Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding.

PubMed

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-05-01

The P2X(7) receptor exhibits complex pharmacological properties. In this study, binding of a [(3)H]-labelled P2X(7) receptor antagonist to human P2X(7) receptors has been examined to further understand ligand interactions with this receptor. The P2X(7) receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.1(3,7)]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X(7) receptors. Binding of [(3)H]-compound-17 was higher in membranes prepared from cells expressing P2X(7) receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X(7) receptors. Binding was reversible, saturable and modulated by P2X(7) receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. These data demonstrate that human P2X(7) receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X(7) receptor complex enhances subsequent binding to other P2X(7) subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X(7) receptor.

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding

PubMed Central

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-01-01

Background and Purpose: The P2X7 receptor exhibits complex pharmacological properties. In this study, binding of a [3H]-labelled P2X7 receptor antagonist to human P2X7 receptors has been examined to further understand ligand interactions with this receptor. Experimental Approach: The P2X7 receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X7 receptors. Key Results: Binding of [3H]-compound-17 was higher in membranes prepared from cells expressing P2X7 receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X7 receptors. Binding was reversible, saturable and modulated by P2X7 receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. Conclusions: These data demonstrate that human P2X7 receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X7 receptor complex enhances subsequent binding to other P2X7 subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X7 receptor. PMID:17339830

Activation of Basal Gluconeogenesis by Coactivator p300 Maintains Hepatic Glycogen Storage

PubMed Central

Cao, Jia; Meng, Shumei; Ma, Anlin; Radovick, Sally; Wondisford, Fredric E.

2013-01-01

Because hepatic glycogenolysis maintains euglycemia during early fasting, proper hepatic glycogen synthesis in the fed/postprandial states is critical. It has been known for decades that gluconeogenesis is essential for hepatic glycogen synthesis; however, the molecular mechanism remains unknown. In this report, we show that depletion of hepatic p300 reduces glycogen synthesis, decreases hepatic glycogen storage, and leads to relative hypoglycemia. We previously reported that insulin suppressed gluconeogenesis by phosphorylating cAMP response element binding protein-binding protein (CBP) at S436 and disassembling the cAMP response element-binding protein-CBP complex. However, p300, which is closely related to CBP, lacks the corresponding S436 phosphorylation site found on CBP. In a phosphorylation-competent p300G422S knock-in mouse model, we found that mutant mice exhibited reduced hepatic glycogen content and produced significantly less glycogen in a tracer incorporation assay in the postprandial state. Our study demonstrates the important and unique role of p300 in glycogen synthesis through maintaining basal gluconeogenesis. PMID:23770612

Donor-π-Acceptor Polymer with Alternating Triarylborane and Triphenylamine Moieties.

PubMed

Li, Haiyan; Jäkle, Frieder

2010-05-12

A luminescent main chain donor-π-acceptor-type polymer (4) was prepared via organometallic polycondensation reaction followed by post modification. With both electron-rich amine and electron-deficient borane moieties embedded in the main chain, 4 exhibits an interesting ambipolar character: it can be reduced and oxidized electrochemically at moderate potentials and shows a strong solvatochromic effect in the emission spectra. Complexation studies show that 4 selectively binds to fluoride and cyanide; quantitative titration with cyanide reveals a two-step binding process. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The role of attention in binding visual features in working memory: evidence from cognitive ageing.

PubMed

Brown, Louise A; Brockmole, James R

2010-10-01

Two experiments were conducted to assess the costs of attentional load during a feature (colour-shape) binding task in younger and older adults. Experiment 1 showed that a demanding backwards counting task, which draws upon central executive/general attentional resources, reduced binding to a greater extent than individual feature memory, but the effect was no greater in older than in younger adults. Experiment 2 showed that presenting memory items sequentially rather than simultaneously, such that items are required to be maintained while new representations are created, selectively affects binding performance in both age groups. Although this experiment exhibited an age-related binding deficit overall, both age groups were affected by the attention manipulation to an equal extent. While a role for attentional processes in colour-shape binding was apparent across both experiments, manipulations of attention exerted equal effects in both age groups. We therefore conclude that age-related binding deficits neither emerge nor are exacerbated under conditions of high attentional load. Implications for theories of visual working memory and cognitive ageing are discussed.

The D-galactose specific lectin of field bean (Dolichos lablab) seed binds sugars with extreme negative cooperativity and half-of-the-sites binding.

PubMed

Rao, Devavratha H; Gowda, Lalitha R

2012-08-15

The field bean (Dolichos lablab) lectin designated as PPO-haemagglutinin (DLL-II) is bifunctional, exhibiting both polyphenol oxidase and haemagglutinating activity. The lectin is unusual in that it binds galactose (Gal), lactose (Lac) and N-acetylgalactosamine (GalNAc) only in the presence of (NH₄)₂SO₄ and exhibits negative cooperativity and half-of-the-sites binding. Circular dichroism, isothermal titration calorimetry and fluorescence quenching were used to assess the sugar binding in the presence of (NH₄)₂O₄. Comparison of the near-UV CD spectra with and without bound sugar revealed ligand induced conformational changes. The intrinsic fluorescence quenching data indicate that DLL-II exhibits weak binding to Gal in the presence of (NH₄)₂SO₄ with a stoichiometry of one bound ligand per dimer. ITC data fitted using a two sets of sites binding model presented a similar picture. The K(a)'s for Gal, Lac and GalNAc in the presence of (NH₄)₂SO₄ were 0.16±0.002, 0.21±0.004 and 8.45±0.78 (×10⁻³) M⁻¹ respectively. The Hill plot for the binding of these sugars to DLL-II was curvilinear with a tangent slope <1.0 indicating negative cooperativity. DLL-II thus exhibits half-of-the-site binding, an extreme form of negative cooperativity in which the second ligand does not bind at all. This is the first report of a legume lectin, exhibiting half-of-the-sites binding. Copyright © 2012 Elsevier Inc. All rights reserved.

A label-free aptamer-fluorophore assembly for rapid and specific detection of cocaine in biofluids.

PubMed

Roncancio, Daniel; Yu, Haixiang; Xu, Xiaowen; Wu, Shuo; Liu, Ran; Debord, Joshua; Lou, Xinhui; Xiao, Yi

2014-11-18

We report a rapid and specific aptamer-based method for one-step cocaine detection with minimal reagent requirements. The feasibility of aptamer-based detection has been demonstrated with sensors that operate via target-induced conformational change mechanisms, but these have generally exhibited limited target sensitivity. We have discovered that the cocaine-binding aptamer MNS-4.1 can also bind the fluorescent molecule 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) and thereby quench its fluorescence. We subsequently introduced sequence changes into MNS-4.1 to engineer a new cocaine-binding aptamer (38-GC) that exhibits higher affinity to both ligands, with reduced background signal and increased signal gain. Using this aptamer, we have developed a new sensor platform that relies on the cocaine-mediated displacement of ATMND from 38-GC as a result of competitive binding. We demonstrate that our sensor can detect cocaine within seconds at concentrations as low as 200 nM, which is 50-fold lower than existing assays based on target-induced conformational change. More importantly, our assay achieves successful cocaine detection in body fluids, with a limit of detection of 10.4, 18.4, and 36 μM in undiluted saliva, urine, and serum samples, respectively.

DNA mutagenic activity and capacity for HIV-1 restriction of the cytidine deaminase APOBEC3G depend on whether DNA or RNA binds to tyrosine 315

PubMed Central

Polevoda, Bogdan; Joseph, Rebecca; Friedman, Alan E.; Bennett, Ryan P.; Greiner, Rebecca; De Zoysa, Thareendra; Stewart, Ryan A.; Smith, Harold C.

2017-01-01

APOBEC3G (A3G) belongs to the AID/APOBEC protein family of cytidine deaminases (CDA) that bind to nucleic acids. A3G mutates the HIV genome by deamination of dC to dU, leading to accumulation of virus-inactivating mutations. Binding to cellular RNAs inhibits A3G binding to substrate single-stranded (ss) DNA and CDA activity. Bulk RNA and substrate ssDNA bind to the same three A3G tryptic peptides (amino acids 181–194, 314–320, and 345–374) that form parts of a continuously exposed protein surface extending from the catalytic domain in the C terminus of A3G to its N terminus. We show here that the A3G tyrosines 181 and 315 directly cross-linked ssDNA. Binding experiments showed that a Y315A mutation alone significantly reduced A3G binding to both ssDNA and RNA, whereas Y181A and Y182A mutations only moderately affected A3G nucleic acid binding. Consistent with these findings, the Y315A mutant exhibited little to no deaminase activity in an Escherichia coli DNA mutator reporter, whereas Y181A and Y182A mutants retained ∼50% of wild-type A3G activity. The Y315A mutant also showed a markedly reduced ability to assemble into viral particles and had reduced antiviral activity. In uninfected cells, the impaired RNA-binding capacity of Y315A was evident by a shift of A3G from high-molecular-mass ribonucleoprotein complexes to low-molecular-mass complexes. We conclude that Tyr-315 is essential for coordinating ssDNA interaction with or entry to the deaminase domain and hypothesize that RNA bound to Tyr-315 may be sufficient to competitively inhibit ssDNA deaminase-dependent antiviral activity. PMID:28381554

Identification of a Second Substrate-binding Site in Solute-Sodium Symporters*

PubMed Central

Li, Zheng; Lee, Ashley S. E.; Bracher, Susanne; Jung, Heinrich; Paz, Aviv; Kumar, Jay P.; Abramson, Jeff; Quick, Matthias; Shi, Lei

2015-01-01

The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ∼1. In addition, the related and more experimentally tractable SSS member PutP (the Na+/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport. PMID:25398883

Engineering Ascorbate Peroxidase Activity Into Cytochrome C Peroxidase

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

Meharenna, Y.T.; Oertel, P.; Bhaskar, B.

2009-05-26

Cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX) have very similar structures, and yet neither CCP nor APX exhibits each others activities with respect to reducing substrates. APX has a unique substrate binding site near the heme propionates where ascorbate H-bonds with a surface Arg and one heme propionate (Sharp et al. (2003) Nat. Struct. Biol. 10, 303--307). The corresponding region in CCP has a much longer surface loop, and the critical Arg residue that is required for ascorbate binding in APX is Asn in CCP. In order to convert CCP into an APX, the ascorbate-binding loop and critical argininemore » were engineered into CCP to give the CCP2APX mutant. The mutant crystal structure shows that the engineered site is nearly identical to that found in APX. While wild-type CCP shows no APX activity, CCP2APX catalyzes the peroxidation of ascorbate at a rate of {approx}12 min{sup -1}, indicating that the engineered ascorbate-binding loop can bind ascorbate.« less

Cows with follicular fluid androgen excess exhibit anovulation and have altered circulating sex hormone binding globulin, gonadotropin secretion and plasma and follicular fluid composition

USDA-ARS?s Scientific Manuscript database

Our laboratory identified a group of cows with excess intrafollicular concentrations of androstenedione (A4; >30 fold), reduced calving rates, and theca gene expression profiles similar to women with Polycystic Ovary Syndrome (PCOS). Based on these previous studies, we hypothesized that High A4 cows...

Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length

PubMed Central

Zheng, Xiangdong; Ramani, Anand; Soni, Komal; Gottardo, Marco; Zheng, Shuangping; Ming Gooi, Li; Li, Wenjing; Feng, Shan; Mariappan, Aruljothi; Wason, Arpit; Widlund, Per; Pozniakovsky, Andrei; Poser, Ina; Deng, Haiteng; Ou, Guangshuo; Riparbelli, Maria; Giuliano, Callaini; Hyman, Anthony A.; Sattler, Michael; Gopalakrishnan, Jay; Li, Haitao

2016-01-01

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAPF375A, with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAPEE343RR that unmasks the β-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a ‘clutch-like' mechanism. PMID:27306797

Electrostatic redesign of the [myoglobin, cytochrome b5] interface to create a well-defined docked complex with rapid interprotein electron transfer.

PubMed

Xiong, Peng; Nocek, Judith M; Griffin, Amanda K K; Wang, Jingyun; Hoffman, Brian M

2009-05-27

Cyt b(5) is the electron-carrier "repair" protein that reduces met-Mb and met-Hb to their O(2)-carrying ferroheme forms. Studies of electron transfer (ET) between Mb and cyt b(5) revealed that they react on a "Dynamic Docking" (DD) energy landscape on which binding and reactivity are uncoupled: binding is weak and involves an ensemble of nearly isoenergetic configurations, only a few of which are reactive; those few contribute negligibly to binding. We set the task of redesigning the surface of Mb so that its reaction with cyt b(5) instead would occur on a conventional "simple docking" (SD) energy landscape, on which a complex exhibits a well-defined (set of) reactive binding configuration(s), with binding and reactivity thus no longer being decoupled. We prepared a myoglobin (Mb) triple mutant (D44K/D60K/E85K; Mb(+6)) substituted with Zn-deuteroporphyrin and monitored cytochrome b(5) (cyt b(5)) binding and electron transfer (ET) quenching of the (3)ZnMb(+6) triplet state. In contrast, to Mb(WT), the three charge reversals around the "front-face" heme edge of Mb(+6) have directed cyt b(5) to a surface area of Mb adjacent to its heme, created a well-defined, most-stable structure that supports good ET pathways, and apparently coupled binding and ET: both K(a) and k(et) are increased by the same factor of approximately 2 x 10(2), creating a complex that exhibits a large ET rate constant, k(et) = 10(6 1) s(-1), and is in slow exchange (k(off) < k(et)). In short, these mutations indeed appear to have created the sought-for conversion from DD to simple docking (SD) energy landscapes.

Mucin-like Region of Herpes Simplex Virus Type 1 Attachment Protein Glycoprotein C (gC) Modulates the Virus-Glycosaminoglycan Interaction*

PubMed Central

Altgärde, Noomi; Eriksson, Charlotta; Peerboom, Nadia; Phan-Xuan, Tuan; Moeller, Stephanie; Schnabelrauch, Matthias; Svedhem, Sofia; Trybala, Edward; Bergström, Tomas; Bally, Marta

2015-01-01

Glycoprotein C (gC) mediates the attachment of HSV-1 to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying a HSV-1 mutant lacking the mucin-like domain in gC and the corresponding purified mutant protein (gCΔmuc) in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared with native HSV-1 (i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells and reduced release of viral particles from the surface of infected cells). Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared with native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells. PMID:26160171

Adjudin attenuates lipopolysaccharide (LPS)- and ischemia-induced microglial activation

PubMed Central

Shao, Jiaxiang; Liu, Tengyuan; Xie, Qian Reuben; Zhang, Tingting; Yu, Hemei; Wang, Boshi; Ying, Weihai; Mruk, Dolores D.; Silvestrini, Bruno; Cheng, C. Yan; Xia, Weiliang

2014-01-01

Neuroinflammation caused by microglial activation plays a key role in ischemia, neurodegeneration and many other CNS diseases. In this study, we found that Adjudin, a potential non-hormonal male contraceptive, exhibits additional function to reduce the production of proinflammatory mediators. Adjudin significantly inhibited LPS-induced IL-6 release and IL-6, IL-1β, TNF-α expression in BV2 microglial cells. Furthermore, Adjudin exhibited anti-inflammatory properties by suppression of NF-κB p65 nuclear translocation and DNA binding activity as well as ERK MAPK phosphorylation. To determine the in vivo effect of Adjudin, we used a permanent middle cerebral artery occlusion (pMCAO) mouse model and found that Adjudin could reduce ischemia-induced CD11b expression, a marker of microglial activation. Furthermore, Adjudin treatment attenuated brain edema and neurological deficits after ischemia but did not reduce infarct volume. Thus, our data suggest that Adjudin may be useful for mitigating neuroinflammation. PMID:23084372

Dynamic Conformational Changes in MUNC18 Prevent Syntaxin Binding

PubMed Central

Bar-On, Dana; Nachliel, Esther; Gutman, Menachem; Ashery, Uri

2011-01-01

The Sec1/munc18 protein family is essential for vesicle fusion in eukaryotic cells via binding to SNARE proteins. Protein kinase C modulates these interactions by phosphorylating munc18a thereby reducing its affinity to one of the central SNARE members, syntaxin-1a. The established hypothesis is that the reduced affinity of the phosphorylated munc18a to syntaxin-1a is a result of local electrostatic repulsion between the two proteins, which interferes with their compatibility. The current study challenges this paradigm and offers a novel mechanistic explanation by revealing a syntaxin-non-binding conformation of munc18a that is induced by the phosphomimetic mutations. In the present study, using molecular dynamics simulations, we explored the dynamics of the wild-type munc18a versus phosphomimetic mutant munc18a. We focused on the structural changes that occur in the cavity between domains 3a and 1, which serves as the main syntaxin-binding site. The results of the simulations suggest that the free wild-type munc18a exhibits a dynamic equilibrium between several conformations differing in the size of its cavity (the main syntaxin-binding site). The flexibility of the cavity's size might facilitate the binding or unbinding of syntaxin. In silico insertion of phosphomimetic mutations into the munc18a structure induces the formation of a conformation where the syntaxin-binding area is rigid and blocked as a result of interactions between residues located on both sides of the cavity. Therefore, we suggest that the reduced affinity of the phosphomimetic mutant/phosphorylated munc18a is a result of the closed-cavity conformation, which makes syntaxin binding energetically and sterically unfavorable. The current study demonstrates the potential of phosphoryalation, an essential biological process, to serve as a driving force for dramatic conformational changes of proteins modulating their affinity to target proteins. PMID:21390273

Organic Sulfur Associated with Aquatic Humic Substances

NASA Astrophysics Data System (ADS)

Aiken, G.; Vairavamurthy, M. A.; Ravichandran, M.

2003-12-01

This study examines the speciation and reactivity of organic sulfur associated with dissolved organic matter isolated from a variety of freshwater environments and the Pacific Ocean. The isolates, which included aquatic humic substances, were obtained using XAD resins and exhibited a wide range of elemental compositions, aromatic carbon contents, and molecular weights. Organic sulfur contents for the samples ranged from 0.4% to 1.9% of the atomic composition and were strongly dependent on the redox chemistry of the environments whence the samples originated, especially with regard to potential interactions with sulfide in sulfate reducing environments. The speciation of the sulfur associated with these samples was investigated using X-ray adsorption near edge spectroscopy (XANES). The samples, all obtained from oxic environments, contained reduced sulfur moieties. Reduced sulfur content (thiophene, organic sulfides and thiols) ranged from 22-70%. In general, humic acid fractions were found to have the largest percentage of reduced sulfur, followed by the fulvic acid and hydrophobic acid fractions. Hydrophilic fractions of the DOC contained a large percentage of oxidized organic sulfur (sulfonate and sulfate moieties). To assess the significance of reduced S content on interactions with soft metals, an environmentally significant process, the binding strength and binding capacity of Hg with organic matter isolated from the Florida Everglades were determined using equilibrium dialysis ligand exchange. Based on elemental analyses and XANES, the DOM sample from the Everglades used in our binding experiments had a reduced-S content of approximately 1.0%. Very strong interactions (KDOM' = 1023.2+/-0.5 L kg-1) were observed at Hg/DOM ratios below approximately 1 μ g Hg per mg DOM. Only a small fraction (approximately 2%) of the reduced-S groups were involved with the strongest interactions between Hg and DOM, suggesting that the binding of Hg to DOM under natural conditions (very low Hg/DOM ratios ranging from 0.01 to 10 ng of Hg/mg of DOM) is controlled by a small fraction of DOM molecules containing reactive sulfur functional groups.

Cupiennin 1a exhibits a remarkably broad, non-stereospecific cytolytic activity on bacteria, protozoan parasites, insects, and human cancer cells.

PubMed

Kuhn-Nentwig, Lucia; Willems, Jean; Seebeck, Thomas; Shalaby, Tarek; Kaiser, Marcel; Nentwig, Wolfgang

2011-01-01

Cupiennin 1a, a cytolytic peptide isolated from the venom of the spider Cupiennius salei, exhibits broad membranolytic activity towards bacteria, trypanosomes, and plasmodia, as well as human blood and cancer cells. In analysing the cytolytic activity of synthesised all-D: - and all-L: -cupiennin 1a towards pro- and eukaryotic cells, a stereospecific mode of membrane destruction could be excluded. The importance of negatively charged sialic acids on the outer leaflet of erythrocytes for the binding and haemolytic activity of L: -cupiennin 1a was demonstrated. Reducing the overall negative charges of erythrocytes by partially removing their sialic acids or by protecting them with tri- or pentalysine results in reduced haemolytic activity of the peptide.

Effect of Disease-Related Changes in Plasma Albumin on the Pharmacokinetics of Naproxen in Male and Female Arthritic Rats

PubMed Central

Li, Xiaonan; DuBois, Debra C.; Almon, Richard R.

2017-01-01

Naproxen (NPX) is used in the treatment of rheumatoid arthritis (RA) for alleviation of pain and inflammation. In view of the extensive albumin binding of NPX, this study investigates whether chronic inflammation and sex influence the physiologic albumin concentrations, plasma protein binding, and pharmacokinetics (PK) of NPX. The PK of NPX was evaluated in a rat model of RA [collagen-induced arthritis (CIA) in Lewis rats] and in healthy controls. These PK studies included 1) NPX in female and male CIA rats that received 10, 25, or 50 mg/kg NPX i.p.; and 2) NPX in healthy female and male rats after i.p. dosing of NPX at 50 mg/kg. Plasma albumin concentrations were quantified by enzyme-linked immunosorbent assay, and protein binding was assessed using ultrafiltration. The NPX concentrations in plasma and filtrates were determined by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Plasma concentration-time data of NPX were first assessed by noncompartmental analysis (NCA). Nonlinear PK as indicated by dose-dependent NCA clearances and distribution volumes was observed. A two-compartment model with a first-order absorption process incorporating nonlinear protein binding in plasma and tissues jointly described the PK data of all groups. Saturable albumin binding accounts for the nonlinearity of NPX PK in all rats as well as part of the PK differences in arthritic rats. The CIA rats exhibited reduced albumin concentrations, reduced overall protein binding, and reduced clearances of unbound NPX, consistent with expectations during inflammation. The net effect of chronic inflammation was an elevation of the Cmax and area under the plasma concentration-time curve (AUC) of unbound drug. PMID:28246126

AmrZ Beta-Sheet Residues Are Essential for DNA Binding and Transcriptional Control of Pseudomonas aeruginosa Virulence Genes ▿ †

PubMed Central

Waligora, Elizabeth A.; Ramsey, Deborah M.; Pryor, Edward E.; Lu, Haiping; Hollis, Thomas; Sloan, Gina P.; Deora, Rajendar; Wozniak, Daniel J.

