRA. Gadolinium(Gd)-based contrast agents are used for CE-MRA of the peripheral arteries. Extracellular Gd agents have a pharmacokinetic profile similar to iodinated contrast media. Accordingly, these agents are employed for first-pass MRA. Blood-pool Gd-based agents are characterized by prolonged intravascular stay, due to macromolecular structure or protein binding. These agents can be used for first-pass, as well as steady-state MRA. Some Gd-based contrast agents with low thermodynamic stability have been linked to development of nephrogenic systemic fibrosis in patients with severe renal insufficiency. Using optimized technique and a stable MRI contrast agent, peripheral CE-MRA is a safe procedure with diagnostic accuracy close to that of conventional catheter X

  1. Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin

    NASA Technical Reports Server (NTRS)

    Schauer-Vukasinovic, Vesna; Deo, Sapna K.; Daunert, Sylvia

    2002-01-01

    Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.

  2. Targeted proteins for diabetes drug design

    NASA Astrophysics Data System (ADS)

    Doan Trang Nguyen, Ngoc; Thi Le, Ly

    2012-03-01

    Type 2 diabetes mellitus is a common metabolism disorder characterized by high glucose in the bloodstream, especially in the case of insulin resistance and relative insulin deficiency. Nowadays, it is very common in middle-aged people and involves such dangerous symptoms as increasing risk of stroke, obesity and heart failure. In Vietnam, besides the common treatment of insulin injection, some herbal medication is used but no unified optimum remedy for the disease yet exists and there is no production of antidiabetic drugs in the domestic market yet. In the development of nanomedicine at the present time, drug design is considered as an innovative tool for researchers to study the mechanisms of diseases at the molecular level. The aim of this article is to review some common protein targets involved in type 2 diabetes, offering a new idea for designing new drug candidates to produce antidiabetic drugs against type 2 diabetes for Vietnamese people.

  3. Peripheral Serotonin: a New Player in Systemic Energy Homeostasis

    PubMed Central

    Namkung, Jun; Kim, Hail; Park, Sangkyu

    2015-01-01

    Whole body energy balance is achieved through the coordinated regulation of energy intake and energy expenditure in various tissues including liver, muscle and adipose tissues. A positive energy imbalance by excessive energy intake or insufficient energy expenditure results in obesity and related metabolic diseases. Although there have been many obesity treatment trials aimed at the reduction of energy intake, these strategies have achieved only limited success because of their associated adverse effects. An ancient neurotransmitter, serotonin is among those traditional pharmacological targets for anti-obesity treatment because it exhibits strong anorectic effect in the brain. However, recent studies suggest the new functions of peripheral serotonin in energy homeostasis ranging from the endocrine regulation by gut-derived serotonin to the autocrine/paracrine regulation by adipocyte-derived serotonin. Here, we discuss the role of serotonin in the regulation of energy homeostasis and introduce peripheral serotonin as a possible target for anti-obesity treatment. PMID:26628041

  4. Cutting Edge: Rag deletion in peripheral T cells blocks TCR revision.

    PubMed

    Hale, J Scott; Ames, Kristina T; Boursalian, Tamar E; Fink, Pamela J

    2010-06-01

    Mature CD4(+)Vbeta5(+) T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or TCR revision. In Vbeta5 transgenic mice, this latter tolerance pathway results in the appearance of CD4(+)Vbeta5(-)TCRbeta(+) T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of V(beta)-DJ(beta) recombination intermediates in peripheral CD4(+) T cells. Because postthymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We show in this study that Rag deletion in post-positive selection T cells in Vbeta5 transgenic mice blocks TCR revision in vivo and that mature peripheral T cells sorted to remove cells bearing endogenous TCRbeta-chains can express newly generated TCRbeta molecules in adoptive hosts. These findings unambiguously demonstrate postthymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4(+) T cells.