2010-01-01

AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the β-sheet for DNA binding, while the α-helices promote oligomerization. AmrZ is of interest due to its dual roles as a transcriptional activator and as a repressor, regulating genes encoding virulence factors associated with both chronic and acute Pseudomonas aeruginosa infection. In this study, cross-linking revealed that AmrZ forms oligomers in solution but that the amino terminus, containing an unordered region and a β-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA binding. Mutagenesis of the AmrZ β-sheet demonstrated that residues 18, 20, and 22 were essential for DNA binding at both activation and repressor sites, suggesting that AmrZ utilizes a similar mechanism for binding to these sites. Mice infected with amrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Direct in vivo competition assays showed a 5-fold competitive advantage for the wild type over an isogenic amrZ mutant. Finally, the reduced infection phenotype of the amrZ-null strain was similar to that of a strain expressing a DNA-binding-deficient AmrZ variant, indicating that DNA binding and transcriptional regulation by AmrZ is responsible for the in vivo virulence defect. These recent infection data, along with previously identified AmrZ-regulated virulence factors, suggest the necessity of AmrZ transcriptional regulation for optimal virulence during acute infection. PMID:20709902

Tandem UIMs confer Lys48 ubiquitin chain substrate preference to deubiquitinase USP25

PubMed Central

Kawaguchi, Kohei; Uo, Kazune; Tanaka, Toshiaki; Komada, Masayuki

2017-01-01

Ubiquitin-specific protease (USP) 25, belonging to the USP family of deubiquitinases, harbors two tandem ubiquitin-interacting motifs (UIMs), a ~20-amino-acid α-helical stretch that binds to ubiquitin. However, the role of the UIMs in USP25 remains unclear. Here we show that the tandem UIM region binds to Lys48-, but not Lys63-, linked ubiquitin chains, where the two UIMs played a critical and cooperative role. Purified USP25 exhibited higher ubiquitin isopeptidase activity to Lys48-, than to Lys63-, linked ubiquitin chains. Mutations that disrupted the ubiquitin-binding ability of the tandem UIMs resulted in a reduced ubiquitin isopeptidase activity of USP25, suggesting a role for the UIMs in exerting the full catalytic activity of USP25. Moreover, when mutations that convert the binding preference from Lys48- to Lys63-linked ubiquitin chains were introduced into the tandem UIM region, the USP25 mutants acquired elevated and reduced isopeptidase activity toward Lys63- and Lys48-linked ubiquitin chains, respectively. These results suggested that the binding preference of the tandem UIMs toward Lys48-linked ubiquitin chains contributes not only to the full catalytic activity but also to the ubiquitin chain substrate preference of USP25, possibly by selectively holding the Lys48-linked ubiquitin chain substrates in the proximity of the catalytic core. PMID:28327663

[Pathomechanism and therapeutic strategy of Fukuyama congenital muscular dystrophy and related disorders].

PubMed

Toda, Tatsushi

2009-11-01

Hypoglycosylation and reduced laminin-binding activity of alpha-dystroglycan are common characteristics of dystroglycanopathy, which is a group of congenital and limb-girdle muscular dystrophies. We previously identified the genes for Fukuyama congenital muscular dystrophy (FCMD) and muscle-eye-brain disease (MEB). FCMD, caused by a mutation in the fukutin gene, is a severe form of dystroglycanopathy. Knock-in mice carrying the founder retrotransposal insertion exhibited hypoglycosylated alpha-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact alpha-dystroglycan, and solid-phase assays determined laminin binding levels to be approximately 50% of normal. In contrast, intact alpha-dystroglycan is undetectable in the dystrophic Large mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact alpha-dystroglycan is sufficient to maintain muscle cell integrity in knock-in mice, suggesting that the treatment of dystroglycanopathies might not require the full recovery of glycosylation. Transfer of fukutin into knock-in mice restored glycosylation of alpha-dystroglycan. Transfer of LARGE produced laminin-binding forms of alpha-dystroglycan in both knock-in mice and the POMGnT1 mutant mouse. These data suggest that even partial restoration of alpha-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits.

Liver Fatty acid binding protein (L-Fabp) modulates murine stellate cell activation and diet induced nonalcoholic fatty liver disease

PubMed Central

Chen, Anping; Tang, Youcai; Davis, Victoria; Hsu, Fong-Fu; Kennedy, Susan M.; Song, Haowei; Turk, John; Brunt, Elizabeth M.; Newberry, Elizabeth P.; Davidson, Nicholas O.

2013-01-01

Activation of hepatic stellate cells (HSCs) is crucial to the development of fibrosis in nonalcoholic fatty liver disease. Quiescent HSCs contain lipid droplets (LDs), whose depletion upon activation induces a fibrogenic gene program. Here we show that liver fatty acid-binding protein (L-Fabp), an abundant cytosolic protein that modulates fatty acid (FA) metabolism in enterocytes and hepatocytes also modulates HSC FA utilization and in turn regulates the fibrogenic program. L-Fabp expression decreased 10-fold following HSC activation, concomitant with depletion of LDs. Primary HSCs isolated from L-FABP−/− mice contain fewer LDs than wild type (WT) HSCs, and exhibit upregulated expression of genes involved in HSC activation. Adenoviral L-Fabp transduction inhibited activation of passaged WT HSCs and increased both the expression of prolipogenic genes and also augmented intracellular lipid accumulation, including triglyceride and FA, predominantly palmitate. Freshly isolated HSCs from L-FABP−/− mice correspondingly exhibited decreased palmitate in the free FA pool. To investigate whether L-FABP deletion promotes HSC activation in vivo, we fed L-FABP−/− and WT mice a high fat diet supplemented with trans-fatty acids and fructose (TFF). TFF-fed L-FABP−/− mice exhibited reduced hepatic steatosis along with decreased LD abundance and size compared to WT mice. In addition, TFF-fed L-FABP−/− mice exhibited decreased hepatic fibrosis, with reduced expression of fibrogenic genes, compared to WT mice. Conclusion L-FABP deletion attenuates both diet-induced hepatic steatosis and fibrogenesis, despite the observation that L-Fabp paradoxically promotes FA and LD accumulation and inhibits HSC activation in vitro. These findings highlight the importance of cell-specific modulation of hepatic lipid metabolism in promoting fibrogenesis in nonalcoholic fatty liver disease. PMID:23401290

Freeze-fracture studies of photoreceptor membranes: new observations bearing upon the distribution of cholesterol

PubMed Central

1983-01-01

We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces presumptive cholesterol detection in the vertebrate photoreceptor cell. In agreement with the pattern seen in our previous study (Andrews, L.D., and A. I. Cohen, 1979, J. Cell Biol., 81:215-228), filipin- binding in membranes exhibiting particle-free patches seemed largely confined to these patches. Favorably fractured photoreceptors exhibited marked filipin-binding in apical inner segment plasma membrane topologically confluent with and proximate to the outer segment plasma membrane, which was comparatively free of filipin binding. A possible boundary between these differing membrane domains was suggested in a number of replicas exhibiting lower filipin binding to the apical plasma membrane of the inner segment in the area surrounding the cilium. This area contains a structure (Andrews, L. D., 1982, Freeze- fracture studies of vertebrate photoreceptors, In Structure of the Eye, J. G. Hollyfield and E. Acosta Vidrio, editors, Elsevier/North-Holland, New York, 11-23) that resembles the active zones of the nerve terminals for the frog neuromuscular junction. These observations lead us to hypothesize that these structures may function to direct vesicle fusion to occur near them, in a domain of membrane more closely resembling outer than inner segment plasma membrane. The above evidence supports the views that (a) all disk membranes contain cholesterol, but the particle-free patches present in some disks trap cholesterol from contiguous particulate membrane regions; (b) contiguous inner and outer segment membranes may greatly differ in cholesterol content; and (c) the suggested higher cholesterol in the inner segment than in the outer segment plasma membrane may help direct newly inserted photopigment molecules to the outer segment. PMID:6411740

Structural characterization of human galectin-4 C-terminal domain: elucidating the molecular basis for recognition of glycosphingolipids, sulfated saccharides and blood group antigens.

PubMed

Bum-Erdene, Khuchtumur; Leffler, Hakon; Nilsson, Ulf J; Blanchard, Helen

2015-09-01

Human galectin-4 is a lectin that is expressed mainly in the gastrointestinal tract and exhibits metastasis-promoting roles in some cancers. Its tandem-repeat nature exhibits two distinct carbohydrate recognition domains allowing crosslinking by simultaneous binding to sulfated and non-sulfated (but not sialylated) glycosphingolipids and glycoproteins, facilitating stabilization of lipid rafts. Critically, galectin-4 exerts favourable or unfavourable effects depending upon the cancer. Here we report the first X-ray crystallographic structural information on human galectin-4, specifically the C-terminal carbohydrate recognition domain of human (galectin-4C) in complex with lactose, lactose-3'-sulfate, 2'-fucosyllactose, lacto-N-tetraose and lacto-N-neotetraose. These structures enable elucidation of galectin-4C binding fine-specificity towards sulfated and non-sulfated lacto- and neolacto-series sphingolipids as well as to human blood group antigens. Analysis of the lactose-3'-sulfate complex structure shows that galectin-4C does not recognize the sulfate group using any specific amino acid, but binds the ligand nonetheless. Complex structures with lacto-N-tetraose and lacto-N-neotetraose displayed differences in binding interactions exhibited by the non-reducing-end galactose. That of lacto-N-tetraose points outward from the protein surface whereas that of lacto-N-neotetraose interacts directly with the protein. Recognition patterns of human galectin-4C towards lacto- and neolacto-series glycosphingolipids are similar to those of human galectin-3; however, detailed scrutiny revealed differences stemming from the extended binding site that offer distinction in ligand profiles of these two galectins. Structural characterization of the complex with 2'-fucosyllactose, a carbohydrate with similarity to the H antigen, and molecular dynamics studies highlight structural features that allow specific recognition of A and B antigens, whilst a lack of interaction with the 2'-fucose of blood group antigens was revealed. 4YLZ, 4YM0, 4YM1, 4YM2, 4YM3. © 2015 FEBS.

Slow ligand-induced conformational switch increases the catalytic rate in Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase.

PubMed

Roy, Sourav; Karmakar, Tarak; Prahlada Rao, Vasudeva S; Nagappa, Lakshmeesha K; Balasubramanian, Sundaram; Balaram, Hemalatha

2015-05-01

P. falciparum (Pf) hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) exhibits a unique mechanism of activation where the enzyme switches from a low activity (unactivated) to a high activity (activated) state upon pre-incubation with substrate/products. Xanthine phosphoribosylation by unactivated PfHGXPRT exhibits a lag phase, the duration of which reduces with an increase in concentration of the enzyme or substrate, PRPP·Mg(2+). Activated PfHGXPRT does not display the lag phase and exhibits a ten-fold drop in the Km value for PRPP·Mg(2+). These observations suggest the involvement of ligand-mediated oligomerization and conformational changes in the process of activation. The dipeptide Leu-Lys in the PPi binding site of human and T. gondii HG(X)PRT that facilitates PRPP·Mg(2+) binding by isomerization from trans to cis conformation is conserved in PfHGXPRT. Free energy calculations using the well-tempered metadynamics technique show the ligand-free enzyme to be more stable when this dipeptide is in the trans conformation than in the cis conformation. The high rotational energy barrier observed for the conformational change from experimental and computational studies permits delineation of the activation mechanism.

Regulation of Hippocampal Glutamate Receptors: Evidence for the Involvement of a Calcium-Activated Protease

NASA Astrophysics Data System (ADS)

Baudry, Michel; Lynch, Gary

1980-04-01

Specific [3H]glutamate binding to rat hippocampal membranes and the calcium-induced increase in this binding are markedly temperature-sensitive and are inhibited by alkylating or reducing agents as well as by various protease inhibitors. N-Ethylmaleimide, chloromethyl ketone derivatives of lysine and phenylalanine, and tosylarginine methyl ester decrease the maximum number of [3H]glutamate binding sites without changing their affinity for glutamate. Preincubation of the membranes with glutamate does not protect the glutamate ``receptors'' from the suppressive effects of these agents. The proteases trypsin and α -chymotrypsin increase the maximum number of [3H]glutamate binding sites. The effects of calcium on glutamate binding are different across brain regions. Cerebellar membranes are almost insensitive whereas hippocampal and striatal membranes exhibit a strong increase in the number of binding sites after exposure to even low concentrations of calcium. These results suggest that an endogenous membrane-associated thiol protease regulates the number of [3H]glutamate binding sites in hippocampal membranes and that this is the mechanism by which calcium stimulates glutamate binding. The possibility is discussed that the postulated mechanisms participate in synaptic physiology and in particular may be related to the long-term potentiation of transmission found in hippocampus under certain conditions.

Aerosol Stable Peptide-Coated Liposome Nanoparticles: A Proof-of-Concept Study with Opioid Fentanyl in Enhancing Analgesic Effects and Reducing Plasma Drug Exposure

PubMed Central

HOEKMAN, JOHN D; SRIVASTAVA, PRAMOD; HO, RODNEY J Y

2014-01-01

Previous we reported a novel pressurized olfactory drug (POD) delivery device that deposit aerosolized drug preferentially to upper nasal cavity. This POD device provided sustained CNS levels of soluble morphine analgesic effects. However, analgesic onset of less soluble fentanyl was more rapid but brief, likely due to hydrophobic fentanyl redistribution readily back to blood. To determine whether fentanyl incorporated into an aerosol stable liposome that binds to nasal epithelial cells will enhance CNS drug exposure and analgesic effects and reduce plasma exposure, we constructed RGD liposomes anchored with acylated integrin binding peptides (palmitoyl-GRGDS). The RGD liposomes, which assume gel-phase membrane structure at 25°C were stable under the stress of aerosolization as only 2.2 ± 0.5 % calcein leakage detected. The RGD mediated integrin binding of liposome is also verified to be unaffected by aerosolization. Rats treated with fentanyl in RGD-liposome and POD device exhibited greater analgesic effect, compared to the free drug counterpart (AUCeffect = 1387.l vs. 760.1 %MPE*min); while ~20% reduced plasma drug exposure was noted (AUC0-120 = 208.2 vs 284.8 ng*min/ml). Collectively, fentanyl incorporated in RGD-liposomes are physically and biologically stable under aerosolization, enhanced the overall analgesic effects and reduced plasma drug exposure for the first 2 hours. PMID:24909764

High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R.

PubMed

Shields, R L; Namenuk, A K; Hong, K; Meng, Y G; Rae, J; Briggs, J; Xie, D; Lai, J; Stadlen, A; Li, B; Fox, J A; Presta, L G

2001-03-02

Immunoglobulin G (IgG) Fc receptors play a critical role in linking IgG antibody-mediated immune responses with cellular effector functions. A high resolution map of the binding site on human IgG1 for human Fc gamma RI, Fc gamma RIIA, Fc gamma RIIB, Fc gamma RIIIA, and FcRn receptors has been determined. A common set of IgG1 residues is involved in binding to all Fc gamma R; Fc gamma RII and Fc gamma RIII also utilize residues outside this common set. In addition to residues which, when altered, abrogated binding to one or more of the receptors, several residues were found that improved binding only to specific receptors or simultaneously improved binding to one type of receptor and reduced binding to another type. Select IgG1 variants with improved binding to Fc gamma RIIIA exhibited up to 100% enhancement in antibody-dependent cell cytotoxicity using human effector cells; these variants included changes at residues not found at the binding interface in the IgG/Fc gamma RIIIA co-crystal structure (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). These engineered antibodies may have important implications for improving antibody therapeutic efficacy.

Tuning the autophagy-inducing activity of lanthanide-based nanocrystals through specific surface-coating peptides

NASA Astrophysics Data System (ADS)

Zhang, Yunjiao; Zheng, Fang; Yang, Tianlong; Zhou, Wei; Liu, Yun; Man, Na; Zhang, Li; Jin, Nan; Dou, Qingqing; Zhang, Yong; Li, Zhengquan; Wen, Long-Ping

2012-09-01

The induction of autophagy on exposure of cells to a variety of nanoparticles represents both a safety concern and an application niche for engineered nanomaterials. Here, we show that a short synthetic peptide, RE-1, identified by means of phage display, binds to lanthanide (LN) oxide and upconversion nanocrystals (UCN), forms a stable coating layer on the nanoparticles’ surface, and effectively abrogates their autophagy-inducing activity. Furthermore, RE-1 peptide variants exhibit a differentially reduced binding capability, and correspondingly, a varied ability to reduce the autophagic response. We also show that the addition of an arginine-glycine-aspartic acid (RGD) motif to RE-1 enhances autophagy for LN UCN through the interaction with integrins. RE-1 and its variants provide a versatile tool for tuning material-cell interactions to achieve the desired level of autophagy, and may prove useful for the various diagnostic and therapeutic applications of LN-based nanomaterials and nanodevices.

Superabsorbent polymer-containing wound dressings have a beneficial effect on wound healing by reducing PMN elastase concentration and inhibiting microbial growth.

PubMed

Wiegand, C; Abel, M; Ruth, P; Hipler, U C

2011-11-01

A comprehensive in vitro approach was used to assess the effects of superabsorbent polymer (SAP) containing wound dressings in treatment of non-healing wounds. A slight negative effect on HaCaT cells was noted in vitro which is most likely due to the Ca(2+) deprivation of the medium by binding to the SAP. It could be shown that SAP wound dressings are able to bind considerable amounts of elastase reducing enzyme activity significantly. Furthermore, SAP's inhibit the formation of free radicals. The SAP-containing wound dressings tested also exhibited a significant to strong antimicrobial activity effectively impeding the growth of gram-negative and gram-positive bacteria as well as yeasts. In conclusion, in vitro data confirm the positive effect of SAP wound dressings observed in vivo and suggest that they should be specifically useful for wound cleansing.

Putative bovine topological association domains and CTCF binding motifs can reduce the search space for causative regulatory variants of complex traits.

PubMed

Wang, Min; Hancock, Timothy P; Chamberlain, Amanda J; Vander Jagt, Christy J; Pryce, Jennie E; Cocks, Benjamin G; Goddard, Mike E; Hayes, Benjamin J

2018-05-24

Topological association domains (TADs) are chromosomal domains characterised by frequent internal DNA-DNA interactions. The transcription factor CTCF binds to conserved DNA sequence patterns called CTCF binding motifs to either prohibit or facilitate chromosomal interactions. TADs and CTCF binding motifs control gene expression, but they are not yet well defined in the bovine genome. In this paper, we sought to improve the annotation of bovine TADs and CTCF binding motifs, and assess whether the new annotation can reduce the search space for cis-regulatory variants. We used genomic synteny to map TADs and CTCF binding motifs from humans, mice, dogs and macaques to the bovine genome. We found that our mapped TADs exhibited the same hallmark properties of those sourced from experimental data, such as housekeeping genes, transfer RNA genes, CTCF binding motifs, short interspersed elements, H3K4me3 and H3K27ac. We showed that runs of genes with the same pattern of allele-specific expression (ASE) (either favouring paternal or maternal allele) were often located in the same TAD or between the same conserved CTCF binding motifs. Analyses of variance showed that when averaged across all bovine tissues tested, TADs explained 14% of ASE variation (standard deviation, SD: 0.056), while CTCF explained 27% (SD: 0.078). Furthermore, we showed that the quantitative trait loci (QTLs) associated with gene expression variation (eQTLs) or ASE variation (aseQTLs), which were identified from mRNA transcripts from 141 lactating cows' white blood and milk cells, were highly enriched at putative bovine CTCF binding motifs. The linearly-furthermost, and most-significant aseQTL and eQTL for each genic target were located within the same TAD as the gene more often than expected (Chi-Squared test P-value < 0.001). Our results suggest that genomic synteny can be used to functionally annotate conserved transcriptional components, and provides a tool to reduce the search space for causative regulatory variants in the bovine genome.

Identification of DNA-Binding Proteins Using Mixed Feature Representation Methods.

PubMed

Qu, Kaiyang; Han, Ke; Wu, Song; Wang, Guohua; Wei, Leyi

2017-09-22

DNA-binding proteins play vital roles in cellular processes, such as DNA packaging, replication, transcription, regulation, and other DNA-associated activities. The current main prediction method is based on machine learning, and its accuracy mainly depends on the features extraction method. Therefore, using an efficient feature representation method is important to enhance the classification accuracy. However, existing feature representation methods cannot efficiently distinguish DNA-binding proteins from non-DNA-binding proteins. In this paper, a multi-feature representation method, which combines three feature representation methods, namely, K-Skip-N-Grams, Information theory, and Sequential and structural features (SSF), is used to represent the protein sequences and improve feature representation ability. In addition, the classifier is a support vector machine. The mixed-feature representation method is evaluated using 10-fold cross-validation and a test set. Feature vectors, which are obtained from a combination of three feature extractions, show the best performance in 10-fold cross-validation both under non-dimensional reduction and dimensional reduction by max-relevance-max-distance. Moreover, the reduced mixed feature method performs better than the non-reduced mixed feature technique. The feature vectors, which are a combination of SSF and K-Skip-N-Grams, show the best performance in the test set. Among these methods, mixed features exhibit superiority over the single features.

Coulomb and CH-π interactions in (6-4) photolyase-DNA complex dominate DNA binding and repair abilities.

PubMed

Terai, Yuma; Sato, Ryuma; Yumiba, Takahiro; Harada, Ryuhei; Shimizu, Kohei; Toga, Tatsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Iwai, Shigenori; Shigeta, Yasuteru; Yamamoto, Junpei

2018-05-14

(6-4) Photolyases ((6-4)PLs) are flavoenzymes that repair the carcinogenic UV-induced DNA damage, pyrimidine(6-4)pyrimidone photoproducts ((6-4)PPs), in a light-dependent manner. Although the reaction mechanism of DNA photorepair by (6-4)PLs has been intensively investigated, the molecular mechanism of the lesion recognition remains obscure. We show that a well-conserved arginine residue in Xenopus laevis (6-4)PL (Xl64) participates in DNA binding, through Coulomb and CH-π interactions. Fragment molecular orbital calculations estimated attractive interaction energies of -80-100 kcal mol-1 for the Coulomb interaction and -6 kcal mol-1 for the CH-π interaction, and the loss of either of them significantly reduced the affinity for (6-4)PP-containing oligonucleotides, as well as the quantum yield of DNA photorepair. From experimental and theoretical observations, we formulated a DNA binding model of (6-4)PLs. Based on the binding model, we mutated this Arg in Xl64 to His, which is well conserved among the animal cryptochromes (CRYs), and found that the CRY-type mutant exhibited reduced affinity for the (6-4)PP-containing oligonucleotides, implying the possible molecular origin of the functional diversity of the photolyase/cryptochrome superfamily.

In vitro hypoglycemic and cholesterol lowering effects of dietary fiber prepared from cocoa (Theobroma cacao L.) shells.

PubMed

Nsor-Atindana, John; Zhong, Fang; Mothibe, Kebitsamang Joseph

2012-10-01

Three dietary fiber (DF) powders; soluble dietary fiber (SDF), insoluble dietary fiber (IDF) and total dietary fiber (TDF) were prepared from cocoa bean shells (CBS) by enzymatic treatment. These DFs were evaluated for their effects on glucose adsorption, glucose diffusion, starch hydrolysis, cholesterol binding, sodium cholate binding and oil binding capacities using in vitro model systems by simulating gastric intestinal conditions. The results showed that SDF generally exhibited significantly (p < 0.05) higher glucose adsorption capacity (GAC), α-amylase inhibition activity, cholesterol and sodium cholate binding capacity, but less significant (>0.05) glucose dialysis retardation index (GDRI) and oil binding capacity, when compared with IDF and TDF which both showed similar effects. Moreover, it was discovered that the three CBS dietary fiber powders contained intrinsic antioxidants (phenolic compounds). The study suggested that CBS could be an alternative cheap source of DF with additional benefits. Thus, CBS fibers could be incorporated as low calorie bulk ingredients in high-fiber diet to reduce calorie and cholesterol levels and control blood glucose level.

Different inactivating mutations of the mineralocorticoid receptor in fourteen families affected by type I pseudohypoaldosteronism.

PubMed

Sartorato, Paola; Lapeyraque, Anne-Laure; Armanini, Decio; Kuhnle, Ursula; Khaldi, Yasmina; Salomon, Rémi; Abadie, Véronique; Di Battista, Eliana; Naselli, Arturo; Racine, Alain; Bosio, Maurizio; Caprio, Massimiliano; Poulet-Young, Véronique; Chabrolle, Jean-Pierre; Niaudet, Patrick; De Gennes, Christiane; Lecornec, Marie-Hélène; Poisson, Elodie; Fusco, Anna Maria; Loli, Paola; Lombès, Marc; Zennaro, Maria-Christina

2003-06-01

We have analyzed the human mineralocorticoid receptor (hMR) gene in 14 families with autosomal dominant or sporadic pseudohypoaldosteronism (PHA1), a rare form of mineralocorticoid resistance characterized by neonatal renal salt wasting and failure to thrive. Six heterozygous mutations were detected. Two frameshift mutations in exon 2 (insT1354, del8bp537) and one nonsense mutation in exon 4 (C2157A, Cys645stop) generate truncated proteins due to premature stop codons. Three missense mutations (G633R, Q776R, L979P) differently affect hMR function. The DNA binding domain mutant R633 exhibits reduced maximal transactivation, although its binding characteristics and ED(50) of transactivation are comparable with wild-type hMR. Ligand binding domain mutants R776 and P979 present reduced or absent aldosterone binding, respectively, which is associated with reduced or absent ligand-dependent transactivation capacity. Finally, P979 possesses a transdominant negative effect on wild-type hMR activity, whereas mutations G633R and Q776R probably result in haploinsufficiency in PHA1 patients. We conclude that hMR mutations are a common feature of autosomal dominant PHA1, being found in 70% of our familial cases. Their absence in some families underscores the importance of an extensive investigation of the hMR gene and the role of precise diagnostic procedures to allow for identification of other genes potentially involved in the disease.

The Agr quorum-sensing system regulates fibronectin binding but not hemolysis in the absence of a functional electron transport chain.

PubMed

Pader, Vera; James, Ellen H; Painter, Kimberley L; Wigneshweraraj, Sivaramesh; Edwards, Andrew M

2014-10-01

Staphylococcus aureus is responsible for numerous chronic and recurrent infections, which are frequently associated with the emergence of small-colony variants (SCVs) that lack a functional electron transport chain. SCVs exhibit enhanced expression of fibronectin-binding protein (FnBP) and greatly reduced hemolysin production, although the basis for this is unclear. One hypothesis is that these phenotypes are a consequence of the reduced Agr activity of SCVs, while an alternative is that the lack of a functional electron transport chain and the resulting reduction in ATP production are responsible. Disruption of the electron transport chain of S. aureus genetically (hemB and menD) or chemically, using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibited both growth and Agr activity and conferred an SCV phenotype. Supplementation of the culture medium with synthetic autoinducing peptide (sAIP) significantly increased Agr expression in both hemB mutant strains and S. aureus grown with HQNO and significantly reduced staphylococcal adhesion to fibronectin. However, sAIP did not promote hemolysin expression in hemB mutant strains or S. aureus grown with HQNO. Therefore, while Agr regulates fibronectin binding in SCVs, it cannot promote hemolysin production in the absence of a functional electron transport chain. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The Agr Quorum-Sensing System Regulates Fibronectin Binding but Not Hemolysis in the Absence of a Functional Electron Transport Chain

PubMed Central

Pader, Vera; James, Ellen H.; Painter, Kimberley L.; Wigneshweraraj, Sivaramesh

2014-01-01

Staphylococcus aureus is responsible for numerous chronic and recurrent infections, which are frequently associated with the emergence of small-colony variants (SCVs) that lack a functional electron transport chain. SCVs exhibit enhanced expression of fibronectin-binding protein (FnBP) and greatly reduced hemolysin production, although the basis for this is unclear. One hypothesis is that these phenotypes are a consequence of the reduced Agr activity of SCVs, while an alternative is that the lack of a functional electron transport chain and the resulting reduction in ATP production are responsible. Disruption of the electron transport chain of S. aureus genetically (hemB and menD) or chemically, using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibited both growth and Agr activity and conferred an SCV phenotype. Supplementation of the culture medium with synthetic autoinducing peptide (sAIP) significantly increased Agr expression in both hemB mutant strains and S. aureus grown with HQNO and significantly reduced staphylococcal adhesion to fibronectin. However, sAIP did not promote hemolysin expression in hemB mutant strains or S. aureus grown with HQNO. Therefore, while Agr regulates fibronectin binding in SCVs, it cannot promote hemolysin production in the absence of a functional electron transport chain. PMID:25092909

Structural basis of redox-dependent substrate binding of protein disulfide isomerase

PubMed Central

Yagi-Utsumi, Maho; Satoh, Tadashi; Kato, Koichi

2015-01-01

Protein disulfide isomerase (PDI) is a multidomain enzyme, operating as an essential folding catalyst, in which the b′ and a′ domains provide substrate binding sites and undergo an open–closed domain rearrangement depending on the redox states of the a′ domain. Despite the long research history of this enzyme, three-dimensional structural data remain unavailable for its ligand-binding mode. Here we characterize PDI substrate recognition using α-synuclein (αSN) as the model ligand. Our nuclear magnetic resonance (NMR) data revealed that the substrate-binding domains of PDI captured the αSN segment Val37–Val40 only in the oxidized form. Furthermore, we determined the crystal structure of an oxidized form of the b′–a′ domains in complex with an undecapeptide corresponding to this segment. The peptide-binding mode observed in the crystal structure with NMR validation, was characterized by hydrophobic interactions on the b′ domain in an open conformation. Comparison with the previously reported crystal structure indicates that the a′ domain partially masks the binding surface of the b′ domain, causing steric hindrance against the peptide in the reduced form of the b′–a′ domains that exhibits a closed conformation. These findings provide a structural basis for the mechanism underlying the redox-dependent substrate binding of PDI. PMID:26350503

Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome.

PubMed

Jetzt, Amanda E; Li, Xiao-Ping; Tumer, Nilgun E; Cohick, Wendie S

2016-11-01

Ricin is a potent ribotoxin that is considered a bioterror threat due to its ease of isolation and possibility of aerosolization. In yeast, mutation of arginine residues away from the active site results in a ricin toxin A chain (RTA) variant that is unable to bind the ribosome and exhibits reduced cytotoxicity. The goal of the present work was to determine if these residues contribute to ribosome binding and cytotoxicity of RTA in mammalian cells. The RTA mutant R193A/R235A did not interact with mammalian ribosomes, while a G212E variant with a point mutation near its active site bound ribosomes similarly to wild-type (WT) RTA. R193A/R235A retained full catalytic activity on naked RNA but had reduced activity on mammalian ribosomes. To determine the effect of this mutant in intact cells, pre R193A/R235A containing a signal sequence directing it to the endoplasmic reticulum and mature R193A/R235A that directly targeted cytosolic ribosomes were each expressed. Depurination and protein synthesis inhibition were reduced by both pre- and mature R193A/R235A relative to WT. Protein synthesis inhibition was reduced to a greater extent by R193A/R235A than by G212E. Pre R193A/R235A caused a greater reduction in caspase activation and loss of mitochondrial membrane potential than G212E relative to WT RTA. These findings indicate that an RTA variant with reduced ribosome binding is less toxic than a variant with less catalytic activity but normal ribosome binding activity. The toxin-ribosome interaction represents a novel target for the development of therapeutics to prevent or treat ricin intoxication. Copyright © 2016 Elsevier Inc. All rights reserved.

Novel soluble, high-affinity gastrin-releasing peptide binding proteins in Swiss 3T3 fibroblasts.

PubMed

Kane, M A; Portanova, L B; Kelley, K; Holley, M; Ross, S E; Boose, D; Escobedo-Morse, A; Alvarado, B

1994-01-01

Swiss 3T3 cells contained substantial amounts of soluble and specific [125I]GRP binders. Like the membrane-associated GRP receptor, they were of high affinity, saturable, bound to GRP(14-27) affinity gels, and exhibited specificity for GRP(14-27) binding. They differed in that acid or freezing destroyed specific binding, specific binding exhibited different time and temperature effects, no detergent was required for their solubilization, ammonium sulfate fractionation yielded different profiles, the M(rs) were lower, GRP(1-16) also blocked binding, and a polyclonal anti-GRP receptor antiserum did not bind on Western blots. The isolated, soluble GRP binding protein(s) rapidly degraded [125I]GRP. These soluble GRP binding proteins may play a role in the regulation of the mitogenic effects of GRP on these cells.

Alteration of lung atrial natriuretic peptide receptors in genetic cardiomyopathy.

PubMed

Mukaddam-Daher, S; Tremblay, J; Fujio, N; Koch, C; Jankowski, M; Quillen, E W; Gutkowska, J

1996-07-01

These studies were designed to characterize the atrial natriuretic peptide (ANF) receptor subtypes [guanylyl cyclase natriuretic peptide receptors (NPR-A, NPR-B) and NPR-C] in lungs of normal hamsters and to evaluate alterations in receptor kinetics in genetic cardiomyopathy (CMO), a model of human congestive heart failure. Lung membranes were obtained from normal and CMO 200-to 230-day-old hamsters. Cross-linking and competitive binding receptor assays using 125I-labeled human ANF showed that lung membranes exhibit NPR, mainly guanylyl cyclase NPR-A and clearance NPR-C receptors. Stimulation of guanylyl cyclase by ANF and C-type natriuretic peptide (CNP) confirmed the presence of NPR-A and NPR-B. The maximum binding capacity of total ANF binding sites (442 +/- 68 vs. 271 +/- 57 fmol/mg protein, P < 0.05) was reduced, but dissociation constant (0.26 +/- 0.04 vs. 0.41 +/- 0.08 nM) was not altered in CMO animals. Similar reductions were observed in the binding sites for brain natriuretic peptide (BNP; 438 +/- 83 vs. 236 +/- 53 fmol/mg protein) and CNP (321 +/- 80 vs. 165 +/- 56 fmol/mg protein, P < 0.05) which may reflect a decline in NPR-A and NPR-B and/or NPR-C. Acid wash improved binding of 125I-labeled rat ANF to lung membranes of both normal and CMO hamsters, but the tendency towards reduced binding in CMO hamsters did not reach statistical significance, implying that downregulation may not have been due only to prior occupancy of the receptors. Transcripts of NPR-A, NPR-B, and NPR-C receptors in hamster lungs were detected by quantitative polymerase chain reaction. Compared with normal controls, the CMO hamster lung NPR-A mRNA was reduced by 50%, but NPR-B mRNA and NPR-C mRNA were not altered. Moreover, CMO hamster lungs showed less activation of guanylyl cyclase by ANF. These studies demonstrate that lung NPR are downregulated in hamster CMO.

Evidence for Osteocalcin Binding and Activation of GPRC6A in β-Cells

DOE PAGES

Pi, Min; Kapoor, Karan; Ye, Ruisong; ...

2016-05-01

The possibility that G protein-coupled receptor family C member A (GPRC6A) is the osteocalcin (Ocn)-sensing G protein-coupled receptor that directly regulates pancreatic β-cell functions is controversial. In the current study, we found that Ocn and an Ocn-derived C-terminal hexapeptide directly activate GPRC6A-dependent ERK signaling in vitro. Computational models probe the structural basis of Ocn binding to GPRC6A and predict that the C-terminal hexapeptide docks to the extracellular side of the transmembrane domain of GPRC6A. Consistent with the modeling, mutations in the computationally identified binding pocket of GPRC6A reduced Ocn and C-terminal hexapeptide activation of this receptor. In addition, selective deletionmore » of Gprc6a in β-cells (Gprc6a β-cell-cko) by crossing Gprc6a flox/flox mice with Ins2-Cre mice resulted in reduced pancreatic weight, islet number, insulin protein content, and insulin message expression. Both islet size and β-cell proliferation were reduced in Gprc6a β-cell-cko compared with control mice. Gprc6a β-cell-cko exhibited abnormal glucose tolerance, but normal insulin sensitivity. Islets isolated from Gprc6a β-cell-cko mice showed reduced insulin simulation index in response to Ocn. Here, these data establish the structural basis for Ocn direct activation of GPRC6A and confirm a role for GPRC6A in regulating β-cell proliferation and insulin secretion.« less

Evidence for Osteocalcin Binding and Activation of GPRC6A in β-Cells

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

Pi, Min; Kapoor, Karan; Ye, Ruisong

The possibility that G protein-coupled receptor family C member A (GPRC6A) is the osteocalcin (Ocn)-sensing G protein-coupled receptor that directly regulates pancreatic β-cell functions is controversial. In the current study, we found that Ocn and an Ocn-derived C-terminal hexapeptide directly activate GPRC6A-dependent ERK signaling in vitro. Computational models probe the structural basis of Ocn binding to GPRC6A and predict that the C-terminal hexapeptide docks to the extracellular side of the transmembrane domain of GPRC6A. Consistent with the modeling, mutations in the computationally identified binding pocket of GPRC6A reduced Ocn and C-terminal hexapeptide activation of this receptor. In addition, selective deletionmore » of Gprc6a in β-cells (Gprc6a β-cell-cko) by crossing Gprc6a flox/flox mice with Ins2-Cre mice resulted in reduced pancreatic weight, islet number, insulin protein content, and insulin message expression. Both islet size and β-cell proliferation were reduced in Gprc6a β-cell-cko compared with control mice. Gprc6a β-cell-cko exhibited abnormal glucose tolerance, but normal insulin sensitivity. Islets isolated from Gprc6a β-cell-cko mice showed reduced insulin simulation index in response to Ocn. Here, these data establish the structural basis for Ocn direct activation of GPRC6A and confirm a role for GPRC6A in regulating β-cell proliferation and insulin secretion.« less

Twisted cyanines: a non-planar fluorogenic dye with superior photostability and its use in a protein-based fluoromodule.

PubMed

Shank, Nathaniel I; Pham, Ha H; Waggoner, Alan S; Armitage, Bruce A

2013-01-09

The cyanine dye thiazole orange (TO) is a well-known fluorogenic stain for DNA and RNA, but this property precludes its use as an intracellular fluorescent probe for non-nucleic acid biomolecules. Further, as is the case with many cyanines, the dye suffers from low photostability. Here, we report the synthesis of a bridge-substituted version of TO named α-CN-TO, where the central methine hydrogen of TO is replaced by an electron withdrawing cyano group, which was expected to decrease the susceptibility of the dye toward singlet oxygen-mediated degradation. An X-ray crystal structure shows that α-CN-TO is twisted drastically out of plane, in contrast to TO, which crystallizes in the planar conformation. α-CN-TO retains the fluorogenic behavior of the parent dye TO in viscous glycerol/water solvent, but direct irradiation and indirect bleaching studies showed that α-CN-TO is essentially inert to visible light and singlet oxygen. In addition, the twisted conformation of α-CN-TO mitigates nonspecific binding and fluorescence activation by DNA and a previously selected TO-binding protein and exhibits low background fluorescence in HeLa cell culture. α-CN-TO was then used to select a new protein that binds and activates fluorescence from the dye. The new α-CN-TO/protein fluoromodule exhibits superior photostability to an analogous TO/protein fluoromodule. These properties indicate that α-CN-TO will be a useful fluorogenic dye in combination with specific RNA and protein binding partners for both in vitro and cell-based applications. More broadly, structural features that promote nonplanar conformations can provide an effective method for reducing nonspecific binding of cationic dyes to nucleic acids and other biomolecules.

Twisted Cyanines: A Non-Planar Fluorogenic Dye with Superior Photostability and its Use in a Protein-Based Fluoromodule

PubMed Central

Shank, Nathaniel I.; Pham, Ha; Waggoner, Alan S.; Armitage, Bruce A.

2013-01-01

The cyanine dye thiazole orange (TO) is a well-known fluorogenic stain for DNA and RNA, but this property precludes its use as an intracellular fluorescent probe for non-nucleic acid biomolecules. Further, as is the case with many cyanines, the dye suffers from low photostability. Here we report the synthesis of a bridge-substituted version of TO named α-CN-TO, where the central methine hydrogen of TO is replaced by an electron withdrawing cyano group, which was expected to decrease the susceptibility of the dye toward singlet oxygen-mediated degradation. An X-ray crystal structure shows that α-CN-TO is twisted drastically out of plane, in contrast to TO, which crystallizes in the planar conformation. α-CN-TO retains the fluorogenic behavior of the parent dye TO in viscous glycerol/water solvent, but direct irradiation and indirect bleaching studies showed that α-CN-TO is essentially inert to visible light and singlet oxygen. In addition, the twisted conformation of α-CN-TO mitigates non-specific binding and fluorescence activation by DNA and a previously selected TO-binding protein and exhibits low background fluorescence in HeLa cell culture. α-CN-TO was then used to select a new protein that binds and activates fluorescence from the dye. The new α-CN-TO/protein fluoromodule exhibits superior photostability to an analogous TO/protein fluoromodule. These properties indicate that α-CN-TO will be a useful fluorogenic dye in combination with specific RNA and protein binding partners for both in vitro and cell-based applications. More broadly, structural features that promote nonplanar conformations can provide an effective method for reducing nonspecific binding of cationic dyes to nucleic acids and other biomolecules. PMID:23252842

An antibody reactive to the Gly63–Lys68 epitope of NT-proBNP exhibits O-glycosylation-independent binding

PubMed Central

Lee, Yujean; Kim, Hyori; Chung, Junho

2014-01-01

The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63–Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63–Lys68 of NT-proBNP exhibits O-glycosylation-independent binding. PMID:25236766

Small Cell Lung Cancer Exhibits Frequent Inactivating Mutations in the Histone Methyltransferase KMT2D/MLL2: CALGB 151111 (Alliance)

PubMed Central

Augert, Arnaud; Zhang, Qing; Bates, Breanna; Cui, Min; Wang, Xiaofei; Wildey, Gary; Dowlati, Afshin; MacPherson, David

2017-01-01

Introduction SCLC is a lethal neuroendocrine tumor type that is highly prone to metastasis. There is an urgency to understand the mutated genes that promote SCLC, as there are no approved targeted therapies yet available. SCLC is rarely resected, limiting the number of samples available for genomic analyses of somatic mutations. Methods To identify potential driver mutations in human SCLC we sequenced the whole exomes of 18 primary SCLCs and seven cell lines along with matched normal controls. We extended these data by resequencing a panel of genes across 40 primary SCLCs and 48 cell lines. Results We report frequent mutations in the lysine methyltransferase 2D gene (KMT2D) (also known as MLL2), a key regulator of transcriptional enhancer function. KMT2D exhibited truncating nonsense/frameshift/splice site mutations in 8% of SCLC tumors and 17% of SCLC cell lines. We found that KMT2D mutation in human SCLC cell lines was associated with reduced lysine methyltransferase 2D protein levels and reduced monomethylation of histone H3 lysine 4, a mark associated with transcriptional enhancers. We also found mutations in other genes associated with transcriptional enhancer control, including CREB binding protein gene (CREBBP), E1A binding protein p300 gene (EP300), and chromodomain helicase DNA binding protein 7 gene (CHD7), and we report mutations in additional chromatin remodeling genes such as polybromo 1 gene (PBRM1). Conclusions These data indicate that KMT2D is one of the major mutated genes in SCLC, and they point to perturbation of transcriptional enhancer control as potentially contributing to SCLC. PMID:28007623

Chl1 DNA helicase and Scc2 function in chromosome condensation through cohesin deposition.

PubMed

Shen, Donglai; Skibbens, Robert V

2017-01-01

Chl1 DNA helicase promotes sister chromatid cohesion and associates with both the cohesion establishment acetyltransferase Eco1/Ctf7 and the DNA polymerase processivity factor PCNA that supports Eco1/Ctf7 function. Mutation in CHL1 results in precocious sister chromatid separation and cell aneuploidy, defects that arise through reduced levels of chromatin-bound cohesins which normally tether together sister chromatids (trans tethering). Mutation of Chl1 family members (BACH1/BRIP/FANCJ and DDX11/ChlR1) also exhibit genotoxic sensitivities, consistent with a role for Chl1 in trans tethering which is required for efficient DNA repair. Chl1 promotes the recruitment of Scc2 to DNA which is required for cohesin deposition onto DNA. There is limited evidence, however, that Scc2 also directs the deposition onto DNA of condensins which promote tethering in cis (intramolecular DNA links). Here, we test the ability of Chl1 to promote cis tethering and the role of both Chl1 and Scc2 to promote condensin recruitment to DNA. The results reveal that chl1 mutant cells exhibit significant condensation defects both within the rDNA locus and genome-wide. Importantly, chl1 mutant cell condensation defects do not result from reduced chromatin binding of condensin, but instead through reduced chromatin binding of cohesin. We tested scc2-4 mutant cells and similarly found no evidence of reduced condensin recruitment to chromatin. Consistent with a role for Scc2 specifically in cohesin deposition, scc2-4 mutant cell condensation defects are irreversible. We thus term Chl1 a novel regulator of both chromatin condensation and sister chromatid cohesion through cohesin-based mechanisms. These results reveal an exciting interface between DNA structure and the highly conserved cohesin complex.

Chl1 DNA helicase and Scc2 function in chromosome condensation through cohesin deposition

PubMed Central

Shen, Donglai

2017-01-01

Chl1 DNA helicase promotes sister chromatid cohesion and associates with both the cohesion establishment acetyltransferase Eco1/Ctf7 and the DNA polymerase processivity factor PCNA that supports Eco1/Ctf7 function. Mutation in CHL1 results in precocious sister chromatid separation and cell aneuploidy, defects that arise through reduced levels of chromatin-bound cohesins which normally tether together sister chromatids (trans tethering). Mutation of Chl1 family members (BACH1/BRIP/FANCJ and DDX11/ChlR1) also exhibit genotoxic sensitivities, consistent with a role for Chl1 in trans tethering which is required for efficient DNA repair. Chl1 promotes the recruitment of Scc2 to DNA which is required for cohesin deposition onto DNA. There is limited evidence, however, that Scc2 also directs the deposition onto DNA of condensins which promote tethering in cis (intramolecular DNA links). Here, we test the ability of Chl1 to promote cis tethering and the role of both Chl1 and Scc2 to promote condensin recruitment to DNA. The results reveal that chl1 mutant cells exhibit significant condensation defects both within the rDNA locus and genome-wide. Importantly, chl1 mutant cell condensation defects do not result from reduced chromatin binding of condensin, but instead through reduced chromatin binding of cohesin. We tested scc2-4 mutant cells and similarly found no evidence of reduced condensin recruitment to chromatin. Consistent with a role for Scc2 specifically in cohesin deposition, scc2-4 mutant cell condensation defects are irreversible. We thus term Chl1 a novel regulator of both chromatin condensation and sister chromatid cohesion through cohesin-based mechanisms. These results reveal an exciting interface between DNA structure and the highly conserved cohesin complex. PMID:29186203

Free Energy Landscape of Lipid Interactions with Regulatory Binding Sites on the Transmembrane Domain of the EGF Receptor.

PubMed

Hedger, George; Shorthouse, David; Koldsø, Heidi; Sansom, Mark S P

2016-08-25

Lipid molecules can bind to specific sites on integral membrane proteins, modulating their structure and function. We have undertaken coarse-grained simulations to calculate free energy profiles for glycolipids and phospholipids interacting with modulatory sites on the transmembrane helix dimer of the EGF receptor within a lipid bilayer environment. We identify lipid interaction sites at each end of the transmembrane domain and compute interaction free energy profiles for lipids with these sites. Interaction free energies ranged from ca. -40 to -4 kJ/mol for different lipid species. Those lipids (glycolipid GM3 and phosphoinositide PIP2) known to modulate EGFR function exhibit the strongest binding to interaction sites on the EGFR, and we are able to reproduce the preference for interaction with GM3 over other glycolipids suggested by experiment. Mutation of amino acid residues essential for EGFR function reduce the binding free energy of these key lipid species. The residues interacting with the lipids in the simulations are in agreement with those suggested by experimental (mutational) studies. This approach provides a generalizable tool for characterizing the interactions of lipids that bind to specific sites on integral membrane proteins.