  5. Comparing sixteen scoring functions for predicting biological activities of ligands for protein targets.

    PubMed

    Xu, Weijun; Lucke, Andrew J; Fairlie, David P

    2015-04-01

    Accurately predicting relative binding affinities and biological potencies for ligands that interact with proteins remains a significant challenge for computational chemists. Most evaluations of docking and scoring algorithms have focused on enhancing ligand affinity for a protein by optimizing docking poses and enrichment factors during virtual screening. However, there is still relatively limited information on the accuracy of commercially available docking and scoring software programs for correctly predicting binding affinities and biological activities of structurally related inhibitors of different enzyme classes. Presented here is a comparative evaluation of eight molecular docking programs (Autodock Vina, Fitted, FlexX, Fred, Glide, GOLD, LibDock, MolDock) using sixteen docking and scoring functions to predict the rank-order activity of different ligand series for six pharmacologically important protein and enzyme targets (Factor Xa, Cdk2 kinase, Aurora A kinase, COX-2, pla2g2a, β Estrogen receptor). Use of Fitted gave an excellent correlation (Pearson 0.86, Spearman 0.91) between predicted and experimental binding only for Cdk2 kinase inhibitors. FlexX and GOLDScore produced good correlations (Pearson>0.6) for hydrophilic targets such as Factor Xa, Cdk2 kinase and Aurora A kinase. By contrast, pla2g2a and COX-2 emerged as difficult targets for scoring functions to predict ligand activities. Although possessing a high hydrophobicity in its binding site, β Estrogen receptor produced reasonable correlations using LibDock (Pearson 0.75, Spearman 0.68). These findings can assist medicinal chemists to better match scoring functions with ligand-target systems for hit-to-lead optimization using computer-aided drug design approaches. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Emergence of Protein Kinase CK2 as a Key Target in Cancer Therapy

    PubMed Central

    Trembley, Janeen H.; Chen, Zhong; Unger, Gretchen; Slaton, Joel; Kren, Betsy T.; Van Waes, Carter; Ahmed, Khalil

    2010-01-01

    Protein kinase CK2, a protein serine/threonine kinase, plays a global role in activities related to cell growth, cell death and cell survival. CK2 has a large number of potential substrates localized in diverse locations in the cell including, e.g., NF-κB as an important downstream target of the kinase. In addition to its involvement in cell growth and proliferation it is also a potent suppressor of apoptosis, raising its key importance in cancer cell phenotype. CK2 interacts with diverse pathways which illustrates the breadth of its impact on the cellular machinery of both cell growth and cell death giving it the status of a “master regulator” in the cell. With respect to cancer, CK2 has been found to be dysregulated in all cancers examined demonstrating increased protein expression levels and nuclear localization in cancer cells compared with their normal counterparts. We originally proposed CK2 as a potentially important target for cancer therapy. Given the ubiquitous and essential for cell survival nature of the kinase, an important consideration would be to target it specifically in cancer cells while sparing normal cells. Towards that end, our design of a tenascin based sub-50 nm (i.e., less than 50 nm size) nanocapsule in which an anti-CK2 therapeutic agent can be packaged is highly promising because this formulation can specifically deliver the cargo intracellularly to the cancer cells in vivo. Thus, appropriate strategies to target CK2 especially by molecular approaches may lead to a highly feasible and effective approach to eradication of a given cancer. PMID:20533398

  7. Searching target sites on DNA by proteins: Role of DNA dynamics under confinement

    PubMed Central

    Mondal, Anupam; Bhattacherjee, Arnab

    2015-01-01

    DNA-binding proteins (DBPs) rapidly search and specifically bind to their target sites on genomic DNA in order to trigger many cellular regulatory processes. It has been suggested that the facilitation of search dynamics is achieved by combining 3D diffusion with one-dimensional sliding and hopping dynamics of interacting proteins. Although, recent studies have advanced the knowledge of molecular determinants that affect one-dimensional search efficiency, the role of DNA molecule is poorly understood. In this study, by using coarse-grained simulations, we propose that dynamics of DNA molecule and its degree of confinement due to cellular crowding concertedly regulate its groove geometry and modulate the inter-communication with DBPs. Under weak confinement, DNA dynamics promotes many short, rotation-decoupled sliding events interspersed by hopping dynamics. While this results in faster 1D diffusion, associated probability of missing targets by jumping over them increases. In contrast, strong confinement favours rotation-coupled sliding to locate targets but lacks structural flexibility to achieve desired specificity. By testing under physiological crowding, our study provides a plausible mechanism on how DNA molecule may help in maintaining an optimal balance between fast hopping and rotation-coupled sliding dynamics, to locate target sites rapidly and form specific complexes precisely. PMID:26400158