Free Energy Landscape of Lipid Interactions with Regulatory Binding Sites on the Transmembrane Domain of the EGF Receptor

PubMed Central

2016-01-01

Lipid molecules can bind to specific sites on integral membrane proteins, modulating their structure and function. We have undertaken coarse-grained simulations to calculate free energy profiles for glycolipids and phospholipids interacting with modulatory sites on the transmembrane helix dimer of the EGF receptor within a lipid bilayer environment. We identify lipid interaction sites at each end of the transmembrane domain and compute interaction free energy profiles for lipids with these sites. Interaction free energies ranged from ca. −40 to −4 kJ/mol for different lipid species. Those lipids (glycolipid GM3 and phosphoinositide PIP2) known to modulate EGFR function exhibit the strongest binding to interaction sites on the EGFR, and we are able to reproduce the preference for interaction with GM3 over other glycolipids suggested by experiment. Mutation of amino acid residues essential for EGFR function reduce the binding free energy of these key lipid species. The residues interacting with the lipids in the simulations are in agreement with those suggested by experimental (mutational) studies. This approach provides a generalizable tool for characterizing the interactions of lipids that bind to specific sites on integral membrane proteins. PMID:27109430

Molecular evolution and expression of oxygen transport genes in livebearing fishes (Poeciliidae) from hydrogen sulfide rich springs.

PubMed

Barts, Nicholas; Greenway, Ryan; Passow, Courtney N; Arias-Rodriguez, Lenin; Kelley, Joanna L; Tobler, Michael

2018-04-01

Hydrogen sulfide (H 2 S) is a natural toxicant in some aquatic environments that has diverse molecular targets. It binds to oxygen transport proteins, rendering them non-functional by reducing oxygen-binding affinity. Hence, organisms permanently inhabiting H 2 S-rich environments are predicted to exhibit adaptive modifications to compensate for the reduced capacity to transport oxygen. We investigated 10 lineages of fish of the family Poeciliidae that have colonized freshwater springs rich in H 2 S-along with related lineages from non-sulfidic environments-to test hypotheses about the expression and evolution of oxygen transport genes in a phylogenetic context. We predicted shifts in the expression of and signatures of positive selection on oxygen transport genes upon colonization of H 2 S-rich habitats. Our analyses indicated significant shifts in gene expression for multiple hemoglobin genes in lineages that have colonized H 2 S-rich environments, and three hemoglobin genes exhibited relaxed selection in sulfidic compared to non-sulfidic lineages. However, neither changes in gene expression nor signatures of selection were consistent among all lineages in H 2 S-rich environments. Oxygen transport genes may consequently be predictable targets of selection during adaptation to sulfidic environments, but changes in gene expression and molecular evolution of oxygen transport genes in H 2 S-rich environments are not necessarily repeatable across replicated lineages.

Site-directed mutagenesis of mouse glutathione transferase P1-1 unlocks masked cooperativity, introduces a novel mechanism for 'ping pong' kinetic behaviour, and provides further structural evidence for participation of a water molecule in proton abstraction from glutathione.

PubMed

McManus, Gavin; Costa, Marta; Canals, Albert; Coll, Miquel; Mantle, Timothy J

2011-01-01

Mouse liver glutathione transferase P1-1 has three cysteine residues at positions 14, 47 and 169. We have constructed the single, double and triple cysteine to alanine mutants to define the behaviour of all three thiols. We confirm that C47 is the 'fast' thiol (pK 7.4), and define C169 as the alkaline reactive residue with a pK(a) of 8.6. Only a small proportion of C14 is reactive with 5,5'-dithiobis-(2-nitrobenoic acid) (DTNB) at pH 9 in the C47A/C169A double mutant. The native enzyme and the C169A mutant exhibited Michaelis-Menten kinetics, but all other thiol to alanine mutants exhibited sigmoidal kinetics to varying degrees. The C169A mutant exhibited 'ping pong' kinetics, consistent with a mechanism whereby liberation of a proton from a reduced enzyme-glutathione (GSH) complex to form an enzyme-GS(-) (unprotonated) complex is essentially irreversible. Intriguingly, similar behaviour has recently been reported for a mutant of the yeast prion Ure2p. This cooperative behaviour is 'mirrored' in the crystal structure of the C47A mutant, which binds the p-nitrobenzyl moiety of p-nitrobenzyglutathione in distinct orientations in the two crystallographic subunits. The asymmetry seen in this structure for product binding is associated with absence of a water molecule W0 in the standard wild-type conformation of product binding that is clearly identifiable in the new structure, which may represent a structural model for binding of incoming GSH prior to displacement of W0. Elimination of W0 as a hydroxonium ion may be the mechanism for the initial proton extrusion from the active site. © 2010 The Authors Journal compilation © 2010 FEBS.

Developing a Novel Hydrogen Sponge with Ideal Binding Energy and High Surface Area for Practical Hydrogen Storage

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

Chung, T. C. Mike

This Phase I (5 quarters) research project was to examine the validity of a new class of boron-containing polymer (B-polymer) frameworks, serving as the adsorbents for the practical onboard H2 storage applications. Three B-polymer frameworks were synthesized and investigated, which include B-poly(butyenylstyrene) (B-PBS) framework (A), B-poly(phenyldiacetyene) (B-PPDA) framework (B), and B-poly(phenyltriacetylene) (B-PPTA) framework (C). They are 2-D polymer structures with the repeating cyclic units that spontaneously form open morphology and the B-doped (p-type) π-electrons delocalized surfaces. The ideal B-polymer framework shall exhibit open micropores (pore size in the range of 1-1.5nm) with high surface area (>3000 m 2/g), and themore » B-dopants in the conjugated framework shall provide high surface energy for interacting with H 2 molecules (an ideal H 2 binding energy in the range of 15-25 kJ/mol). The pore size distribution and H2 binding energy were investigated at both Penn State and NREL laboratories. So far, the experimental results show the successful synthesis of B-polymer frameworks with the relatively well-defined planar (2-D) structures. The intrinsically formed porous morphology exhibits a broad pore size distribution (in the range of 0.5-10 nm) with specific surface area (~1000 m 2/g). The miss-alignment between 2-D layers may block some micropore channels and limit gas diffusion throughout the entire matrix. In addition, the 2-D planar conjugated structure may also allow free π-electrons delocalization throughout the framework, which significantly reduces the acidity of B-moieties (electron-deficiency).The resulting 2-D B-polymer frameworks only exhibit a small increase of H 2 binding energy in the range of 8-9 KJ/mole (quite constant over the whole sorption range).« less

Differences in Organizational Structure of Insulin Receptor on Rat Adipocyte and Liver Plasma Membranes: Role of Disulfide Bonds

NASA Astrophysics Data System (ADS)

Schweitzer, John B.; Smith, Robert M.; Jarett, Leonard

1980-08-01

Binding of 125I-labeled insulin to rat liver and adipocyte plasma membranes has been investigated after treatment of the membranes with agents that modify disulfide bonds or sulfhydryl groups. Dithiothreitol, a disulfide-reducing agent, produced a bimodal response in adipocyte plasma membranes with dose-dependent increases in binding occurring over the range of 0-1 mM dithiothreitol; 5 mM dithiothreitol produced decreased binding. Insulin binding reached its maximal increase at 1 mM and was 3 times control values. Scatchard analysis of the 1 mM dithiothreitol effect revealed a straight line plot indicative of one class of sites with a Ka of 1.0× 108 M-1 which is intermediate between the two Kas obtained from the curvilinear Scatchard plot of control membranes. There was a 20-fold increase in the number of intermediate-affinity receptors compared to high-affinity receptors. The increased 125I-labeled insulin binding after dithiothreitol treatment was reversed by oxidized glutathione in a dose-dependent manner. Interposition of treatment with N-ethylmaleimide, an alkylating agent, prevented oxidized glutathione from reversing the dithiothreitol effect. Reduced glutathione produced the same effect as dithiothreitol. Liver plasma membranes treated with up to 1 mM dithiothreitol exhibited a maximum increase in insulin binding of 20% compared to control. Dithiothreitol at 5 mM decreased insulin binding below that of control membranes. The results indicate that the dithiothreitol effect on insulin binding to adipocyte plasma membranes is due to disruption of disulfide bonds, and that the structural organization of the insulin receptor on the plasma membranes is different for liver and for adipose tissue. The data imply that the insulin receptors on the plasma membrane of adipocytes possess at least two functionally distinct subclasses of disulfide bond but liver insulin receptors do not.

Characterization of sarcoplasmic reticulum Ca{sup 2+} ATPase nucleotide binding domain mutants using NMR spectroscopy

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

Myint, Wazo; Gong, Qingguo; Ahn, Jinwoo

2011-02-04

Research highlights: {yields} Structural consequence by substitution mutations on the isolated SERCA-nucleotide binding (SERCA-N) domain was studied. {yields} The study fills a gap between the previous clinical, physiological, and biochemical data and the molecular basis of SERCA-N. {yields} The E412G mutation, known to be seen in patients with Darier's disease, was found to maintain the active conformation but exhibit reduced protein stability. -- Abstract: Sarcoplasmic reticulum Ca{sup 2+} ATPase (SERCA) is essential for muscle function by transporting Ca{sup 2+} from the cytosol into the sarcoplasmic reticulum through ATP hydrolysis. In this report, the effects of substitution mutations on the isolatedmore » SERCA-nucleotide binding domain (SERCA-N) were studied using NMR. {sup 15}N-{sup 1}H HSQC spectra of substitution mutants at the nucleotide binding site, T441A, R560V, and C561A, showed chemical shift changes, primarily in residues adjacent to the mutation sites, indicating only local effects. Further, the patterns of chemical shift changes upon AMP-PNP binding to these mutants were similar to that of the wild type SERCA-N (WT). In contrast to these nucleotide binding site mutants, a mutant found in patients with Darier's disease, E412G, showed small but significant chemical shift changes throughout the protein and rapid precipitation. However, the AMP-PNP dissociation constant ({approx}2.5 mM) was similar to that of WT ({approx}3.8 mM). These results indicate that the E412G mutant retains its catalytic activity but most likely reduces its stability. Our findings provide molecular insight into previous clinical, physiological, and biochemical observations.« less

How actin binds and assembles onto plasma membranes from Dictyostelium discoideum

PubMed Central

1988-01-01

We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross- linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell. PMID:3392099

Characterization of 18F-FPyKYNE-Losartan for Imaging AT1 Receptors.

PubMed

Hachem, Maryam; Tiberi, Mario; Ismail, Basma; Hunter, Chad R; Arksey, Natasha; Hadizad, Tayebeh; Beanlands, Rob S; deKemp, Robert A; DaSilva, Jean N

2016-10-01

Most physiologic effects of the renin angiotensin system (RAS) are mediated via the angiotensin (Ang) type 1 receptor (AT 1 R). The 18 F-FPyKYNE derivative of the clinically used AT 1 R blocker losartan exhibits high binding selectivity for kidney AT 1 R and rapid metabolism in rats. The aim of this study was to further assess the binding profile of this novel PET agent for imaging AT 1 R in rats and pigs. In vitro binding assays were performed with 18 F-FPyKYNE-losartan in rat kidneys. Male Sprague-Dawley rats were used to assess dosimetry, antagonistic efficacy via blood pressure measurements, and presence of labeled metabolites in kidneys. Test-retest PET imaging, blocking with AT 1 R antagonist candesartan (10 mg/kg), and plasma metabolism analysis were performed in female Yorkshire pigs. 18 F-FPyKYNE-losartan bound with high affinity (dissociation constant of 49.4 ± 18.0 nM and maximal binding of 348 ± 112 fmol/mm 2 ) to rat kidney AT 1 R. It bound strongly to plasma proteins in rats (97%), and its labeled metabolites displayed minimal interference on renal AT 1 R binding. FPyKYNE-losartan fully antagonized the Ang II pressor effect, albeit with 4-fold potency reduction (the effective dose inhibiting 50% of the Ang II-induced maximal pressor response of 25.5 mg/kg) relative to losartan. PET imaging exhibited high kidney-to-blood contrast and slow renal clearance, with an SUV of 14.1 ± 6.2. Excellent reproducibility was observed in the calculated test-retest variability (7.2% ± 0.75%). Only hydrophilic-labeled metabolites were present in plasma samples, and renal retention was reduced (-60%) at 10-15 min after blockade with candesartan. 18 F-FPyKYNE-losartan has a favorable binding profile and displays high potential for translational work in humans as an AT 1 R PET imaging agent. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches

PubMed Central

Walavalkar, Ninad M.; Cramer, Jason M.; Buchwald, William A.; Scarsdale, J. Neel; Williams, David C.

2014-01-01

Unlike other members of the methyl-cytosine binding domain (MBD) family, MBD4 serves as a potent DNA glycosylase in DNA mismatch repair specifically targeting mCpG/TpG mismatches arising from spontaneous deamination of methyl-cytosine. The protein contains an N-terminal MBD (MBD4MBD) and a C-terminal glycosylase domain (MBD4GD) separated by a long linker. This arrangement suggests that the MBD4MBD either directly augments enzymatic catalysis by the MBD4GD or targets the protein to regions enriched for mCpG/TpG mismatches. Here we present structural and dynamic studies of MBD4MBD bound to dsDNA. We show that MBD4MBD binds with a modest preference formCpG as compared to mismatch, unmethylated and hydroxymethylated DNA. We find that while MBD4MBD exhibits slow exchange between molecules of DNA (intermolecular exchange), the domain exhibits fast exchange between two sites in the same molecule of dsDNA (intramolecular exchange). Introducing a single-strand defect between binding sites does not greatly reduce the intramolecular exchange rate, consistent with a local hopping mechanism for moving along the DNA. These results support a model in which the MBD4MBD4 targets the intact protein to mCpG islands and promotes scanning by rapidly exchanging between successive mCpG sites which facilitates repair of nearby mCpG/TpG mismatches by the glycosylase domain. PMID:25183517

FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation.

PubMed

Ge, Xiao Na; Bastan, Idil; Dileepan, Mythili; Greenberg, Yana; Ha, Sung Gil; Steen, Kaylee A; Bernlohr, David A; Rao, Savita P; Sriramarao, P

2018-04-26

Fatty acid binding protein 4 (FABP4), a member of a family of lipid-binding proteins, is known to play a role in inflammation by virtue of its ability to regulate intracellular events such as lipid fluxes and signaling. Studies have indicated a pro-inflammatory role for FABP4 in allergic asthma, although its expression and function in eosinophils, the predominant inflammatory cells recruited to allergic airways, was not investigated. We examined expression of FABP4 in murine eosinophils and its role in regulating cell recruitment in vitro as well as in cockroach antigen (CRA)-induced allergic airway inflammation. CRA exposure led to airway recruitment of FABP4-expressing inflammatory cells, specifically eosinophils, in wild type (WT) mice. FABP4 expression in eosinophils was induced by TNF-α as well as IL-4 and IL-13. FABP4-deficient eosinophils exhibited markedly decreased cell spreading/formation of leading edges on vascular cell adhesion molecule-1 and significantly decreased adhesion to intercellular adhesion molecule-1 associated with reduced β2 integrin expression relative to WT cells. Further, FABP4-deficient eosinophils exhibited decreased migration, F-actin polymerization, calcium flux and ERK (1/2) phosphorylation in response to eotaxin-1. In vivo, CRA-challenged FABP4-deficient mice exhibited attenuated eosinophilia and significantly reduced airway inflammation (improved airway reactivity, lower IL-5, IL-13, TNFα and LTC4 levels, decreased airway structural changes) compared to WT mice. In conclusion, expression of FABP4 in eosinophils is induced during conditions of inflammation and plays a pro-inflammatory role in the development of allergic asthma by promoting eosinophil adhesion and migration and contributing to the development of various aspects of airway inflammation.

Involvement of two classes of binding sites in the interactions of cyclophilin B with peripheral blood T-lymphocytes.

PubMed

Denys, A; Allain, F; Carpentier, M; Spik, G

1998-12-15

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes. We first provide evidence that the CyPB binding to heparin-Sepharose is prevented by soluble sulphated glycosaminoglycans (GAG), raising the interesting possibility that such interactions may occur on the T-cell surface. We then characterized CyPB binding to T-cell surface GAG and found that these interactions involved the N-terminal extension of CyPB, but not its conserved CsA-binding domain. In addition, we determined the presence of a second CyPB binding site, which we termed a type I site, in contrast with type II for GAG interactions. The two binding sites exhibit a similar affinity but the expression of the type I site was 3-fold lower. The conclusion that CyPB binding to the type I site is distinct from the interactions with GAG was based on the findings that it was (1) resistant to NaCl wash and GAG-degrading enzyme treatments, (2) reduced in the presence of CsA or cyclophilin C, and (3) unmodified in the presence of either the N-terminal peptide of CyPB or protamine. Finally, we showed that the type I binding sites were involved in an endocytosis process, supporting the hypothesis that they may correspond to a functional receptor for CyPB.

Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome

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

Jetzt, Amanda E.

Ricin is a potent ribotoxin that is considered a bioterror threat due to its ease of isolation and possibility of aerosolization. In yeast, mutation of arginine residues away from the active site results in a ricin toxin A chain (RTA) variant that is unable to bind the ribosome and exhibits reduced cytotoxicity. The goal of the present work was to determine if these residues contribute to ribosome binding and cytotoxicity of RTA in mammalian cells. The RTA mutant R193A/R235A did not interact with mammalian ribosomes, while a G212E variant with a point mutation near its active site bound ribosomes similarlymore » to wild-type (WT) RTA. R193A/R235A retained full catalytic activity on naked RNA but had reduced activity on mammalian ribosomes. To determine the effect of this mutant in intact cells, pre R193A/R235A containing a signal sequence directing it to the endoplasmic reticulum and mature R193A/R235A that directly targeted cytosolic ribosomes were each expressed. Depurination and protein synthesis inhibition were reduced by both pre- and mature R193A/R235A relative to WT. Protein synthesis inhibition was reduced to a greater extent by R193A/R235A than by G212E. Pre R193A/R235A caused a greater reduction in caspase activation and loss of mitochondrial membrane potential than G212E relative to WT RTA. These findings indicate that an RTA variant with reduced ribosome binding is less toxic than a variant with less catalytic activity but normal ribosome binding activity. The toxin-ribosome interaction represents a novel target for the development of therapeutics to prevent or treat ricin intoxication. - Highlights: • Arginines 193 and 235 of RTA are critical for binding to the mammalian ribosome. • R193A/R235A has full catalytic activity on RNA but not on mammalian ribosomes. • R193A/R235A is less toxic than a mutant that targets the active site. • The toxin-ribosome interaction is a therapeutic target for ricin intoxication.« less

Electroabsorption Spectroscopy Measurements of the Exciton Binding Energy, ElectronHole Reduced Effective Mass, and Band Gap in the Perovskite CHsub3NHsub3PbIsub3

DTIC Science & Technology

2016-07-28

can essentially be described in terms of free carrier generation . KEYWORDS: solar cell, photovoltaic, Franz−Keldysh effect, Wannier exciton, Stark...optoelectronic devices such as lasers,1,2 LEDs,3 and solar cells,4−6 despite requiring only inexpensive and relatively crude processing conditions...compared to current high-performance crystalline semiconductors. The archetypal material, CH3NH3PbI3, has exhibited excellent power con- version

Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes

NASA Astrophysics Data System (ADS)

Weinberger, Andreas; Walter, Vivien; MacEwan, Sarah R.; Schmatko, Tatiana; Muller, Pierre; Schroder, André P.; Chilkoti, Ashutosh; Marques, Carlos M.

2017-03-01

Although cationic cell-penetrating peptides (CPPs) are able to bind to cell membranes, thus promoting cell internalization by active pathways, attachment of cargo molecules to CPPs invariably reduces their cellular uptake. We show here that CPP binding to lipid bilayers, a simple model of the cell membrane, can be recovered by designing cargo molecules that self-assemble into spherical micelles and increase the local interfacial density of CPP on the surface of the cargo. Experiments performed on model giant unilamellar vesicles under a confocal laser scanning microscope show that a family of thermally responsive elastin-like polypeptides that exhibit temperature-triggered micellization can promote temperature triggered attachment of the micelles to membranes, thus rescuing by self-assembly the cargo-induced loss of the CPP affinity to bio-membranes.

Antiviral drug acyclovir exhibits antitumor activity via targeting βTrCP1: Molecular docking and dynamics simulation study.

PubMed

Shafique, Shagufta; Rashid, Sajid

2017-03-01

The critical role of βTrCP1 in cancer development makes it a discerning target for the development of small drug like molecules. Currently, no inhibitor exists that is able to target its substrate binding site. Through molecular docking and dynamics simulation assays, we explored the comparative binding pattern of βTrCP1-WD40 domain with ACV and its phospho-derivatives (ACVMP, ACVDP and ACVTP). Consequently, through principal component analysis, βTrCP1-ACVTP was found to be more stable complex by obscuring a reduced conformational space than other systems. Thus based on the residual contribution and hydrogen bonding pattern, ACVTP was considered as a noteworthy inhibitor which demarcated binding in the cleft formed by βTrCP1-WD40 specific β-propeller. The outcomes of this study may provide a platform for rational design of specific and potent inhibitor against βTrCP1, with special emphasis on anticancer activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Binding of alkylphenols and alkylated non-phenolics to rainbow trout (Oncorhynchus mykiss) hepatic estrogen receptors.

PubMed

Tollefsen, Knut-Erik; Julie Nilsen, Anja

2008-02-01

Alkylphenols are well-known endocrine disrupters, mediating effects through the estrogen receptor (ER). In the present work, the interaction of alkylphenols and alkylated non-phenolics with hepatic rainbow trout (Oncorhynchus mykiss) estrogen receptors (rtERs) was determined. The role of alkyl chain length and branching, substituent position, number of alkylated groups, and the requirement of a phenolic ring structure was assessed. The results showed that the rtERs bound most alkylphenols, although with 20,000 to 2 million times lower affinity than the endogenous estrogen 17beta-estradiol. Mono-substituted alkylphenols with moderate (C4-C6) and long (C8 and C12) alkyl chain length in the para position exhibited the highest affinity for the rtERs. Substitution with multiple alkyl groups, presence of substituents in the ortho- and meta-position, and lack of a hydroxyl group on the benzene ring reduced the binding affinity. The rtERs resembled the reported binding specificity of the human ER for alkylphenols, although some exceptions were identified.

Elucidation of the Human Serum Albumin (HSA) Binding Site for the Cu-PTSM and Cu-ATSM Radiopharmaceuticals

PubMed Central

Basken, Nathan E.; Mathias, Carla J.; Green, Mark A.

2008-01-01

The Cu-PTSM (pyruvaldehyde bis(N4-methylthiosemicarbazonato)copper(II)) and Cu-ATSM (diacetyl bis(N4-methylthiosemicarbazonato)copper(II)) radiopharmaceuticals exhibit strong, species-dependent binding to human serum albumin (HSA), while Cu-ETS (ethylglyoxal bis(thiosemicarbazonato)copper(II)) appears to only exhibit non-specific binding to human and animal serum albumins. This study examines the structural basis for HSA binding of Cu-PTSM and Cu-ATSM via competition with drugs having known albumin binding sites. Warfarin, furosemide, ibuprofen, phenylbutazone, benzylpenicillin, and cephmandole were added to HSA solutions at drug:HSA mole ratios from 0 to 8:1, followed by quantification of radiopharmaceutical binding to HSA by ultrafiltration. Warfarin, a site IIA drug, progressively displaced both [64Cu]Cu-PTSM and [64Cu]Cu-ATSM from HSA. At 8:1 warfarin:HSA mole ratios, free [64Cu]Cu-PTSM and [64Cu]Cu-ATSM levels increased 300–500%. This was in contrast to solutions containing ibuprofen, a site IIIA drug; no increase in free [64Cu]Cu-PTSM or [64Cu]Cu-ATSM was observed except at high ibuprofen:HSA ratios, where secondary ibuprofen binding to the IIA site may cause modest radiopharmaceutical displacement. By contrast, and consistent with earlier findings suggesting Cu-ETS exhibits only non-specific associations, [64Cu]Cu-ETS binding to HSA was unaffected by the addition of drugs that bind in either site. We conclude that the species-dependence of Cu-PTSM and Cu-ATSM albumin binding arises from interaction(s) with the IIA site of HSA. PMID:18937368

Mechanism of pathogen recognition by human dectin-2.

PubMed

Feinberg, Hadar; Jégouzo, Sabine A F; Rex, Maximus J; Drickamer, Kurt; Weis, William I; Taylor, Maureen E

2017-08-11

Dectin-2, a C-type lectin on macrophages and other cells of the innate immune system, functions in response to pathogens, particularly fungi. The carbohydrate-recognition domain (CRD) in dectin-2 is linked to a transmembrane sequence that interacts with the common Fc receptor γ subunit to initiate immune signaling. The molecular mechanism by which dectin-2 selectively binds to pathogens has been investigated by characterizing the CRD expressed in a bacterial system. Competition binding studies indicated that the CRD binds to monosaccharides with modest affinity and that affinity was greatly enhanced for mannose-linked α1-2 or α1-4 to a second mannose residue. Glycan array analysis confirmed selective binding of the CRD to glycans that contain Manα1-2Man epitopes. Crystals of the CRD in complex with a mammalian-type high-mannose Man 9 GlcNAc 2 oligosaccharide exhibited interaction with Manα1-2Man on two different termini of the glycan, with the reducing-end mannose residue ligated to Ca 2+ in a primary binding site and the nonreducing terminal mannose residue occupying an adjacent secondary site. Comparison of the binding sites in DC-SIGN and langerin, two other pathogen-binding receptors of the innate immune system, revealed why these two binding sites accommodate only terminal Manα1-2Man structures, whereas dectin-2 can bind Manα1-2Man in internal positions in mannans and other polysaccharides. The specificity and geometry of the dectin-2-binding site provide the molecular mechanism for binding of dectin-2 to fungal mannans and also to bacterial lipopolysaccharides, capsular polysaccharides, and lipoarabinomannans that contain the Manα1-2Man disaccharide unit. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Insight into the binding modes of Lassa nucleoprotein complexed with ssRNA by molecular dynamic simulations and free energy calculations.

PubMed

Zhang, Ying; Chen, Hang; Han, Ju-Guang

2015-01-01

Lassa virus (LASV), an arenavirus known to be responsible for a severe hemorrhagic fever, causes thousands of deaths annually and there is no effective vaccine for it so far. The nucleoprotein (NP) of LASV plays an essential role in the replication and transcription of the viral genome. Recent research shows that viral RNA binds in a deep crevice located within the N-terminal domain of NP and suggests a gating mechanism in which NP transforms from a "closed" position to an "open" position to bind RNA. To characterize the molecular mechanisms of how RNA binds to N-terminal domain of NP, two molecular dynamic (MD) simulations of RNA-binding structure and RNA-free structure have been performed. The simulation results show that an important helix α6 interacts with RNA in the "open" conformation, while helix α6 rotates toward the binding crevice and reduces the space of RNA-binding pocket in the "closed" conformation; it appears that helix α6 would clash with RNA while NP is in a "closed" state. In addition, to characterize the role of residues involved in the binding of RNA, the MD simulations of the double-mutant (W164A/F176A) and the single-mutant (G243P) RNA-binding NP complexes have been performed. Our MD simulations and molecular mechanics-generalized born surface area (MM-GBSA) energy calculations exhibit that the three mutant residues increase the binding affinity. Furthermore, we infer that the defect of the replication and transcription of viral genome is possibly due to the change of structural integrity rather than the reduction of RNA-binding affinity.

Ultra-sensitive detection of kanamycin for food safety using a reduced graphene oxide-based fluorescent aptasensor

NASA Astrophysics Data System (ADS)

Ha, Na-Reum; Jung, In-Pil; La, Im-Joung; Jung, Ho-Sup; Yoon, Moon-Young

2017-01-01

Overuse of antibiotics has caused serious problems, such as appearance of super bacteria, whose accumulation in the human body through the food chain is a concern. Kanamycin is a common antibiotic used to treat diverse infections; however, residual kanamycin can cause many side effects in humans. Thus, development of an ultra-sensitive, precise, and simple detection system for residual kanamycin in food products is urgently needed for food safety. In this study, we identified kanamycin-binding aptamers via a new screening method, and truncated variants were analyzed for optimization of the minimal sequence required for target binding. We found various aptamers with high binding affinity from 34.7 to 669 nanomolar Kdapp values with good specificity against kanamycin. Furthermore, we developed a reduced graphene oxide (RGO)-based fluorescent aptasensor for kanamycin detection. In this system, kanamycin was detected at a concentration as low as 1 pM (582.6 fg/mL). In addition, this method could detect kanamycin accurately in kanamycin-spiked blood serum and milk samples. Consequently, this simple, rapid, and sensitive kanamycin detection system with newly structural and functional analysis aptamer exhibits outstanding detection compared to previous methods and provides a new possibility for point of care testing and food safety.

The correlation between ouabain binding and potassium pump inhibition in human and sheep erythrocytes.

PubMed Central

Joiner, C H; Lauf, P K

1978-01-01

1. [3H]Ouabain binding to human and sheep red blood cells was shown to be specific for receptors associated with Na/K transport. Virtually all tritium binding was abolished by dilution with unlabelled drug. Saturation levels of binding were independent of glycoside concentration and were identical to those associated with 100% inhibition of K pumping. 2. [3H]Ouabain binding and 42K influx were measured simultaneously in order to correlate the degree of K pump inhibition with the amount of glycoside bound. Results by this method agreed exactly with those obtained by pre-exposing cells to drug, followed by washing and then measuring K influx. 3. Plots of [3H]oubain binding vs. K pump inhibition were rectilinear for human and low K (LK) sheep red cells, indicating one glycoside receptor per K pump site and functional homogeneity of pump sites. High K (HK) sheep red cells exhibited curved plots of binding versus inhibition, which were best explained in terms of one receptor per pump, but a heterogeneous population of pump sites. 4. External K reduced the rate of glycoside binding, but did not alter the relationship between binding and inhibition. 5. The number of K pump sites was estimated as 450--500 per human cell and 30--50 per LK sheep cell. HK sheep cells had 90--130 sites per cell, of which eighty to ninety were functionally dominant. The number of K pump sites on LK sheep cells was not changed by anti-L, although the maximum velocity of pump turnover was increased. PMID:722573

Recombinant mouse sperm ZP3-binding protein (ZP3R/sp56) forms a high order oligomer that binds eggs and inhibits mouse fertilization in vitro.

PubMed

Buffone, Mariano G; Zhuang, Tiangang; Ord, Teri S; Hui, Ling; Moss, Stuart B; Gerton, George L

2008-05-02

Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.

Aqueous Synthesis of PEGylated Quantum Dots with Increased Colloidal Stability and Reduced Cytotoxicity.

PubMed

Ulusoy, Mehriban; Jonczyk, Rebecca; Walter, Johanna-Gabriela; Springer, Sergej; Lavrentieva, Antonina; Stahl, Frank; Green, Mark; Scheper, Thomas

2016-02-17

Ligands used on the surface of colloidal nanoparticles (NPs) have a significant impact on physiochemical properties of NPs and their interaction in biological environments. In this study, we report a one-pot aqueous synthesis of 3-mercaptopropionic acid (MPA)-functionalized CdTe/CdS/ZnS quantum dots (Qdots) in the presence of thiol-terminated methoxy polyethylene glycol (mPEG) molecules as a surface coordinating ligand. The resulting mPEG-Qdots were characterized by using ζ potential, FTIR, thermogravimetric (TG) analysis, and microscale thermophoresis (MST) studies. We investigated the effect of mPEG molecules and their grafting density on the Qdots photophysical properties, colloidal stability, protein binding affinity, and in vitro cellular toxicity. Moreover, cellular binding features of the resulting Qdots were examined by using three-dimensional (3D) tumor-like spheroids, and the results were discussed in detail. Promisingly, mPEG ligands were found to increase colloidal stability of Qdots, reduce adsorption of proteins to the Qdot surface, and mitigate Qdot-induced side effects to a great extent. Flow cytometry and confocal microscopy studies revealed that PEGylated Qdots exhibited distinctive cellular interactions with respect to their mPEG grafting density. As a result, mPEG molecules demonstrated a minimal effect on the ZnS shell deposition and the Qdot fluorescence efficiency at a low mPEG density, whereas they showed pronounced effect on Qdot colloidal stability, protein binding affinity, cytotoxicity, and nonspecific binding at a higher mPEG grafting amount.

Laminar and regional distribution of galanin binding sites in cat and monkey visual cortex determined by in vitro receptor autoradiography

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

Rosier, A.M.; Vandesande, F.; Orban, G.A.

1991-03-08

The distribution of galanin (GAL) binding sites in the visual cortex of cat and monkey was determined by autoradiographic visualization of ({sup 125}I)-GAL binding to tissue sections. Binding conditions were optimized and, as a result, the binding was saturable and specific. In cat visual cortex, GAL binding sites were concentrated in layers I, IVc, V, and VI. Areas 17, 18, and 19 exhibited a similar distribution pattern. In monkey primary visual cortex, the highest density of GAL binding sites was observed in layers II/III, lower IVc, and upper V. Layers IVA and VI contained moderate numbers of GAL binding sites,more » while layer I and the remaining parts of layer IV displayed the lowest density. In monkey secondary visual cortex, GAL binding sites were mainly concentrated in layers V-VI. Layer IV exhibited a moderate density, while the supragranular layers contained the lowest proportion of GAL binding sites. In both cat and monkey, we found little difference between regions subserving central and those subserving peripheral vision. Similarities in the distribution of GAL and acetylcholine binding sites are discussed.« less

Isolation of copper-binding proteins from activated sludge culture.

PubMed

Fukushi, K; Kato, S; Antsuki, T; Omura, T

2001-01-01

Six copper-binding microbial proteins were isolated from activated sludge cultures grown on media containing copper at various concentrations. Molecular weights among isolated proteins were ranged from 1.3k to 1 74k dalton. Isolated proteins were compared for their copper binding capabilities. Proteins isolated from cultures grown in the presence of copper in the growth media exhibited higher copper binding capabilities than those isolated from the culture grown in the absence of copper. The highest metal uptake of 61.23 (mol copper/mol protein) was observed by a protein isolated from a culture grown with copper at a concentration of 0.25 mM. This isolated protein (CBP2) had a molecular weight of 24k dalton. Other protein exhibited copper binding capability of 4.8-32.5 (mol copper/mol protein).

Analysis of the hormone-binding domain of steroid receptors using chimeras generated by homologous recombination

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

Martinez, Elisabeth D.; Pattabiraman, Nagarajan; Department of Oncology, Georgetown University School of Medicine, Washington, DC 20057

2005-08-15

The glucocorticoid receptor and the mineralocorticoid receptor are members of the steroid receptor family that exhibit ligand cross-reactivity. Specificity of steroid receptor action is investigated in the present work by the construction and characterization of chimeras between the glucocorticoid receptor and the mineralocorticoid receptor. We used an innovative approach to make novel steroid receptor proteins in vivo that in general, contrary to our expectations, show increased ligand specificity compared to the parental receptors. We describe a receptor that is specific for the potent synthetic glucocorticoid triamcinolone acetonide and does not bind aldosterone. A further set of chimeras has an increasedmore » ability to discriminate between ligands, responding potently to mineralocorticoids and only very weakly to synthetic glucocorticoids. A chimera with the fusion site in the hinge highlights the importance of the region between the DNA-binding and the hormone-binding domains since, unlike both the glucocorticoid and mineralocorticoid receptors, it only responds to mineralocorticoids. One chimera has reduced specificity in that it acts as a general corticoid receptor, responding to glucocorticoids and mineralocorticoids with similar potency and efficacy. Our data suggest that regions of the glucocorticoid and mineralocorticoid receptor hormone-binding domains are functionally non-reciprocal. We present transcriptional, hormone-binding, and structure-modeling evidence that suggests that receptor-specific interactions within and across domains mediate aspects of specificity in transcriptional responses to steroids.« less

In vitro and in silico assessment of the structure-dependent binding of bisphenol analogues to glucocorticoid receptor.

PubMed

Zhang, Jie; Zhang, Tiehua; Guan, Tianzhu; Yu, Hansong; Li, Tiezhu

2017-03-01

Widespread use of bisphenol A (BPA) and other bisphenol analogues has attracted increasing attention for their potential adverse effects. As environmental endocrine-disrupting compounds (EDCs), bisphenols (BPs) may activate a variety of nuclear receptors, including glucocorticoid receptor (GR). In this work, the binding of 11 BPs to GR was investigated by fluorescence polarization (FP) assay in combination with molecular dynamics simulations. The human glucocorticoid receptor was prepared as a soluble recombinant protein. A fluorescein-labeled dexamethasone derivative (Dex-fl) was employed as tracer. Competitive displacement of Dex-fl from GR by BPs showed that the binding affinities of bisphenol analogues were largely dependent on their characteristic functional groups. In order to further understand the relationship between BPs structures and their GR-mediated activities, molecular docking was utilized to explore the binding modes at the atomic level. The results confirmed that structural variations of bisphenol analogues contributed to different interactions of BPs with GR, potentially causing distinct toxic effects. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation (R 2  = 0.8266), which might be helpful for the design of environmentally benign materials with reduced toxicities. In addition, the established FP assay based on GR exhibited the potential to offer an alternative to traditional methods for the detection of bisphenols.

Computer simulation of protein systems

NASA Technical Reports Server (NTRS)

Osguthorpe, D. J.; Dauber-Osguthorpe, P.; Wolff, J.; Kitson, D. H.; Hagler, A. T.

1984-01-01

Ligand binding to dihydrofolate reductase (DHFR) is discussed. This is an extremely important enzyme, as it is the target of several drugs (inhibitors) which are used clinically as antibacterials, antiprotozoals and in cancer chemotherapy. DHFR catalyzes the NADPH (reduced nicotinamide adenine dinucleotide phosphate) dependent reduction of dihydrofolate to tetrahydrofolate, which is used in several pathways of purine and pyrimidine iosynthesis, including that of thymidylate. Since DNA synthesis is dependent on a continuing supply of thymidylate, a blockade of DHFR resulting in a depletion of thymidylate can lead to the cessation of growth of a rapidly proliferating cell line. DHFR exhibits a significant species to species variability in its sensitivity to various inhibitors. For example, trimethoprim, an inhibitor of DHFR, binds to bacterial DHFR's 5 orders of magnitude greater than to vertebrate DHFR's. The structural mechanics, dynamics and energetics of a family of dihydrofolate reductases are studied to rationalize the basis for the inhibitor of these enyzmes and to understand the molecular basis of the difference in the binding constants between the species. This involves investigating the conformational changes induced in the protein on binding the ligand, the internal strain imposed by the enzyme on the ligand, the restriction of fluctuations in atom positions due to binding and the consequent change in entropy.

Salt-soluble proteins from wheat-derived foodstuffs show lower allergenic potency than those from raw flour.

PubMed

de Gregorio, Marta; Armentia, Alicia; Díaz-Perales, Araceli; Palacín, Arantxa; Dueñas-Laita, Antonio; Martín, Blanca; Salcedo, Gabriel; Sánchez-Monge, Rosa

2009-04-22

Salt-soluble proteins from wheat flour have been described as main allergens associated with both baker's asthma and food allergy. However, most studies have used raw flour as starting material, thus not considering potential changes in allergenic properties induced by the heat treatment and other industrial processing to produce wheat-derived foodstuffs. Salt extracts from different commercial wheat-derived products were obtained and their allergenic properties investigated by IgE-immunodetection, ELISA assays, and skin prick test. The IgE-binding capacity of salt-soluble proteins from commercial breads and cooked pastas was reduced around 50% compared with that of raw flour, the reduction being less dramatic in noncooked pastas and biscuits. Several wheat-derived foodstuffs showed major IgE-binding components of 20 and 35 kDa, identified as avenin-like and globulin proteins, respectively. These proteins, as well as most flour and bread salt-soluble proteins, were hydrolyzed when subjected to simulated gastrointestinal digestion. However, the digested products still exhibited a residual IgE-binding capacity. Therefore, processing of wheat flour to obtain derived foodstuffs decreases the IgE binding-capacity of the major salt-soluble wheat proteins. Moreover, simulated gastric fluid digestion further inactivates some heat-resistant IgE-binding proteins.

Atrial natriuretic peptide receptor heterogeneity and effects on cyclic GMP accumulation

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

Leitman, D.C.

1988-01-01

The effects of atrial natriuretic peptide (ANP), oxytocin (OT) and vasopressin (AVP) on guanylate cyclase activity and cyclic GMP accumulation were examined, since these hormones appear to be intimately associated with blood pressure and intravascular volume homeostasis. ANP was found to increase cyclic GMP accumulation in ten cell culture systems, which were derived from blood vessels, adrenal cortex, kidney, lung, testes and mammary gland. ANP receptors were characterized in intact cultured cells using {sup 125}I-ANP{sub 8-33}. Specific {sup 125}I-ANP binding was saturable and of high affinity. Scratchard analysis of the binding data for all cell types exhibited a straight line,more » indicating that these cells possessed a single class of binding sites. Despite the presence of linear Scatchard plots, these studies demonstrated that cultured cells possess two functionally and physically distinct ANP-binding sites. Most of the ANP-binding sites in cultured cells have a molecular size of 66,000 daltons under reducing conditions. The identification of cultured cell types in which hormones (ANP and oxytocin) regulate guanylate cyclase activity and increase cyclic GMP synthesis will provide valuable systems to determine the mechanisms of hormone-receptor coupling to guanylate cyclase and the cellular processes regulated by cyclic GMP.« less

Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid-Activated Receptor

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

Kruse, Schoen W; Suino-Powell, Kelly; Zhou, X Edward

2010-01-12

The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 {angstrom} crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix {alpha}10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site,more » thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.« less

Potent and Selective Amidopyrazole Inhibitors of IRAK4 That Are Efficacious in a Rodent Model of Inflammation

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

McElroy, William T.; Tan, Zheng; Ho, Ginny

IRAK4 is a critical upstream kinase in the IL-1R/TLR signaling pathway. Inhibition of IRAK4 is hypothesized to be beneficial in the treatment of autoimmune related disorders. A screening campaign identified a pyrazole class of IRAK4 inhibitors that were determined by X-ray crystallography to exhibit an unusual binding mode. SAR efforts focused on the identification of a potent and selective inhibitor with good aqueous solubility and rodent pharmacokinetics. Pyrazole C-3 piperidines were well tolerated, with N-sulfonyl analogues generally having good rodent oral exposure but poor solubility. N-Alkyl piperidines exhibited excellent solubility and reduced exposure. Pyrazoles possessing N-1 pyridine and fluorophenyl substituentsmore » were among the most active. Piperazine 32 was a potent enzyme inhibitor with good cellular activity. Compound 32 reduced the in vivo production of proinflammatory cytokines and was orally efficacious in a mouse antibody induced arthritis disease model of inflammation.« less

Characterization of the Vibrio vulnificus 1-Cys peroxiredoxin Prx3 and regulation of its expression by the Fe-S cluster regulator IscR in response to oxidative stress and iron starvation.

PubMed

Lim, Jong Gyu; Bang, Ye-Ji; Choi, Sang Ho

2014-12-26

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes that reduce toxic peroxides. A new Vibrio vulnificus Prx, named Prx3, was identified and characterized in this study. Biochemical and mutational analyses revealed that Prx3 reduces H2O2, utilizing glutaredoxin 3 (Grx3) and glutathione (GSH) as reductants, and requires only N-terminal peroxidatic cysteine for its catalysis. These results, combined with the monomeric size of Prx3 observed under non-reducing conditions, suggested that Prx3 is a Grx3/GSH-dependent 1-Cys Prx and oxidized without forming intermolecular disulfide bonds. The prx3 mutation impaired growth in the medium containing peroxides and reduced virulence in mice, indicating that Prx3 is essential for survival under oxidative stress and pathogenesis of V. vulnificus. The Fe-S cluster regulator IscR activates prx3 by direct binding to a specific binding sequence centered at -44 from the transcription start site. The binding sequence was homologous to the Type 2 IscR-binding sequence, most likely recognized by the Fe-S clusterless apo-IscR in Escherichia coli. The iscR3CA mutant, chromosomally encoding the apo-locked IscR, exhibited 3-fold higher levels of activation of prx3 than the wild type and accumulated more IscR3CA protein in cells. The IscR-dependent activation of prx3 by aerobic growth and iron starvation was also associated with the increase in cellular levels of IscR protein. Taken together, the results suggested that IscR senses iron starvation as well as reactive oxygen species and shifts to the apo-form, which leads to the increase of cellular IscR and in turn prx3 expression, contributing to the survival and virulence of V. vulnificus during pathogenesis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

CCAAT/enhancer-binding protein beta inhibits proliferation in monocytic cells by affecting the retinoblastoma protein/E2F/cyclin E pathway but is not directly required for macrophage morphology.

PubMed

Gutsch, Romina; Kandemir, Judith D; Pietsch, Daniel; Cappello, Christian; Meyer, Johann; Simanowski, Kathrin; Huber, René; Brand, Korbinian

2011-07-01

Monocytic differentiation is orchestrated by complex networks that are not fully understood. This study further elucidates the involvement of transcription factor CCAAT/enhancer-binding protein β (C/EBPβ). Initially, we demonstrated a marked increase in nuclear C/EBPβ-liver-enriched activating protein* (LAP*)/liver-enriched activating protein (LAP) levels and LAP/liver-enriched inhibiting protein (LIP) ratios in phorbol 12-myristate 13-acetate (PMA)-treated differentiating THP-1 premonocytic cells accompanied by reduced proliferation. To directly study C/EBPβ effects on monocytic cells, we generated novel THP-1-derived (low endogenous C/EBPβ) cell lines stably overexpressing C/EBPβ isoforms. Most importantly, cells predominantly overexpressing LAP* (C/EBPβ-long), but not those overexpressing LIP (C/EBPβ-short), exhibited a reduced proliferation, with no effect on morphology. PMA-induced inhibition of proliferation was attenuated in C/EBPβ-short cells. In C/EBPβ(WT) macrophage-like cells (high endogenous C/EBPβ), we measured a reduced proliferation/cycling index compared with C/EBPβ(KO). The typical macrophage morphology was only observed in C/EBPβ(WT), whereas C/EBPβ(KO) stayed round. C/EBPα did not compensate for C/EBPβ effects on proliferation/morphology. Serum reduction, an independent approach known to inhibit proliferation, induced macrophage morphology in C/EBPβ(KO) macrophage-like cells but not THP-1. In PMA-treated THP-1 and C/EBPβ-long cells, a reduced phosphorylation of cell cycle repressor retinoblastoma was found. In addition, C/EBPβ-long cells showed reduced c-Myc expression accompanied by increased CDK inhibitor p27 and reduced cyclin D1 levels. Finally, C/EBPβ-long and C/EBPβ(WT) cells exhibited low E2F1 and cyclin E levels, and C/EBPβ overexpression was found to inhibit cyclin E1 promoter-dependent transcription. Our results suggest that C/EBPβ reduces monocytic proliferation by affecting the retinoblastoma/E2F/cyclin E pathway and that it may contribute to, but is not directly required for, macrophage morphology. Inhibition of proliferation by C/EBPβ may be important for coordinated monocytic differentiation.