  8. From laptop to benchtop to bedside: Structure-based Drug Design on Protein Targets

    PubMed Central

    Chen, Lu; Morrow, John K.; Tran, Hoang T.; Phatak, Sharangdhar S.; Du-Cuny, Lei; Zhang, Shuxing

    2013-01-01

    As an important aspect of computer-aided drug design, structure-based drug design brought a new horizon to pharmaceutical development. This in silico method permeates all aspects of drug discovery today, including lead identification, lead optimization, ADMET prediction and drug repurposing. Structure-based drug design has resulted in fruitful successes drug discovery targeting protein-ligand and protein-protein interactions. Meanwhile, challenges, noted by low accuracy and combinatoric issues, may also cause failures. In this review, state-of-the-art techniques for protein modeling (e.g. structure prediction, modeling protein flexibility, etc.), hit identification/optimization (e.g. molecular docking, focused library design, fragment-based design, molecular dynamic, etc.), and polypharmacology design will be discussed. We will explore how structure-based techniques can facilitate the drug discovery process and interplay with other experimental approaches. PMID:22316152

  9. Bystander protein protects potential vaccine-targeting ligands against intestinal proteolysis.

    PubMed

    Reuter, Fabian; Bade, Steffen; Hirst, Timothy R; Frey, Andreas

    2009-07-20

    Endowing mucosal vaccines with ligands that target antigen to mucosal lymphoid tissues may improve immunization efficacy provided that the ligands withstand the proteolytic environment of the gastro-intestinal tract until they reach their destination. Our aim was to investigate whether and how three renowned ligands - Ulex europaeus agglutinin I and the B subunits of cholera toxin and E. coli heat-labile enterotoxin - master this challenge. We assessed the digestive power of natural murine intestinal fluid (natIF) using assays for trypsin, chymotrypsin and pancreatic elastase along with a test for nonspecific proteolysis. The natIF was compared with simulated murine intestinal fluid (simIF) that resembled the trypsin, chymotrypsin and elastase activities of its natural counterpart but lacked or contained albumins as additional protease substrates. The ligands were exposed to the digestive fluids and degradation was determined. The studies revealed that (i) the three pancreatic endoproteases constitute only one third of the total protease activity of natIF and (ii) the ligands resist proteolysis in natIF and protein-enriched simIF over 3 h but (iii) are partially destroyed in simIF that lacks additional protease substrate. We assume that the proteins of natIF are preferred substrates for the intestinal proteases and thus can protect vaccine-targeting ligands from destruction.

  10. Proteome Dynamics in Biobanked Horse Peripheral Blood Derived Lymphocytes (PBL) with Induced Autoimmune Uveitis.

    PubMed

    Hauck, Stefanie M; Lepper, Marlen F; Hertl, Michael; Sekundo, Walter; Deeg, Cornelia A

    2017-10-01

    Equine recurrent uveitis is the only spontaneous model for recurrent autoimmune uveitis in humans, where T cells target retinal proteins. Differences between normal and autoaggressive lymphocytes were identified in this study by analyzing peripheral blood derived lymphocytes (PBL) proteomes from the same case with interphotoreceptor retinoid binding protein induced uveitis sampled before (Day 0), during (Day 15), and after uveitic attack (Day 23). Relative protein abundances of PBL were investigated in a quantitative, label-free differential proteome analysis in cells that were kept frozen for 14 years since the initial experiment. Quantitative data could be acquired for 2632 proteins at all three time points. Profound changes (≥2-fold change) in PBL protein abundance were observed when comparing Day 0 with 15, representing acute inflammation (1070 regulated proteins) and Day 0 with 23 (cessation; 1571 regulated). Significant differences applied to proteins with functions in integrin signaling during active uveitis, involving "Erk and pi-3 kinase are necessary for collagen binding in corneal epithelia," "integrins in angiogenesis," and "integrin-linked kinase signaling" pathways. In contrast, at cessation of uveitic attack, significantly changed proteins belonged to pathways of "nongenotropic androgen signaling," "classical complement pathway," and "Amb2 integrin signaling." Several members of respective pathways were earlier shown to be changed in naturally occurring uveitis, underscoring the significance of these findings here and proofing the value of the induced model in mimicking spontaneous autoimmune uveitis. All MS data have been deposited to the ProteomeXchange consortium via the PRIDE partner repository (dataset identifier PXD005580). © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Redox proteomic analysis of serum from aortic anerurysm patients: insights on oxidation of specific protein target.

    PubMed

    Spadaccio, Cristiano; Coccia, Raffaella; Perluigi, Marzia; Pupo, Gilda; Schininà, Maria Eugenia; Giorgi, Alessandra; Blarzino, Carla; Nappi, Francesco; Sutherland, Fraser W; Chello, Massimo; Di Domenico, Fabio

    2016-06-21

    oxidative stress is undoubtedly one of the main players in abdominal aortic aneurysm (AAA) pathophysiology. Recent studies in AAA patients reported an increase in the indices of oxidative damage at the tissue level and in biological fluids coupled with the loss of counter-regulatory mechanisms of protection from oxidative stress. We recently reported, in a proteomic analysis of AAA patient sera, changes in the expression of several proteins exerting important modulatory activities on cellular proliferation, differentiation and response to damage. This study aimed to explore the involvement of protein oxidation, at peripheral levels, in AAA. a redox proteomic approach was used to investigate total and specific protein carbonylation and protein-bound 4-hydroxy-2-nonenal (HNE) in the serum of AAA patients compared with age-matched controls. our results show increased oxidative damage to protein as indexed by the total carbonyl levels and total protein-bound HNE. By redox proteomics we identified specific carbonylation of three serum proteins: serum retinol-binding protein, vitamin D-binding protein and fibrinogen α-chain HNE. We also identified increased protein-bound HNE levels for hemopexin, IgK chain C region and IgK chain V-III region SIE. In addition we found a high correlation between specific protein carbonylation and protein-bound HNE and the aortic diameter. Moreover the analysis of serum proteins with antioxidant activity demonstrates the oxidation of albumin together with the overexpression of transferrin, haptoglobin and HSPs 90, 70, 60 and 32. this study support the involvement of oxidative stress in the pathogenesis of AAA and might provide a further degree of knowledge in the cause-effect role of oxidative stress shedding new light on the molecular candidates involved in the disease.

  12. Transcriptomics analysis of lungs and peripheral blood of crystalline silica-exposed rats

    PubMed Central

    Sellamuthu, Rajendran; Umbright, Christina; Roberts, Jenny R.; Chapman, Rebecca; Young, Shih-Houng; Richardson, Diana; Cumpston, Jared; McKinney, Walter; Chen, Bean T.; Frazer, David; Li, Shengqiao; Kashon, Michael; Joseph, Pius