Methanobactin and the Link between Copper and Bacterial Methane Oxidation

PubMed Central

Semrau, Jeremy D.; Murrell, J. Colin; Gallagher, Warren H.; Dennison, Christopher; Vuilleumier, Stéphane

2016-01-01

SUMMARY Methanobactins (mbs) are low-molecular-mass (<1,200 Da) copper-binding peptides, or chalkophores, produced by many methane-oxidizing bacteria (methanotrophs). These molecules exhibit similarities to certain iron-binding siderophores but are expressed and secreted in response to copper limitation. Structurally, mbs are characterized by a pair of heterocyclic rings with associated thioamide groups that form the copper coordination site. One of the rings is always an oxazolone and the second ring an oxazolone, an imidazolone, or a pyrazinedione moiety. The mb molecule originates from a peptide precursor that undergoes a series of posttranslational modifications, including (i) ring formation, (ii) cleavage of a leader peptide sequence, and (iii) in some cases, addition of a sulfate group. Functionally, mbs represent the extracellular component of a copper acquisition system. Consistent with this role in copper acquisition, mbs have a high affinity for copper ions. Following binding, mbs rapidly reduce Cu2+ to Cu1+. In addition to binding copper, mbs will bind most transition metals and near-transition metals and protect the host methanotroph as well as other bacteria from toxic metals. Several other physiological functions have been assigned to mbs, based primarily on their redox and metal-binding properties. In this review, we examine the current state of knowledge of this novel type of metal-binding peptide. We also explore its potential applications, how mbs may alter the bioavailability of multiple metals, and the many roles mbs may play in the physiology of methanotrophs. PMID:26984926

Flightless I interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling

PubMed Central

Arora, Pamma D.; Wang, Yongqiang; Bresnick, Anne; Janmey, Paul A.; McCulloch, Christopher A.

2015-01-01

We examined the role of the actin-capping protein flightless I (FliI) in collagen remodeling by mouse fibroblasts. FliI-overexpressing cells exhibited reduced spreading on collagen but formed elongated protrusions that stained for myosin10 and fascin and penetrated pores of collagen-coated membranes. Inhibition of Cdc42 blocked formation of cell protrusions. In FliI-knockdown cells, transfection with constitutively active Cdc42 did not enable protrusion formation. FliI-overexpressing cells displayed increased uptake and degradation of exogenous collagen and strongly compacted collagen fibrils, which was blocked by blebbistatin. Mass spectrometry analysis of FliI immunoprecipitates showed that FliI associated with nonmuscle myosin IIA (NMMIIA), which was confirmed by immunoprecipitation. GFP-FliI colocalized with NMMIIA at cell protrusions. Purified FliI containing gelsolin-like domains (GLDs) 1–6 capped actin filaments efficiently, whereas FliI GLD 2–6 did not. Binding assays showed strong interaction of purified FliI protein (GLD 1–6) with the rod domain of NMMIIA (kD = 0.146 μM), whereas FliI GLD 2–6 showed lower binding affinity (kD = 0.8584 μM). Cells expressing FliI GLD 2–6 exhibited fewer cell extensions, did not colocalize with NMMIIA, and showed reduced collagen uptake compared with cells expressing FliI GLD 1–6. We conclude that FliI interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling in fibroblasts. PMID:25877872

Structural and Histone Binding Ability Characterizations of Human PWWP Domains

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

Wu, Hong; Zeng, Hong; Lam, Robert

2013-09-25

The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members,more » implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.« less

ADAP interactions with talin and kindlin promote platelet integrin αIIbβ3 activation and stable fibrinogen binding

PubMed Central

Kang, Jian; Kahner, Bryan; Ye, Feng; Ginsberg, Mark H.; Shattil, Sanford J.

2014-01-01

ADAP is a hematopoietic-restricted adapter protein that promotes integrin activation and is a carrier for other adapter proteins, Src kinase–associated phosphoprotein 1 (SKAP1) and SKAP2. In T lymphocytes, SKAP1 is the ADAP-associated molecule that activates integrins through direct linkages with Rap1 effectors (regulator of cell adhesion and polarization enriched in lymphoid tissues; Rap1-interacting adapter molecule). ADAP also promotes integrin αIIbβ3 activation in platelets, which lack SKAP1, suggesting an ADAP integrin–regulatory pathway different from those in lymphocytes. Here we characterized a novel association between ADAP and 2 essential integrin-β cytoplasmic tail-binding proteins involved in αIIbβ3 activation, talin and kindlin-3. Glutathione S-transferase pull-downs identified distinct regions in ADAP necessary for association with kindlin or talin. ADAP was physically proximal to talin and kindlin-3 in human platelets, as assessed biochemically, and by immunofluorescence microscopy and proximity ligation. Relative to wild-type mouse platelets, ADAP-deficient platelets exhibited reduced co-localization of talin with αIIbβ3, and reduced irreversible fibrinogen binding in response to a protease activated receptor 4 (PAR4) thrombin receptor agonist. When ADAP was heterologously expressed in Chinese hamster ovary cells co-expressing αIIbβ3, talin, PAR1, and kindlin-3, it associated with an αIIbβ3/talin complex and enabled kindlin-3 to promote agonist-dependent ligand binding to αIIbβ3. Thus, ADAP uniquely promotes activation of and irreversible fibrinogen binding to platelet αIIbβ3 through interactions with talin and kindlin-3. PMID:24523237

Kindlin-2 directly binds actin and regulates integrin outside-in signaling

PubMed Central

Bledzka, Kamila; Bialkowska, Katarzyna; Sossey-Alaoui, Khalid; Vaynberg, Julia; Pluskota, Elzbieta

2016-01-01

Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2+/− mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK47/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK47/AA mutation were equivalent in their ability to coactivate integrin αIIbβ3 in a CHO cell system when coexpressed with talin. However, K2-LK47/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses. PMID:27044892

Dimensionality of Carbon Nanomaterials Determines the Binding and Dynamics of Amyloidogenic Peptides: Multiscale Theoretical Simulations

PubMed Central

Hine, Nicholas D. M.; Mostofi, Arash A.; Yarovsky, Irene

2013-01-01

Experimental studies have demonstrated that nanoparticles can affect the rate of protein self-assembly, possibly interfering with the development of protein misfolding diseases such as Alzheimer's, Parkinson's and prion disease caused by aggregation and fibril formation of amyloid-prone proteins. We employ classical molecular dynamics simulations and large-scale density functional theory calculations to investigate the effects of nanomaterials on the structure, dynamics and binding of an amyloidogenic peptide apoC-II(60-70). We show that the binding affinity of this peptide to carbonaceous nanomaterials such as C60, nanotubes and graphene decreases with increasing nanoparticle curvature. Strong binding is facilitated by the large contact area available for π-stacking between the aromatic residues of the peptide and the extended surfaces of graphene and the nanotube. The highly curved fullerene surface exhibits reduced efficiency for π-stacking but promotes increased peptide dynamics. We postulate that the increase in conformational dynamics of the amyloid peptide can be unfavorable for the formation of fibril competent structures. In contrast, extended fibril forming peptide conformations are promoted by the nanotube and graphene surfaces which can provide a template for fibril-growth. PMID:24339760

Biophysical Characterization of Nucleophosmin Interactions with Human Immunodeficiency Virus Rev and Herpes Simplex Virus US11

PubMed Central

Nouri, Kazem; Moll, Jens M.; Milroy, Lech-Gustav; Hain, Anika; Dvorsky, Radovan; Amin, Ehsan; Lenders, Michael; Nagel-Steger, Luitgard; Howe, Sebastian; Smits, Sander H. J.; Hengel, Hartmut; Schmitt, Lutz; Münk, Carsten; Brunsveld, Luc; Ahmadian, Mohammad R.

2015-01-01

Nucleophosmin (NPM1, also known as B23, numatrin or NO38) is a pentameric RNA-binding protein with RNA and protein chaperon functions. NPM1 has increasingly emerged as a potential cellular factor that directly associates with viral proteins; however, the significance of these interactions in each case is still not clear. In this study, we have investigated the physical interaction of NPM1 with both human immunodeficiency virus type 1 (HIV-1) Rev and Herpes Simplex virus type 1 (HSV-1) US11, two functionally homologous proteins. Both viral proteins show, in mechanistically different modes, high affinity for a binding site on the N-terminal oligomerization domain of NPM1. Rev, additionally, exhibits low-affinity for the central histone-binding domain of NPM1. We also showed that the proapoptotic cyclic peptide CIGB-300 specifically binds to NPM1 oligomerization domain and blocks its association with Rev and US11. Moreover, HIV-1 virus production was significantly reduced in the cells treated with CIGB-300. Results of this study suggest that targeting NPM1 may represent a useful approach for antiviral intervention. PMID:26624888

Dynamics and lithium binding energies of polyelectrolytes based on functionalized poly(para-phenylene terephthalamide).

PubMed

Grozema, F C; Best, A S; van Eijck, L; Stride, J; Kearley, G J; de Leeuw, S W; Picken, S J

2005-04-28

Polyelectrolyte materials are an interesting class of electrolytes for use in fuel cell and battery applications. Poly(para-phenylene terephthalamide) (PPTA, Kevlar) is a liquid crystalline polymer that, when sulfonated, is a polyelectrolyte that exhibits moderate ion conductivity at elevated temperatures. In this work, quasi-elastic neutron scattering (QENS) experiments were performed to gain insight into the effect of the presence of lithium counterions on the chain dynamics in the material. It was found that the addition of lithium ions decreases the dynamics of the chains. Additionally, the binding of lithium ions to the sulfonic acids groups was investigated by density functional theory (DFT) calculations. It was found that the local surroundings of the sulfonic acid group have very little effect on the lithium-ion binding energy. Binding energies for a variety of different systems were all calculated to be around 150 kcal/mol. The DFT calculations also show the existence of a structure in which a single lithium ion interacts with two sulfonic acid moieties on different chains. The formation of such "electrostatic cross-links" is believed to be the source of the increased tendency to aggregate and the reduced dynamics in the presence of lithium ions.

Exposure of Trypanosoma brucei to an N-acetylglucosamine-Binding Lectin Induces VSG Switching and Glycosylation Defects Resulting in Reduced Infectivity

PubMed Central

Castillo-Acosta, Víctor M.; Ruiz-Pérez, Luis M.; Van Damme, Els J. M.; Balzarini, Jan; González-Pacanowska, Dolores

2015-01-01

Trypanosoma brucei variant surface glycoproteins (VSG) are glycosylated by both paucimannose and oligomannose structures which are involved in the formation of a protective barrier against the immune system. Here, we report that the stinging nettle lectin (UDA), with predominant N-acetylglucosamine-binding specificity, interacts with glycosylated VSGs and kills parasites by provoking defects in endocytosis together with impaired cytokinesis. Prolonged exposure to UDA induced parasite resistance based on a diminished capacity to bind the lectin due to an enrichment of biantennary paucimannose and a reduction of triantennary oligomannose structures. Two molecular mechanisms involved in resistance were identified: VSG switching and modifications in N-glycan composition. Glycosylation defects were correlated with the down-regulation of the TbSTT3A and/or TbSTT3B genes (coding for oligosaccharyltransferases A and B, respectively) responsible for glycan specificity. Furthermore, UDA-resistant trypanosomes exhibited severely impaired infectivity indicating that the resistant phenotype entails a substantial fitness cost. The results obtained further support the modification of surface glycan composition resulting from down-regulation of the genes coding for oligosaccharyltransferases as a general resistance mechanism in response to prolonged exposure to carbohydrate-binding agents. PMID:25746926

Cd and proton adsorption onto bacterial consortia grown from industrial wastes and contaminated geologic settings.

PubMed

Borrok, David M; Fein, Jeremy B; Kulpa, Charles F

2004-11-01

To model the effects of bacterial metal adsorption in contaminated environments, results from metal adsorption experiments involving individual pure stains of bacteria must be extrapolated to systems in which potentially dozens of bacterial species are present. This extrapolation may be made easier because bacterial consortia from natural environments appear to exhibit similar metal binding properties. However, bacteria that thrive in highly perturbed contaminated environments may exhibit significantly different adsorptive behavior. Here we measure proton and Cd adsorption onto a range of bacterial consortia grown from heavily contaminated industrial wastes, groundwater, and soils. We model the results using a discrete site surface complexation approach to determine binding constants and site densities for each consortium. The results demonstrate that bacterial consortia from different contaminated environments exhibit a range of total site densities (approximately a 3-fold difference) and Cd-binding constants (approximately a 10-fold difference). These ranges for Cd binding constants may be small enough to suggest that bacteria-metal adsorption in contaminated environments can be described using relatively few "averaged" bacteria-metal binding constants (in conjunction with the necessary binding constants for competing surfaces and ligands). However, if additional precision is necessary, modeling parameters must be developed separately for each contaminated environment of interest.

DNA interaction studies of new nano metal based anticancer agent: validation by spectroscopic methods

NASA Astrophysics Data System (ADS)

Tabassum, Sartaj; Sharma, Girish Chandra; Arjmand, Farukh; Azam, Ameer

2010-05-01

A new nano dimensional heterobimetallic Cu-Sn containing complex as a potential drug candidate was designed, synthesized and characterized by analytical and spectral methods. The electronic absorption and electron paramagnetic resonance parameters of the complex revealed that the Cu(II) ion exhibits a square pyramidal geometry with the two pyrazole nitrogen atoms, the amine nitrogen atom and the carboxylate oxygen of the phenyl glycine chloride ligand located at the equatorial sites and the coordinated chloride ion occupying an apical position. 119Sn NMR spectral data showed a hexa-coordinated environment around the Sn(IV) metal ion. TEM, AFM and XRD measurements illustrate that the complex could induce the condensation of CT-DNA to a particulate nanostructure. The interaction of the Cu-Sn complex with CT-DNA was investigated by UV-vis absorption and emission spectroscopy, as well as cyclic voltammetric measurements. The results indicated that the complex interacts with DNA through an electrostatic mode of binding with an intrinsic binding constant Kb = 8.42 × 104 M - 1. The Cu-Sn complex exhibits effective cleavage of pBR322 plasmid DNA by an oxidative cleavage mechanism, monitored at different concentrations both in the absence and in the presence of reducing agents.

Discovery and evaluation of triple inhibitors of VEGFR-2, TIE-2 and EphB4 as anti-angiogenic and anti-cancer agents

PubMed Central

Zhang, Lin; Shan, Yuanyuan; Ji, Xingyue; Zhu, Mengyuan; Li, Chuansheng; Sun, Ying; Si, Ru; Pan, Xiaoyan; Wang, Jinfeng; Ma, Weina; Dai, Bingling; Wang, Binghe; Zhang, Jie

2017-01-01

Receptor tyrosine kinases (RTKs), especially VEGFR-2, TIE-2, and EphB4, play a crucial role in both angiogenesis and tumorigenesis. Moreover, complexity and heterogeneity of angiogenesis make it difficult to treat such pathological traits with single-target agents. Herein, we developed two classes of multi-target RTK inhibitors (RTKIs) based on the highly conserved ATP-binding pocket of VEGFR-2/TIE-2/EphB4, using previously reported BPS-7 as a lead compound. These multi-target RTKIs exhibited considerable potential as novel anti-angiogenic and anticancer agents. Among them, QDAU5 displayed the most promising potency and selectivity. It significantly suppressed viability of EA.hy926 and proliferation of several cancer cells. Further investigations indicated that QDAU5 showed high affinity to VEGFR-2 and reduced the phosphorylation of VEGFR-2. We identified QDAU5 as a potent multiple RTKs inhibitor exhibiting prominent anti-angiogenic and anticancer potency both in vitro and in vivo. Moreover, quinazolin-4(3H)-one has been identified as an excellent hinge binding moiety for multi-target inhibitors of angiogenic VEGFR-2, Tie-2, and EphB4. PMID:29285210

Targeted binding of the M13 bacteriophage to thiamethoxam organic crystals.

PubMed

Cho, Whirang; Fowler, Jeffrey D; Furst, Eric M

2012-04-10

Phage display screening with a combinatorial library was used to identify M13-type bacteriophages that express peptides with selective binding to organic crystals of thiamethoxam. The six most strongly binding phages exhibit at least 1000 times the binding affinity of wild-type M13 and express heptapeptide sequences that are rich in hydrophobic, hydrogen-bonding amino acids and proline. Among the peptide sequences identified, M13 displaying the pIII domain heptapeptide ASTLPKA exhibits the strongest binding to thiamethoxam in competitive binding assays. Electron and confocal microscopy confirm the specific binding affinity of ASTLPKA to thiamethoxam. Using atomic force microscope (AFM) probes functionalized with ASTLPKA expressing phage, we found that the average adhesion force between the bacteriophage and a thiamethoxam surface is 1.47 ± 0.80 nN whereas the adhesion force of wild-type M13KE phage is 0.18 ± 0.07 nN. Such a strongly binding bacteriophage could be used to modify the surface chemistry of thiamethoxam crystals and other organic solids with a high degree of specificity. © 2012 American Chemical Society

Identification of a divalent metal cation binding site in herpes simplex virus 1 (HSV-1) ICP8 required for HSV replication.

PubMed

Bryant, Kevin F; Yan, Zhipeng; Dreyfus, David H; Knipe, David M

2012-06-01

Herpes simplex virus 1 (HSV-1) ICP8 is a single-stranded DNA-binding protein that is necessary for viral DNA replication and exhibits recombinase activity in vitro. Alignment of the HSV-1 ICP8 amino acid sequence with ICP8 homologs from other herpesviruses revealed conserved aspartic acid (D) and glutamic acid (E) residues. Amino acid residue D1087 was conserved in every ICP8 homolog analyzed, indicating that it is likely critical for ICP8 function. We took a genetic approach to investigate the functions of the conserved ICP8 D and E residues in HSV-1 replication. The E1086A D1087A mutant form of ICP8 failed to support the replication of an ICP8 mutant virus in a complementation assay. E1086A D1087A mutant ICP8 bound DNA, albeit with reduced affinity, demonstrating that the protein is not globally misfolded. This mutant form of ICP8 was also recognized by a conformation-specific antibody, further indicating that its overall structure was intact. A recombinant virus expressing E1086A D1087A mutant ICP8 was defective in viral replication, viral DNA synthesis, and late gene expression in Vero cells. A class of enzymes called DDE recombinases utilize conserved D and E residues to coordinate divalent metal cations in their active sites. We investigated whether the conserved D and E residues in ICP8 were also required for binding metal cations and found that the E1086A D1087A mutant form of ICP8 exhibited altered divalent metal binding in an in vitro iron-induced cleavage assay. These results identify a novel divalent metal cation-binding site in ICP8 that is required for ICP8 functions during viral replication.

Plasmin substrate binding site cooperativity guides the design of potent peptide aldehyde inhibitors.

PubMed

Swedberg, Joakim E; Harris, Jonathan M

2011-10-04

Perioperative bleeding is a cause of major blood loss and is associated with increased rates of postoperative morbidity and mortality. To combat this, antifibrinolytic inhibitors of the serine protease plasmin are commonly used to reduce bleeding during surgery. The most effective and previously widely used of these is the broad range serine protease inhibitor aprotinin. However, adverse clinical outcomes have led to use of alternative serine lysine analogues to inhibit plasmin. These compounds suffer from low selectivity and binding affinity. Consequently, a concerted effort to discover potent and selective plasmin inhibitors has developed. This study used a noncombinatorial peptide library to define plasmin's extended substrate specificity and guide the design of potent transition state analogue inhibitors. The various substrate binding sites of plasmin were found to exhibit a higher degree of cooperativity than had previously been appreciated. Peptide sequences capitalizing on these features produced high-affinity inhibitors of plasmin. The most potent of these, Lys-Met(sulfone)-Tyr-Arg-H [KM(O(2))YR-H], inhibited plasmin with a K(i) of 3.1 nM while maintaining 25-fold selectivity over plasma kallikrein. Furthermore, 125 nM (0.16 μg/mL) KM(O(2))YR-H attenuated fibrinolysis in vitro with an efficacy similar to that of 15 nM (0.20 μg/mL) aprotinin. To date, this is the most potent peptide inhibitor of plasmin that exhibits selectivity against plasma kallikrein, making this compound an attractive candidate for further therapeutic development.

Amyloid Plaques in PSAPP Mice Bind Less Metal than Plaques in Human Alzheimer's Disease

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

Leskovjan, A.; Lanzirotti, A; Miller, L

2009-01-01

Amyloid beta (A{Beta}) is the primary component of Alzheimer's disease (AD) plaques, a key pathological feature of the disease. Metal ions of zinc (Zn), copper (Cu), iron (Fe), and calcium (Ca) are elevated in human amyloid plaques and are thought to be involved in neurodegeneration. Transgenic mouse models of AD also exhibit amyloid plaques, but fail to exhibit the high degree of neurodegeneration observed in humans. In this study, we imaged the Zn, Cu, Fe, and Ca ion distribution in the PSAPP transgenic mouse model representing end-stage AD (N = 6) using synchrotron X-ray fluorescence (XRF) microprobe. In order tomore » account for differences in density in the plaques, the relative protein content was imaged with synchrotron Fourier transform infrared microspectroscopy (FTIRM) on the same samples. FTIRM results revealed a 61% increase in protein content in the plaques compared to the surrounding tissue. After normalizing to protein density, we found that the PSAPP plaques contained only a 29% increase in Zn and there was actually less Cu, Fe, and Ca in the plaque compared to the surrounding tissue. Since metal binding to A{beta} is thought to induce redox chemistry that is toxic to neurons, the reduced metal binding in PSAPP mice is consistent with the lack of neurodegeneration in these animals. These findings were in stark contrast to the high metal ion content observed in human AD plaques, further implicating the role of metal ions in human AD pathology.« less

VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability.

PubMed

Schwarz, Toni M; Edwards, Megan R; Diederichs, Audrey; Alinger, Joshua B; Leung, Daisy W; Amarasinghe, Gaya K; Basler, Christopher F

2017-02-15

Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Reston ebolavirus (RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA binding affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. The interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of the Ebolavirus genus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA modulates inhibition of IFN signaling and VP24 stability. The effect of KPNA interaction on VP24 stability is a novel functional consequence of this virus-host interaction, and the differences identified between viral species may contribute to differences in pathogenesis. Copyright © 2017 American Society for Microbiology.

Visual feature binding in younger and older adults: encoding and suffix interference effects.

PubMed

Brown, Louise A; Niven, Elaine H; Logie, Robert H; Rhodes, Stephen; Allen, Richard J

2017-02-01

Three experiments investigated younger (18-25 yrs) and older (70-88 yrs) adults' temporary memory for colour-shape combinations (binding). We focused upon estimating the magnitude of the binding cost for each age group across encoding time (Experiment 1; 900/1500 ms), presentation format (Experiment 2; simultaneous/sequential), and interference (Experiment 3; control/suffix) conditions. In Experiment 1, encoding time did not differentially influence binding in the two age groups. In Experiment 2, younger adults exhibited poorer binding performance with sequential relative to simultaneous presentation, and serial position analyses highlighted a particular age-related difficulty remembering the middle item of a series (for all memory conditions). Experiments 1-3 demonstrated small to medium binding effect sizes in older adults across all encoding conditions, with binding less accurate than shape memory. However, younger adults also displayed negative effects of binding (small to large) in two of the experiments. Even when older adults exhibited a greater suffix interference effect in Experiment 3, this was for all memory types, not just binding. We therefore conclude that there is no consistent evidence for a visual binding deficit in healthy older adults. This relative preservation contrasts with the specific and substantial deficits in visual feature binding found in several recent studies of Alzheimer's disease.

NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING

PubMed Central

Zhang, Lu; Mo, Jinyao; Swanson, Karen V.; Wen, Haitao; Petrucelli, Alex; Gregory, Sean M.; Zhang, Zhigang; Schneider, Monika; Jiang, Yan; Fitzgerald, Katherine A.; Ouyang, Songying; Liu, Zhi-Jie; Damania, Blossom A; Shu, Hong-Bing; Duncan, Joseph A.; Ting, Jenny P-Y.

2014-01-01

SUMMARY Stimulator of interferon genes (STING, also named MITA, MYPS or ERIS) is an intracellular DNA sensor that induces type I interferon through its interaction with TANK-binding kinase 1 (TBK1). Here we found that the nucleotide-binding, leucine-rich repeat containing protein, NLRC3, reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP) and DNA viruses. NLRC3 associated with both STING and TBK1, and impeded STING-TBK1 interaction and downstream type I interferon production. Using purified recombinant proteins NLRC3 was found to interact directly with STING. Furthermore, NLRC3 prevented proper trafficking of STING to perinuclear and punctated region, known to be important for its activation. In animals, herpes simplex virus 1 (HSV-1)-infected Nlrc3−/− mice exhibited enhanced innate immunity, reduced morbidity and viral load. This demonstrates the intersection of two key pathways of innate immune regulation, NLR and STING, to fine tune host response to intracellular DNA, DNA virus and c-di-GMP PMID:24560620

Social instability stress in adolescent male rats reduces social interaction and social recognition performance and increases oxytocin receptor binding.

PubMed

Hodges, Travis E; Baumbach, Jennet L; Marcolin, Marina L; Bredewold, Remco; Veenema, Alexa H; McCormick, Cheryl M

2017-09-17

Social experiences in adolescence are essential for displaying context-appropriate social behaviors in adulthood. We previously found that adult male rats that underwent social instability stress (SS) in adolescence had reduced social interactions with unfamiliar peers compared with non-stressed controls (CTL). Here we determined whether SS altered social recognition and social reward and brain oxytocin and vasopressin receptor density in adolescence. We confirmed that SS rats spent less time interacting with unfamiliar peers than did CTL rats (p=0.006). Furthermore, CTL rats showed a preference for novel over familiar conspecifics in a social recognition test whereas SS rats did not, which may reflect reduced recognition, impaired memory, or reduced preference for novelty in SS rats. The reward value of social interactions was not affected by SS based on conditioned place preference tests and based on the greater time SS rats spent investigating stimulus rats than did CTL rats when the stimulus rat was behind wire mesh (p=0.03). Finally, oxytocin receptor binding density was higher in the dorsal lateral septum and nucleus accumbens shell in SS rats compared with CTL rats (p=0.02, p=0.01, respectively). No effect of SS was found for vasopressin 1a receptor binding density in any of the brain regions analyzed. We discuss the extent to which the differences in social behavior exhibited after social instability in adolescence involve changes in social salience and social competency, and the possibility that changes in oxytocin signaling in the brain underlie the differences in social behavior. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

High-oleic canola oil consumption enriches LDL particle cholesteryl oleate content and reduces LDL proteoglycan binding in humans

PubMed Central

Jones, Peter J. H.; MacKay, Dylan. S.; Senanayake, Vijitha K.; Pu, Shuaihua; Jenkins, David J. A.; Connelly, Philip W.; Lamarche, Benoît; Couture, Patrick; Kris-Etherton, Penny M.; West, Sheila G.; Liu, Xiaoran; Fleming, Jennifer A.; Hantgan, Roy R.; Rudel, Lawrence L.

2015-01-01

Oleic acid consumption is considered cardio-protective according to studies conducted examining effects of the Mediterranean diet. However, animal models have shown that oleic acid consumption increases LDL particle cholesteryl oleate content which is associated with increased LDL-proteoglycan binding and atherosclerosis. The objective was to examine effects of varying oleic, linoleic and docosahexaenoic acid consumption on human LDL-proteoglycan binding in a non-random subset of the Canola Oil Multi-center Intervention Trial (COMIT) participants. COMIT employed a randomized, double-blind, five-period, cross-over trial design. Three of the treatment oil diets; 1) a blend of corn/safflower oil (25:75); 2) high oleic canola oil; and 3) DHA-enriched high oleic canola oil were selected for analysis of LDL-proteoglycan binding in 50 participants exhibiting good compliance. LDL particles were isolated from frozen plasma by gel filtration chromatography and LDL cholesteryl esters quantified by mass-spectrometry. LDL-proteoglycan binding was assessed using surface plasmon resonance. LDL particle cholesterol ester fatty acid composition was sensitive to the treatment fatty acid compositions, with the main fatty acids in the treatments increasing in the LDL cholesterol esters. The corn/safflower oil and high-oleic canola oil diets lowered LDL-proteoglycan binding relative to their baseline values (p=0.0005 and p=0.0012, respectively). At endpoint, high-oleic canola oil feeding resulted in lower LDL-proteoglycan binding than corn/safflower oil (p=0.0243) and DHA-enriched high oleic canola oil (p=0.0249), although high-oleic canola oil had the lowest binding at baseline (p=0.0344). Our findings suggest that high-oleic canola oil consumption in humans increases cholesteryl oleate percentage in LDL, but in a manner not associated with a rise in LDL-proteoglycan binding. PMID:25528432

Analysis and prediction of calcium-binding pockets from apo-protein structures exhibiting calcium-induced localized conformational changes

PubMed Central

Wang, Xue; Zhao, Kun; Kirberger, Michael; Wong, Hing; Chen, Guantao; Yang, Jenny J

2010-01-01

Calcium binding in proteins exhibits a wide range of polygonal geometries that relate directly to an equally diverse set of biological functions. The binding process stabilizes protein structures and typically results in local conformational change and/or global restructuring of the backbone. Previously, we established the MUG program, which utilized multiple geometries in the Ca2+-binding pockets of holoproteins to identify such pockets, ignoring possible Ca2+-induced conformational change. In this article, we first report our progress in the analysis of Ca2+-induced conformational changes followed by improved prediction of Ca2+-binding sites in the large group of Ca2+-binding proteins that exhibit only localized conformational changes. The MUGSR algorithm was devised to incorporate side chain torsional rotation as a predictor. The output from MUGSR presents groups of residues where each group, typically containing two to five residues, is a potential binding pocket. MUGSR was applied to both X-ray apo structures and NMR holo structures, which did not use calcium distance constraints in structure calculations. Predicted pockets were validated by comparison with homologous holo structures. Defining a “correct hit” as a group of residues containing at least two true ligand residues, the sensitivity was at least 90%; whereas for a “correct hit” defined as a group of residues containing at least three true ligand residues, the sensitivity was at least 78%. These data suggest that Ca2+-binding pockets are at least partially prepositioned to chelate the ion in the apo form of the protein. PMID:20512971

Analysis of SOX10 mutations identified in Waardenburg-Hirschsprung patients: Differential effects on target gene regulation.

PubMed

Chan, Kwok Keung; Wong, Corinne Kung Yen; Lui, Vincent Chi Hang; Tam, Paul Kwong Hang; Sham, Mai Har

2003-10-15

SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans. A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities. Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems. Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified. We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays. To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter. We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines. The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients. Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter. Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter. While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis, overexpression of genes coding for structural myelin proteins such as P0 due to mutant SOX10 may explain the dysmyelination phenotype observed in the patients with an additional neurological disorder. Copyright 2003 Wiley-Liss, Inc.

Dynamics of the Extended String-Like Interaction of TFIIE with the p62 Subunit of TFIIH.

PubMed

Okuda, Masahiko; Higo, Junichi; Komatsu, Tadashi; Konuma, Tsuyoshi; Sugase, Kenji; Nishimura, Yoshifumi

2016-09-06

General transcription factor II E (TFIIE) contains an acid-rich region (residues 378-393) in its α-subunit, comprising 13 acidic and two hydrophobic (Phe387 and Val390) residues. Upon binding to the p62 subunit of TFIIH, the acidic region adopts an extended string-like structure on the basic groove of the pleckstrin homology domain (PHD) of p62, and inserts Phe387 and Val390 into two shallow pockets in the groove. Here, we have examined the dynamics of this interaction by NMR and molecular dynamics (MD) simulations. Although alanine substitution of Phe387 and/or Val390 greatly reduced binding to PHD, the binding mode of the mutants was similar to that of the wild-type, as judged by the chemical-shift changes of the PHD. NMR relaxation dispersion profiles of the interaction exhibited large amplitudes for residues in the C-terminal half-string in the acidic region (Phe387, Glu388, Val390, Ala391, and Asp392), indicating a two-site binding mode: one corresponding to the final complex structure, and one to an off-pathway minor complex. To probe the off-pathway complex structure, an atomically detailed free-energy landscape of the binding mode was computed by all-atom multicanonical MD. The most thermodynamically stable cluster corresponded to the final complex structure. One of the next stable clusters was the off-pathway structure cluster, showing the reversed orientation of the C-terminal half-string on the PHD groove, as compared with the final structure. MD calculations elucidated that the C-terminal half-acidic-string forms encounter complexes mainly around the positive groove region with nearly two different orientations of the string, parallel and antiparallel to the final structure. Interestingly, the most encountered complexes exhibit a parallel-like orientation, suggesting that the string has a tendency to bind around the groove in the proper orientation with the aid of Phe387 and/or Val390 to proceed smoothly to the final complex structure. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Non-Immune Binding of Human IgG to M-Related Proteins Confers Resistance to Phagocytosis of Group A Streptococci in Blood

PubMed Central

Courtney, Harry S.; Li, Yi

2013-01-01

The non-immune binding of immunoglobulins by bacteria is thought to contribute to the pathogenesis of infections. M-related proteins (Mrp) are group A streptococcal (GAS) receptors for immunoglobulins, but it is not known if this binding has any impact on virulence. To further investigate the binding of immunoglobulins to Mrp, we engineered mutants of an M type 4 strain of GAS by inactivating the genes for mrp, emm, enn, sof, and sfbX and tested these mutants in IgG-binding assays. Inactivation of mrp dramatically decreased the binding of human IgG, whereas inactivation of emm, enn, sof, and sfbx had only minor effects, indicating that Mrp is a major IgG-binding protein. Binding of human immunoglobulins to a purified, recombinant form of Mrp indicated that it selectively binds to the Fc domain of human IgG, but not IgA or IgM and that it preferentially bound subclasses IgG1>IgG4>IgG2>IgG3. Recombinant proteins encompassing different regions of Mrp were engineered and used to map its IgG-binding domain to its A-repeat region and a recombinant protein with 3 A-repeats was a better inhibitor of IgG binding than one with a single A-repeat. A GAS mutant expressing Mrp with an in-frame deletion of DNA encoding the A-repeats had a dramatically reduced ability to bind human IgG and to grow in human blood. Mrp exhibited host specificity in binding IgG; human IgG was the best inhibitor of the binding of IgG followed by pig, horse, monkey, and rabbit IgG. IgG from goat, mouse, rat, cow, donkey, chicken, and guinea pig were poor inhibitors of binding. These findings indicate that Mrp preferentially binds human IgG and that this binding contributes to the ability of GAS to resist phagocytosis and may be a factor in the restriction of GAS infections to the human host. PMID:24205299

The mechanism and high-free-energy transition state of lac repressor–lac operator interaction

PubMed Central

Sengupta, Rituparna; Capp, Michael W.; Shkel, Irina A.

2017-01-01

Abstract Significant, otherwise-unavailable information about mechanisms and transition states (TS) of protein folding and binding is obtained from solute effects on rate constants. Here we characterize TS for lac repressor(R)–lac operator(O) binding by analyzing effects of RO-stabilizing and RO-destabilizing solutes on association (ka) and dissociation (kd) rate constants. RO-destabilizing solutes (urea, KCl) reduce ka comparably (urea) or more than (KCl) they increase kd, demonstrating that they destabilize TS relative to reactants and RO, and that TS exhibits most of the Coulombic interactions between R and O. Strikingly, three solutes which stabilize RO by favoring burial/dehydration of amide oxygens and anionic phosphate oxygens all reduce kd without affecting ka significantly. The lack of stabilization of TS by these solutes indicates that O phosphates remain hydrated in TS and that TS preferentially buries aromatic carbons and amide nitrogens while leaving amide oxygens exposed. In our proposed mechanism, DNA-binding-domains (DBD) of R insert in major grooves of O pre-TS, forming most Coulombic interactions of RO and burying aromatic carbons. Nucleation of hinge helices creates TS, burying sidechain amide nitrogens. Post-TS, hinge helices assemble and the DBD-hinge helix-O-DNA module docks on core repressor, partially dehydrating phosphate oxygens and tightening all interfaces to form RO. PMID:29036376

Two insulin-like peptide family members from the mosquito Aedes aegypti exhibit differential biological and receptor binding activities

PubMed Central

Wen, Zhimou; Gulia, Monika; Clark, Kevin D.; Dhara, Animesh; Crim, Joe W.; Strand, Michael R.; Brown, Mark R.

2010-01-01

Insects encode multiple ILPs but only one homolog of the vertebrate IR that activates the insulin signaling pathway. However, it remains unclear whether all insect ILPs are high affinity ligands for the IR or have similar biological functions. The yellow fever mosquito, Aedes aegypti, encodes eight ILPs with prior studies strongly implicating ILPs from the brain in regulating metabolism and the maturation of eggs following blood feeding. Here we addressed whether two ILP family members expressed in the brain, ILP4 and ILP3, have overlapping functional and receptor binding activities. Our results indicated that ILP3 exhibits strong insulin-like activity by elevating carbohydrate and lipid storage in sugar-fed adult females, whereas ILP4 does not. In contrast, both ILPs exhibited dose-dependent gonadotropic activity in blood-fed females as measured by the stimulation of ovaries to produce ecdysteroids and the uptake of yolk by primary oocytes. Binding studies using ovary membranes indicated that ILP4 and ILP3 do not cross compete; a finding further corroborated by cross-linking and immunoblotting experiments showing that ILP3 binds the MIR while ILP4 binds an unknown 55 kDa membrane protein. In contrast, each ILP activated the insulin signaling pathway in ovaries as measured by enhanced phosphorylation of Akt. RNAi and inhibitor studies further indicated that the gonadotropic activity of ILP4 and ILP3 requires the MIR and a functional insulin signaling pathway. Taken together, our results indicate that two members of the Ae. aegypti ILP family exhibit partially overlapping biological activity and different binding interactions with the MIR. PMID:20643184

Eukaryotic Initiation Factor eIFiso4G1 and eIFiso4G2 Are Isoforms Exhibiting Distinct Functional Differences in Supporting Translation in Arabidopsis*

PubMed Central

Gallie, Daniel R.

2016-01-01

The eukaryotic translation initiation factor (eIF) 4G is required during protein synthesis to promote the assembly of several factors involved in the recruitment of a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, the eIF4G isoforms in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization but both can interact with eIF4A, eIF4B, eIF4E isoforms, and the poly(A)-binding protein. Nevertheless, eIF4G and eIFiso4G from wheat exhibit preferences in the mRNAs they translate optimally. For example, mRNA containing the 5′-leader (called Ω) of tobacco mosaic virus preferentially uses eIF4G in wheat germ lysate. In this study, the eIF4G isoform specificity of Ω was used to examine functional differences of the eIF4G isoforms in Arabidopsis. As in wheat, Ω-mediated translation was reduced in an eif4g null mutant. Loss of the eIFiso4G1 isoform, which is similar in sequence to wheat eIFiso4G, did not substantially affect Ω-mediated translation. However, loss of the eIFiso4G2 isoform substantially reduced Ω-mediated translation. eIFiso4G2 is substantially divergent from eIFiso4G1 and is present only in the Brassicaceae, suggesting a recent evolution. eIFiso4G2 isoforms exhibit sequence-specific differences in regions representing partner protein and RNA binding sites. Loss of any eIF4G isoform also resulted in a substantial reduction in reporter transcript level. These results suggest that eIFiso4G2 appeared late in plant evolution and exhibits more functional similarity with eIF4G than with eIFiso4G1 during Ω-mediated translation. PMID:26578519

Filamin A regulates the organization and remodeling of the pericellular collagen matrix.

PubMed

Mezawa, Masaru; Pinto, Vanessa I; Kazembe, Mwayi P; Lee, Wilson S; McCulloch, Christopher A

2016-10-01

Extracellular matrix remodeling by cell adhesion-related processes is critical for proliferation and tissue homeostasis, but how adhesions and the cytoskeleton interact to organize the pericellular matrix (PCM) is not understood. We examined the role of the actin-binding protein, filamin A (FLNa), in pericellular collagen remodeling. Compared with wild-type (WT), mice with fibroblast-specific deletion of FLNa exhibited higher density but reduced organization of collagen fibers after increased loading of the periodontal ligament for 2 wk. In cultured fibroblasts, FLNa knockdown (KD) did not affect collagen mRNA, but after 24 h of culture, FLNa WT cells exhibited ∼2-fold higher cell-surface collagen KD cells and 13-fold higher levels of activated β1 integrins. In FLNa WT cells, there was 3-fold more colocalization of talin with pericellular cleaved collagen than in FLNa KD cells. MMP-9 mRNA and protein expression were >2-fold higher in FLNa KD cells than in WT cells. Cathepsin B, which is necessary for intracellular collagen digestion, was >3-fold higher in FLNa WT cells than in KD cells. FLNa WT cells exhibited 2-fold more collagen phagocytosis than KD cells, which involved the FLNa actin-binding domain. Evidently, FLNa regulates PCM remodeling through its effects on degradation pathways that affect the abundance and organization of collagen.-Mezawa, M., Pinto, V. I., Kazembe, M. P., Lee, W. S., McCulloch, C. A. Filamin A regulates the organization and remodeling of the pericellular collagen matrix. © FASEB.

Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin*

PubMed Central

Strobel, Kathryn L.; Pfeiffer, Katherine A.; Blanch, Harvey W.; Clark, Douglas S.

2015-01-01

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs. PMID:26209638

Chitin binding proteins act synergistically with chitinases in Serratia proteamaculans 568.

PubMed

Purushotham, Pallinti; Arun, P V Parvati Sai; Prakash, Jogadhenu S S; Podile, Appa Rao

2012-01-01

Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥ 0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues.

Chitin Binding Proteins Act Synergistically with Chitinases in Serratia proteamaculans 568

PubMed Central

Purushotham, Pallinti; Arun, P. V. Parvati Sai; Prakash, Jogadhenu S. S.; Podile, Appa Rao

2012-01-01

Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues. PMID:22590591

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

Pi, Min; Kapoor, Karan P.; Ye, Ruisong

The possibility that G protein-coupled receptor family C member A (GPRC6A) is the osteocalcin (Ocn)-sensing G protein-coupled receptor that directly regulates pancreatic β-cell functions is controversial. In the current study, we found that Ocn and an Ocn-derived C-terminal hexapeptide directly activate GPRC6A-dependent ERK signaling in vitro. Computational models probe the structural basis of Ocn binding to GPRC6A and predict that the C-terminal hexapeptide docks to the extracellular side of the transmembrane domain of GPRC6A. Consistent with the modeling, mutations in the computationally identified binding pocket of GPRC6A reduced Ocn and C-terminal hexapeptide activation of this receptor. In addition, selective deletionmore » of Gprc6a in β-cells (Gprc6aβ-cell-cko) by crossing Gprc6aflox/flox mice with Ins2-Cre mice resulted in reduced pancreatic weight, islet number, insulin protein content, and insulin message expression. Both islet size and β-cell proliferation were reduced in Gprc6aβ-cell-cko compared with control mice. Gprc6aβ-cell-cko exhibited abnormal glucose tolerance, but normal insulin sensitivity. Islets isolated from Gprc6aβ-cell-cko mice showed reduced insulin simulation index in response to Ocn. These data establish the structural basis for Ocn direct activation of GPRC6A and confirm a role for GPRC6A in regulating β-cell proliferation and insulin secretion.« less

Repeated intermittent MDMA binges reduce DAT density in mice and SERT density in rats in reward regions of the adolescent brain.

PubMed

Kindlundh-Högberg, Anna M S; Schiöth, Helgi B; Svenningsson, Per

2007-11-01

The popular recreational drug, 3,4-methylenedioxymethamphetamine (MDMA) is often taken as intermittent binges by adolescents at dance clubs. The neurobiological mechanisms that underlie MDMA-induced psychiatric conditions are still poorly understood. In the present study, mimicking adolescent patterns of administration, repeated intermittent MDMA binges (3x5 mg/(kg day) given 3h apart, every 7th day for 4 weeks) were given to adolescent mice and rats. Behavioral responses in the open-field and autoradiographic ligand-binding to dopamine (DAT) and serotonin (SERT) transporters in reward regions of the brain were measured. In the open-field, total horizontal activity (HA) was significantly increased in both mice and rats following the first and third weekly administered MDMA binge. However, rats, but not mice, exhibited an enhanced activity in the centre of the open-field arena, indicating on reduced anxiety or enhanced impulsivity, which is known to be associated with altered serotonin activity. Specific binding of DAT, but not SERT, was significantly reduced in the mouse AcbSh and CPU using in vitro autoradiography. On the contrary, SERT, but not DAT density was significantly reduced in the AcbSh of rats. Taken together, our data provide evidence for differential regulation of DAT and SERT densities in reward-related brain regions of rats and mice after long-term intermittent administration of MDMA.

Functional characterization of a multi-cancer risk locus on chr5p15.33 reveals regulation of TERT by ZNF148.

PubMed

Fang, Jun; Jia, Jinping; Makowski, Matthew; Xu, Mai; Wang, Zhaoming; Zhang, Tongwu; Hoskins, Jason W; Choi, Jiyeon; Han, Younghun; Zhang, Mingfeng; Thomas, Janelle; Kovacs, Michael; Collins, Irene; Dzyadyk, Marta; Thompson, Abbey; O'Neill, Maura; Das, Sudipto; Lan, Qi; Koster, Roelof; Stolzenberg-Solomon, Rachael S; Kraft, Peter; Wolpin, Brian M; Jansen, Pascal W T C; Olson, Sara; McGlynn, Katherine A; Kanetsky, Peter A; Chatterjee, Nilanjan; Barrett, Jennifer H; Dunning, Alison M; Taylor, John C; Newton-Bishop, Julia A; Bishop, D Timothy; Andresson, Thorkell; Petersen, Gloria M; Amos, Christopher I; Iles, Mark M; Nathanson, Katherine L; Landi, Maria Teresa; Vermeulen, Michiel; Brown, Kevin M; Amundadottir, Laufey T

2017-05-02

Genome wide association studies (GWAS) have mapped multiple independent cancer susceptibility loci to chr5p15.33. Here, we show that fine-mapping of pancreatic and testicular cancer GWAS within one of these loci (Region 2 in CLPTM1L) focuses the signal to nine highly correlated SNPs. Of these, rs36115365-C associated with increased pancreatic and testicular but decreased lung cancer and melanoma risk, and exhibited preferred protein-binding and enhanced regulatory activity. Transcriptional gene silencing of this regulatory element repressed TERT expression in an allele-specific manner. Proteomic analysis identifies allele-preferred binding of Zinc finger protein 148 (ZNF148) to rs36115365-C, further supported by binding of purified recombinant ZNF148. Knockdown of ZNF148 results in reduced TERT expression, telomerase activity and telomere length. Our results indicate that the association with chr5p15.33-Region 2 may be explained by rs36115365, a variant influencing TERT expression via ZNF148 in a manner consistent with elevated TERT in carriers of the C allele.

Streptococcus mutans autolysin AtlA is a fibronectin-binding protein and contributes to bacterial survival in the bloodstream and virulence for infective endocarditis.