    2015-01-01

    Minimally invasive approaches to detect/predict target organ toxicity have significant practical applications in occupational toxicology. The potential application of peripheral blood transcriptomics as a practical approach to study the mechanisms of silica-induced pulmonary toxicity was investigated. Rats were exposed by inhalation to crystalline silica (15 mg/m3, 6 h/day, 5 days) and pulmonary toxicity and global gene expression profiles of lungs and peripheral blood were determined at 32 weeks following termination of exposure. A significant elevation in bronchoalveolar lavage fluid lactate dehydrogenase activity and moderate histological changes in the lungs, including type II pneumocyte hyperplasia and fibrosis, indicated pulmonary toxicity in the rats. Similarly, significant infiltration of neutrophils and elevated monocyte chemotactic protein-1 levels in the lungs showed pulmonary inflammation in the rats. Microarray analysis of global gene expression profiles identified significant differential expression [>1.5-fold change and false discovery rate (FDR) p < 0.01] of 520 and 537 genes, respectively, in the lungs and blood of the exposed rats. Bioinformatics analysis of the differentially expressed genes demonstrated significant similarity in the biological processes, molecular networks, and canonical pathways enriched by silica exposure in the lungs and blood of the rats. Several genes involved in functions relevant to silica-induced pulmonary toxicity such as inflammation, respiratory diseases, cancer, cellular movement, fibrosis, etc, were found significantly differentially expressed in the lungs and blood of the silica-exposed rats. The results of this study suggested the potential application of peripheral blood gene expression profiling as a toxicologically relevant and minimally invasive surrogate approach to study the mechanisms underlying silica-induced pulmonary toxicity. PMID:22861000

  13. Ses proteins as possible targets for vaccine development against Staphylococcus epidermidis infections.

    PubMed

    Hofmans, Dorien; Khodaparast, Laleh; Khodaparast, Ladan; Vanstreels, Els; Shahrooei, Mohammad; Van Eldere, Johan; Van Mellaert, Lieve

    2018-05-09

    The opportunistic pathogen Staphylococcus epidermidis is progressively involved in device-related infections. Since these infections involve biofilm formation, antibiotics are not effective. Conversely, a vaccine can be advantageous to prevent these infections. In view of vaccine development, predicted surface proteins were evaluated on their potential as a vaccine target. Immunoglobulins directed against S. epidermidis surface proteins SesB, M, O, Q and R, were used to firstly affirm their surface location. Further, inhibitory effects of these IgGs on biofilm formation were determined in vitro on polystyrene and polyurethane surfaces and in vivo using a subcutaneous catheter mouse model. We also examined the opsonophagocytic capacity of these IgGs. Surface localization of the five Ses proteins was demonstrated both for planktonic and sessile cells, though to a variable extent. Ses-specific IgGs added to planktonic cells had a variable inhibitory effect on cell adhesion to polystyrene, while only anti-SesO IgGs decreased cell attachment to polyurethane catheters. Although phagocytic killing was only obtained after opsonisation with SesB-specific IgGs, a significant reduction of in vivo formed biofilms was observed after administration of SesB-, SesM- and SesO-specific IgGs. Regardless of their characterization or function, S. epidermidis surface proteins can be adequate targets for vaccine development aiming the prevention of device-related infections caused by invasive S. epidermidis strains. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Targeting functional motifs of a protein family

    NASA Astrophysics Data System (ADS)

    Bhadola, Pradeep; Deo, Nivedita

    2016-10-01

    The structural organization of a protein family is investigated by devising a method based on the random matrix theory (RMT), which uses the physiochemical properties of the amino acid with multiple sequence alignment. A graphical method to represent protein sequences using physiochemical properties is devised that gives a fast, easy, and informative way of comparing the evolutionary distances between protein sequences. A correlation matrix associated with each property is calculated, where the noise reduction and information filtering is done using RMT involving an ensemble of Wishart matrices. The analysis of the eigenvalue statistics of the correlation matrix for the β -lactamase family shows the universal features as observed in the Gaussian orthogonal ensemble (GOE). The property-based approach captures the short- as well as the long-range correlation (approximately following GOE) between the eigenvalues, whereas the previous approach (treating amino acids as characters) gives the usual short-range correlations, while the long-range correlations are the same as that of an uncorrelated series. The distribution of the eigenvector components for the eigenvalues outside the bulk (RMT bound) deviates significantly from RMT observations and contains important information about the system. The information content of each eigenvector of the correlation matrix is quantified by introducing an entropic estimate, which shows that for the β -lactamase family the smallest eigenvectors (low eigenmodes) are highly localized as well as informative. These small eigenvectors when processed gives clusters involving positions that have well-defined biological and structural importance matching with experiments. The approach is crucial for the recognition of structural motifs as shown in β -lactamase (and other families) and selectively identifies the important positions for targets to deactivate (activate) the enzymatic actions.

  15. Thiazolidine-2,4-dione derivatives: programmed chemical weapons for key protein targets of various pathological conditions.

    PubMed

    Chadha, Navriti; Bahia, Malkeet Singh; Kaur, Maninder; Silakari, Om

    2015-07-01

    Thiazolidine-2,4-dione is an extensively explored heterocyclic nucleus for designing of novel agents implicated for a wide variety of pathophysiological conditions, that is, diabetes, diabetic complications, cancer, arthritis, inflammation, microbial infection, and melanoma, etc. The current paradigm of drug development has shifted to the structure-based drug design, since high-throughput screenings have continued to generate disappointing results. The gap between hit generation and drug establishment can be narrowed down by investigation of ligand interactions with its receptor protein. Therefore, it would always be highly beneficial to gain knowledge of molecular level interactions between specific protein target and developed ligands; since this information can be maneuvered to design new molecules with improved protein fitting. Thus, considering this aspect, we have corroborated the information about molecular (target) level implementations of thiazolidine-2,4-diones (TZD) derivatives having therapeutic implementations such as, but not limited to, anti-diabetic (glitazones), anti-cancer, anti-arthritic, anti-inflammatory, anti-oxidant and anti-microbial, etc. The structure based SAR of TZD derivatives for various protein targets would serve as a benchmark for the alteration of existing ligands to design new ones with better binding interactions. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Biophysical characterization of interactions between the C-termini of peripheral nerve claudins and the PDZ₁ domain of zonula occludens.

    PubMed

    Wu, Jiawen; Peng, Dungeng; Zhang, Yang; Lu, Zhenwei; Voehler, Markus; Sanders, Charles R; Li, Jun

    2015-03-27

    Our recent study has shown that cellular junctions in myelin and in the epi-/perineruium that encase nerve fibers regulate the permeability of the peripheral nerves. This permeability may affect propagation of the action potential. Direct interactions between the PDZ₁ domain of zonula occludens (ZO₁ or ZO₂) and the C-termini of claudins are known to be crucial for the formation of tight junctions. Using the purified PDZ₁ domain of ZO₂ and a variety of C-terminal mutants of peripheral nerve claudins (claudin-1, claudin-2, claudin-3, claudin-5 in epi-/perineurium; claudin-19 in myelin), we have utilized NMR spectroscopy to determine specific roles of the 3 C-terminal claudin residues (position -2, -1, 0) for their interactions with PDZ₁ of ZO₂. In contrast to the canonical model that emphasizes the importance of residues at the -2 and 0 positions, our results demonstrate that, for peripheral nerve claudins, the residue at position -1 plays a critical role in association with PDZ₁, while the side-chain of residue 0 plays a significant but lesser role. Surprisingly, claudin-19, the most abundant claudin in myelin, exhibited no binding to ZO₂. These findings reveal that the binding mechanism of claudin/ZO in epi-/perineurium is distinct from the canonical interactions between non-ZO PDZ-containing proteins with their ligands. This observation provides the molecular basis for a strategy to develop drugs that target tight junctions in the epi-/perineurium of peripheral nerves. Published by Elsevier Inc.

  17. [Peripheral paralysis of facial nerve in children].

    PubMed

    Steczkowska-Klucznik, Małgorzata; Kaciński, Marek

    2006-01-01

    Peripheral facial paresis is one of the most common diagnosed neuropathies in adults and also in children. Many factors can trigger facial paresis and most frequent are infectious, carcinoma and demyelinisation diseases. Very important and interesting problem is an idiopathic facial paresis (Bell's palsy). Actually the main target of scientific research is to assess the etiology (infectious, genetic, immunologic) and to find the most appropriate treatment.

  18. Drug-target residence time--a case for G protein-coupled receptors.

    PubMed

    Guo, Dong; Hillger, Julia M; IJzerman, Adriaan P; Heitman, Laura H

    2014-07-01

    A vast number of marketed drugs act on G protein-coupled receptors (GPCRs), the most successful category of drug targets to date. These drugs usually possess high target affinity and selectivity, and such combined features have been the driving force in the early phases of drug discovery. However, attrition has also been high. Many investigational new drugs eventually fail in clinical trials due to a demonstrated lack of efficacy. A retrospective assessment of successfully launched drugs revealed that their beneficial effects in patients may be attributed to their long drug-target residence times (RTs). Likewise, for some other GPCR drugs short RT could be beneficial to reduce the potential for on-target side effects. Hence, the compounds' kinetics behavior might in fact be the guiding principle to obtain a desired and durable effect in vivo. We therefore propose that drug-target RT should be taken into account as an additional parameter in the lead selection and optimization process. This should ultimately lead to an increased number of candidate drugs moving to the preclinical development phase and on to the market. This review contains examples of the kinetics behavior of GPCR ligands with improved in vivo efficacy and summarizes methods for assessing drug-target RT. © 2014 Wiley Periodicals, Inc.

  19. Nmnat mitigates sensory dysfunction in a Drosophila model of paclitaxel-induced peripheral neuropathy.

    PubMed

    Brazill, Jennifer M; Cruz, Beverley; Zhu, Yi; Zhai, R Grace

    2018-06-12

    Chemotherapy-induced peripheral neuropathy (CIPN) is the major dose-limiting side effect of many commonly used chemotherapeutic agents, including paclitaxel. Currently, there are no neuroprotective or effective symptomatic treatments for CIPN. Lack of understanding of the in vivo mechanisms of CIPN has greatly impeded the identification of therapeutic targets. Here, we optimized a model of paclitaxel-induced peripheral neuropathy using Drosophila larvae that recapitulates aspects of chemotherapy-induced sensory dysfunction . We showed that nociceptive sensitivity is associated with disrupted organization of microtubule-associated MAP1B/Futsch and aberrant stabilization of peripheral sensory dendrites. These findings establish a robust and amenable model for studying peripheral mechanisms of CIPN. Using this model, we uncovered a critical role for nicotinamide mononucleotide adenylyltransferase (Nmnat) in maintaining the integrity and function of peripheral sensory neurons and uncovered Nmnat's therapeutic potential against diverse sensory symptoms of CIPN. © 2018. Published by The Company of Biologists Ltd.

  20. Lipid-Mediated Targeting with Membrane Wrapped Nanoparticles in the Presence of Corona Formation

    PubMed Central

    Xu, Fangda; Reiser, Michael; Yu, Xinwei; Gummuluru, Suryaram; Wetzler, Lee; Reinhard, Björn M.

    2016-01-01

    Membrane wrapped nanoparticles represent a versatile platform for utilizing specific lipid-receptor interactions, such as siallyllactose-mediated binding of the ganglioside GM3 to Siglec1 (CD169), for targeting purposes. The membrane wrap around the nanoparticles does not only serve as a matrix to incorporate GM3 as targeting moiety for antigen presenting cells but also offers unique opportunities for constructing a biomimetic surface from lipids with potentially protein repellent properties. We characterize non-specific protein adsorption (corona formation) to membrane wrapped nanoparticles with core diameters of approx. 35 nm and 80 nm and its effect on the GM3-mediated targeting efficacy as function of surface charge through combined in vitro and in vivo studies. The stability and fate of the membrane wrap around the nanoparticles in a simulated biological fluid and after uptake in CD169 expressing antigen presenting cells is experimentally tested. Finally, we demonstrate in hock immunization studies in mice that GM3 decorated membrane wrapped nanoparticles achieve a selective enrichment in the peripheral regions of popliteal lymph nodes that contain high concentrations of CD169 expressing antigen presenting cells. PMID:26720275