PubMed

Jung, Chiau-Jing; Zheng, Quan-Hau; Shieh, Ya-Hsiung; Lin, Chi-Shuan; Chia, Jean-San

2009-11-01

Streptococcus mutans, a commensal of the human oral cavity, can survive in the bloodstream and cause infective endocarditis (IE). However, the virulence factors associated with this manifestation of disease are not known. Here, we demonstrate that AtlA, an autolysin of S. mutans is a newly identified fibronectin (Fn) binding protein and contributes to bacterial resistance to phagocytosis and survival in the bloodstream. Interestingly, prior exposure to plasma at low concentrations was sufficient to enhance bacterial survival in the circulation. Calcium ions at physiological plasma concentrations induced maturation of AtlA from the 104-90 kDa isoform resulting in increased Fn binding and resistance to phagocytosis. An isogenic mutant strain defective in AtlA expression exhibited reduced survival and virulence when tested in a rat model of IE compared with the wild-type and complemented strains. The data presented suggest that plasma components utilized by S. mutans enhanced survival in the circulation and AtlA is a virulence factor associated with infective endocarditis.

Potential of goat probiotic to bind mutagens.

PubMed

Apás, Ana Lidia; González, Silvia Nelina; Arena, Mario Eduardo

2014-08-01

The mutagen binding ability of the goat probiotics (Lactobacillus reuteri DDL 19, Lactobacillus alimentarius DDL 48, Enterococcus faecium DDE 39, and Bifidobacterium bifidum DDBA) was evaluated. The oral administration of these probiotics reduced fecal mutagens and intestinal cancer markers in goats. Secondly, the effects of probiotics against the mutagenesis induced by sodium azide (SA), and Benzopyrene (B[α]P) by performing the modified Ames test using Salmonella typhimurium TA 100 was investigated. The capacity to bind benzopyrene and the stability of the bacterial-mutagen complex was analyzed by HPLC. The dismutagenic potential against both mutagens was proportional to probiotic concentration. Results showed that probiotic antimutagenic capacity against SA was ranging from 13 to 78%. The mixture of four goat probiotics (MGP) displayed higher antimutagenic activity against SA than any individual strains at the same cell concentration. This study shows that the highest diminution of mutagenicity in presence of B[α]P (74%) was observed in presence of MGP. The antimutagenic activity of nearly all the individual probiotic and the MGP were in concordance with the B[α]P binding determined by HPLC. According to our results, the B[α]P binding to probiotic was irreversible still after being washed with DMSO solution. The stability of the toxic compounds-bacterial cell binding is a key consideration when probiotic antimutagenic property is evaluated. MGP exhibits the ability to bind and detoxify potent mutagens, and this property can be useful in supplemented foods for goats since it can lead to the removal of potent mutagens and protect and enhance ruminal health and hence food safety of consumers. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evaluation of iron-binding activity of collagen peptides prepared from the scales of four cultivated fishes in Taiwan.

PubMed

Huang, Chun-Yung; Wu, Chien-Hui; Yang, Jing-Iong; Li, Ying-Han; Kuo, Jen-Min

2015-12-01

Iron deficiency is one of the most concerning deficiency problems in the world. It may generate several adverse effects such as iron deficiency anemia (IDA) and reduced physical and intellectual working capacity. The aim of this study is to evaluate the Fe(II)-binding activity of collagen peptides from fishery by-products. Lates calcarifer, Mugil cephalus, Chanos chanos, and Oreochromis spp are four major cultivated fishes in Taiwan; thousands of scales of these fish are wasted without valuable utilization. In this study, scales of these fish were hydrolyzed by papain plus flavourzyme. Collagen peptides were obtained and compared for their Fe(II)-binding activity. Collagen peptides from Chanos chanos showed the highest Fe(II)-binding activity, followed by those from Lates calcarifer and Mugil cephalus; that from Oreochromis spp exhibited the lowest one. Fe(II)-binding activity of collagen peptides from fish scales was also confirmed with a dialysis method. Molecular weight (MW) distributions of the collagen peptides from scales of four fish are all < 10 kDa, and averaged 1.3 kDa. Hydrolysates of fish scales were further partially purified with ion exchange chromatography. Fractions having Fe(II)-binding activity were obtained and their activity compared. Data obtained showed that collagen peptides from fish scales did have Fe(II)-binding activity. This is the first observation elucidating fish scale collagen possessing this functionality. The results from this study also indicated that collagen peptides from fish scales could be applied in industry as a bioresource. Copyright © 2014. Published by Elsevier B.V.

Dpb11 may function with RPA and DNA to initiate DNA replication.

PubMed

Bruck, Irina; Dhingra, Nalini; Martinez, Matthew P; Kaplan, Daniel L

2017-01-01

Dpb11 is required for the initiation of DNA replication in budding yeast. We found that Dpb11 binds tightly to single-stranded DNA (ssDNA) or branched DNA structures, while its human homolog, TopBP1, binds tightly to branched-DNA structures. We also found that Dpb11 binds stably to CDK-phosphorylated RPA, the eukaryotic ssDNA binding protein, in the presence of branched DNA. A Dpb11 mutant specifically defective for DNA binding did not exhibit tight binding to RPA in the presence of DNA, suggesting that Dpb11-interaction with DNA may promote the recruitment of RPA to melted DNA. We then characterized a mutant of Dpb11 that is specifically defective in DNA binding in budding yeast cells. Expression of dpb11-m1,2,3,5,ΔC results in a substantial decrease in RPA recruitment to origins, suggesting that Dpb11 interaction with DNA may be required for RPA recruitment to origins. Expression of dpb11-m1,2,3,5,ΔC also results in diminished GINS interaction with Mcm2-7 during S phase, while Cdc45 interaction with Mcm2-7 is like wild-type. The reduced GINS interaction with Mcm2-7 may be an indirect consequence of diminished origin melting. We propose that the tight interaction between Dpb11, CDK-phosphorylated RPA, and branched-DNA may be required for the essential function of stabilizing melted origin DNA in vivo. We also propose an alternative model, wherein Dpb11-DNA interaction is required for some other function in DNA replication initiation, such as helicase activation.

Characterization of the growth and auxin physiology of roots of the tomato mutant, diageotropica

NASA Technical Reports Server (NTRS)

Muday, G. K.; Lomax, T. L.; Rayle, D. L.

1995-01-01

Roots of the tomato (Lycopersicon esculentum, Mill.) mutant (diageotropica (dgt) exhibit an altered phenotype. These roots are agravitropic and lack lateral roots. Relative to wild-type (VFN8) roots, dgt roots are less sensitive to growth inhibition by exogenously applied IAA and auxin transport inhibitors (phytotropins), and the roots exhibit a reduction in maximal growth inhibition in response to ethylene. However, IAA transport through roots, binding of the phytotropin, tritiated naphthylphthalamic acid ([3H]NPA), to root microsomal membranes, NPA-sensitive IAA uptake by root segments, and uptake of [3H]NPA into root segments are all similar in mutant and wild-type roots. We speculate that the reduced sensitivity of dgt root growth to auxin-transport inhibitors and ethylene is an indirect result of the reduction in sensitivity to auxin in this single gene, recessive mutant. We conclude that dgt roots, like dgt shoots, exhibit abnormalities indicating they have a defect associated with or affecting a primary site of auxin perception or action.

The Neck Region of the C-type Lectin DC-SIGN Regulates Its Surface Spatiotemporal Organization and Virus-binding Capacity on Antigen-presenting Cells*

PubMed Central

Manzo, Carlo; Torreno-Pina, Juan A.; Joosten, Ben; Reinieren-Beeren, Inge; Gualda, Emilio J.; Loza-Alvarez, Pablo; Figdor, Carl G.; Garcia-Parajo, Maria F.; Cambi, Alessandra

2012-01-01

The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well defined nanopatterning and role in viral binding remain enigmatic. By combining biochemical and advanced biophysical techniques, including optical superresolution and single particle tracking, we demonstrate that DC-SIGN intrinsic nanoclustering strictly depends on its molecular structure. DC-SIGN nanoclusters exhibited free, Brownian diffusion on the cell membrane. Truncation of the extracellular neck region, known to abrogate tetramerization, significantly reduced nanoclustering and concomitantly increased lateral diffusion. Importantly, DC-SIGN nanocluster dissolution exclusively compromised binding to nanoscale size pathogens. Monte Carlo simulations revealed that heterogeneity on nanocluster density and spatial distribution confers broader binding capabilities to DC-SIGN. As such, our results underscore a direct relationship between spatial nanopatterning, driven by intermolecular interactions between the neck regions, and receptor diffusion to provide DC-SIGN with the exquisite ability to dock pathogens at the virus length scale. Insight into how virus receptors are organized prior to virus binding and how they assemble into functional platforms for virus docking is helpful to develop novel strategies to prevent virus entry and infection. PMID:23019323

The Dystrophin Glycoprotein Complex Regulates the Epigenetic Activation of Muscle Stem Cell Commitment.

PubMed

Chang, Natasha C; Sincennes, Marie-Claude; Chevalier, Fabien P; Brun, Caroline E; Lacaria, Melanie; Segalés, Jessica; Muñoz-Cánoves, Pura; Ming, Hong; Rudnicki, Michael A

2018-05-03

Asymmetrically dividing muscle stem cells in skeletal muscle give rise to committed cells, where the myogenic determination factor Myf5 is transcriptionally activated by Pax7. This activation is dependent on Carm1, which methylates Pax7 on multiple arginine residues, to recruit the ASH2L:MLL1/2:WDR5:RBBP5 histone methyltransferase complex to the proximal promoter of Myf5. Here, we found that Carm1 is a specific substrate of p38γ/MAPK12 and that phosphorylation of Carm1 prevents its nuclear translocation. Basal localization of the p38γ/p-Carm1 complex in muscle stem cells occurs via binding to the dystrophin-glycoprotein complex (DGC) through β1-syntrophin. In dystrophin-deficient muscle stem cells undergoing asymmetric division, p38γ/β1-syntrophin interactions are abrogated, resulting in enhanced Carm1 phosphorylation. The resulting progenitors exhibit reduced Carm1 binding to Pax7, reduced H3K4-methylation of chromatin, and reduced transcription of Myf5 and other Pax7 target genes. Therefore, our experiments suggest that dysregulation of p38γ/Carm1 results in altered epigenetic gene regulation in Duchenne muscular dystrophy. Copyright © 2018 Elsevier Inc. All rights reserved.

Estrogenicity of halogenated bisphenol A: in vitro and in silico investigations.

PubMed

Zhang, Jie; Li, Tiezhu; Wang, Tuoyi; Yuan, Cuiping; Zhong, Shuning; Guan, Tianzhu; Li, Zhuolin; Wang, Yongzhi; Yu, Hansong; Luo, Quan; Wang, Yongjun; Zhang, Tiehua

2018-03-01

The binding interactions of bisphenol A (BPA) and its halogenated derivatives (halogenated BPAs) to human estrogen receptor α ligand binding domain (hERα-LBD) was investigated using a combined in vitro and in silico approach. First, the recombinant hERα-LBD was prepared as a soluble protein in Escherichia coli BL21(DE3)pLysS. A native fluorescent phytoestrogen, coumestrol, was employed as tracer for the fluorescence polarization assay. The results of the in vitro binding assay showed that bisphenol compounds could bind to hERα-LBD as the affinity ligands. All the tested halogenated BPAs exhibited weaker receptor binding than BPA, which might be explained by the steric effect of substituents. Molecular docking studies elucidated that the halogenated BPAs adopted different conformations in the flexible hydrophobic ligand binding pocket (LBP), which is mainly dependent on their distinct halogenation patterns. The compounds with halogen substituents on the phenolic rings and on the bridging alkyl moiety acted as agonists and antagonists for hERα, respectively. Interestingly, all the compounds in the agonist conformation of hERα formed a hydrogen bond with His524, while the compounds in the antagonist conformation formed a hydrogen bond with Thr347. These docking results suggested a pivotal role of His524/Thr347 in maintaining the hERα structure in the biologically active agonist/antagonist conformation. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation, which might be applicable for the structure-based design of novel bisphenol compounds with reduced toxicities and for environmental risk assessment. In addition, based on hERα-LBD as a recognition element, the proposed fluorescence polarization assay may offer an alternative to chromatographic techniques for the multi-residue determination of bisphenol compounds.

(3H)WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

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

Norman, A.B.; Battaglia, G.; Creese, I.

1985-12-01

In the presence of a 30 nM prazosin mask, (/sup 3/H)-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ((/sup 3/H)WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for (/sup 3/H) WB4101 binding in cerebral cortex. We have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at (/sup 3/H)WB4101-binding sites in the presence of 30 nM prazosin and (/sup 3/H) lysergic acid diethylamide ((/sup 3/H)LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5more » mM MgSO4, the Bmax of (/sup 3/H)WB4101 is significantly lower than the Bmax of (/sup 3/H)LSD in various brain regions. WB4101 competition for (/sup 3/H) LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of (/sup 3/H)WB4101 binding derived from saturation experiments. This suggests that (/sup 3/H)WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by (/sup 3/H)LSD. The selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for (/sup 3/H)WB4101 but compete for multiple (/sup 3/H)LSD 5-HT1 binding sites. These data indicate that (/sup 3/H)WB4101 selectively labels the 5-HT1A serotonin receptor, whereas (/sup 3/H) LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of (/sup 3/H)WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of (/sup 3/H)WB4101 binding.« less

Isolation and characterization of a dual function protein from Allium sativum bulbs which exhibits proteolytic and hemagglutinating activities.

PubMed

Parisi, Mónica G; Moreno, Silvia; Fernández, Graciela

2008-04-01

A dual function protein was isolated from Allium sativum bulbs and was characterized. The protein had a molecular mass of 25-26 kDa under non-reducing conditions, whereas two polypeptide chains of 12.5+/-0.5 kDa were observed under reducing conditions. E-64 and leupeptin inhibited the proteolytic activity of the protein, which exhibited characteristics similar to cysteine peptidase. The enzyme exhibited substrate specificity and hydrolyzed natural substrates such as alpha-casein (K(m): 23.0 microM), azocasein, haemoglobin and gelatin. It also showed a high affinity for synthetic peptides such as Cbz-Ala-Arg-Arg-OMe-beta-Nam (K(m): 55.24 microM, k(cat): 0.92 s(-1)). The cysteine peptidase activity showed a remarkable stability after incubation at moderate temperatures (40-50 degrees C) over a pH range of 5.5-6.5. The N-terminus of the protein displayed a 100% sequence similarity to the sequences of a mannose-binding lectin isolated from garlic bulbs. Moreover, the purified protein was retained in the chromatographic column when Con-A Sepharose affinity chromatography was performed and the protein was able to agglutinate trypsin-treated rabbit red cells. Therefore, our results indicate the presence of an additional cysteine peptidase activity on a lectin previously described.

Nature of the Active Sites for CO Reduction on Copper Nanoparticles; Suggestions for Optimizing Performance.

PubMed

Cheng, Tao; Xiao, Hai; Goddard, William A

2017-08-30

Recent experiments show that the grain boundaries (GBs) of copper nanoparticles (NPs) lead to an outstanding performance in reducing CO 2 and CO to alcohol products. We report here multiscale simulations that simulate experimental synthesis conditions to predict the structure of a 10 nm Cu NP (158 555 atoms). To identify active sites, we first predict the CO binding at a large number of sites and select four exhibiting CO binding stronger than the (211) step surface. Then, we predict the formation energy of the *OCCOH intermediate as a descriptor for C-C coupling, identifying two active sites, both of which have an under-coordinated surface square site adjacent to a subsurface stacking fault. We then propose a periodic Cu surface (4 by 4 supercell) with a similar site that substantially decreases the formation energy of *OCCOH, by 0.14 eV.

An HMGA2-IGF2BP2 Axis Regulates Myoblast Proliferation and Myogenesis

PubMed Central

Li, Zhizhong; Gilbert, Jason A.; Zhang, Yunyu; Zhang, Minsi; Qiu, Qiong; Ramanujan, Krishnan; Shavlakadze, Tea; Eash, John K.; Scaramozza, Annarita; Goddeeris, Matthew M.; Kirsch, David G.; Campbell, Kevin P.; Brack, Andrew S.; Glass, David J.

2013-01-01

Summary A group of genes that are highly and specifically expressed in proliferating skeletal myoblasts during myogenesis was identified. Expression of one of these genes, Hmga2, increases coincident with satellite cell activation, and later its expression significantly declines correlating with fusion of myoblasts into myotubes. Hmga2 knockout mice exhibit impaired muscle development and reduced myoblast proliferation, while overexpression of HMGA2 promotes myoblast growth. This perturbation in proliferation can be explained by the finding that HMGA2 directly regulates the RNA-binding protein IGF2BP2. Add-back of IGF2BP2 rescues the phenotype. IGF2BP2 in turn binds to and controls the translation of a set of mRNAs, including c-myc, Sp1, and Igf1r. These data demonstrate that the HMGA2-IGF2BP2 axis functions as a key regulator of satellite cell activation and therefore skeletal muscle development. PMID:23177649

Composite Reinforcement using Boron Nitride Nanotubes

DTIC Science & Technology

2016-11-15

atomistic environment, which often results in notable changes of its binding to the system. By contrast, Cu and Ti exhibits quite uniform behaviour ...contrast, Cu and Ti exhibits quite uniform behaviour and hardly change their binding energy with tube size for the systems investigated. Studies on...reinforced Cu is rather brittle and therefore comparison of the behaviour of Al, Ti and Cu is important, and also because Cu is used in alloys to produce

Ebola Virus Binding to Tim-1 on T Lymphocytes Induces a Cytokine Storm.

PubMed

Younan, Patrick; Iampietro, Mathieu; Nishida, Andrew; Ramanathan, Palaniappan; Santos, Rodrigo I; Dutta, Mukta; Lubaki, Ndongala Michel; Koup, Richard A; Katze, Michael G; Bukreyev, Alexander

2017-09-26

Ebola virus (EBOV) disease (EVD) results from an exacerbated immunological response that is highlighted by a burst in the production of inflammatory mediators known as a "cytokine storm." Previous reports have suggested that nonspecific activation of T lymphocytes may play a central role in this phenomenon. T-cell immunoglobulin and mucin domain-containing protein 1 (Tim-1) has recently been shown to interact with virion-associated phosphatidylserine to promote infection. Here, we demonstrate the central role of Tim-1 in EBOV pathogenesis, as Tim-1 -/- mice exhibited increased survival rates and reduced disease severity; surprisingly, only a limited decrease in viremia was detected. Tim-1 -/- mice exhibited a modified inflammatory response as evidenced by changes in serum cytokines and activation of T helper subsets. A series of in vitro assays based on the Tim-1 expression profile on T cells demonstrated that despite the apparent absence of detectable viral replication in T lymphocytes, EBOV directly binds to isolated T lymphocytes in a phosphatidylserine-Tim-1-dependent manner. Exposure to EBOV resulted in the rapid development of a CD4 Hi CD3 Low population, non-antigen-specific activation, and cytokine production. Transcriptome and Western blot analysis of EBOV-stimulated CD4 + T cells confirmed the induction of the Tim-1 signaling pathway. Furthermore, comparative analysis of transcriptome data and cytokine/chemokine analysis of supernatants highlight the similarities associated with EBOV-stimulated T cells and the onset of a cytokine storm. Flow cytometry revealed virtually exclusive binding and activation of central memory CD4 + T cells. These findings provide evidence for the role of Tim-1 in the induction of a cytokine storm phenomenon and the pathogenesis of EVD. IMPORTANCE Ebola virus infection is characterized by a massive release of inflammatory mediators, which has come to be known as a cytokine storm. The severity of the cytokine storm is consistently linked with fatal disease outcome. Previous findings have demonstrated that specific T-cell subsets are key contributors to the onset of a cytokine storm. In this study, we investigated the role of Tim-1, a T-cell-receptor-independent trigger of T-cell activation. We first demonstrated that Tim-1-knockout (KO) mice survive lethal Ebola virus challenge. We then used a series of in vitro assays to demonstrate that Ebola virus directly binds primary T cells in a Tim-1-phosphatidylserine-dependent manner. We noted that binding induces a cytokine storm-like phenomenon and that blocking Tim-1-phosphatidylserine interactions reduces viral binding, T-cell activation, and cytokine production. These findings highlight a previously unknown role of Tim-1 in the development of a cytokine storm and "immune paralysis." Copyright © 2017 Younan et al.

Synthesis and pharmacological evaluation of pyrazolo[4,3-c]cinnoline derivatives as potential anti-inflammatory and antibacterial agents.

PubMed

Tonk, Rajiv Kumar; Bawa, Sandhya; Chawla, Gita; Deora, Girdhar Singh; Kumar, Suresh; Rathore, Vandana; Mulakayala, Naveen; Rajaram, Azad; Kalle, Arunasree M; Afzal, Obaid

2012-11-01

A series of pyrazolo[4,3-c]cinnoline derivatives was synthesized, characterized and evaluated for anti-inflammatory and antibacterial activity. Test compounds that exhibited good anti-inflammatory activity were further screened for their ulcerogenic and lipid peroxidation activity. Compounds 4d and 4l showed promising anti-inflammatory activity with reduced ulcerogenic and lipid peroxidation activity when compared to naproxen. Docking results of these two compounds with COX-2 (PDB ID: 1CX2) also exhibited a strong binding profile. Among the test derivatives, compound 4i displayed significant antibacterial property against gram-negative (Escherichia coli and Pseudomonas aeruginosa) and gram-positive (Staphylococcus aureus) bacteria. However, compound 4b emerged as the best dual anti-inflammatory-antibacterial agent in the present study. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

The influence of SV40 immortalization of human fibroblasts on p53-dependent radiation responses

NASA Technical Reports Server (NTRS)

Kohli, M.; Jorgensen, T. J.

1999-01-01

The simian virus 40 large tumor antigen (SV40 Tag) has been ascribed many functions critical to viral propagation, including binding to the mammalian tumor suppressor p53. Recent studies have demonstrated that SV40-transformed murine cells have functional p53. The status of p53 in SV40-immortalized human cells, however, has not been characterized. We have found that in response to ionizing radiation, p53-dependent p21 transactivation activity is present, albeit reduced, in SV40-immortalized cells and that this activity can be further reduced with either dominant negative p53 expression or higher SV40 Tag expression. Furthermore, overexpression of p53 in SV40-immortalized ataxia-telangiectasia (A-T) cells restores p53-dependent p21 induction to typical A-T levels. All SV40-immortalized cell lines exhibited an absence of G1 arrest. Moreover, all SV40-immortalized cell lines exhibited increased apoptosis relative to primary cells in response to ionizing radiation, suggesting that SV40 immortalization results in a unique phenotype with regard to DNA damage responses. Copyright 1999 Academic Press.

Effects of rutin on the redox reactions of hemoglobin.

PubMed

Lu, Naihao; Ding, Yun; Yang, Zhen; Gao, Pingzhang

2016-08-01

Flavonoids are widely used to attenuate oxidative damage in vitro and in vivo. In this study, we investigated the influence of rutin (quercetin-3-rhamnosylglucoside) on hemoglobin (Hb)- dependent redox reactions, i.e. oxidative stability of Hb and its cytotoxic ferryl intermediate. It was found that rutin induced generation of H2O2, which in turn oxidized Hb rapidly. Meanwhile, rutin exhibited anti-oxidant effect by effectively reducing ferryl intermediate back to ferric Hb at physiological pH. In comparison with quercetin, rutin had stronger capability on reducing ferryl species while lesser pro-oxidant effect on H2O2 generation, thus it exhibited more protective effect on H2O2-induced Hb oxidation. Circular dichroism spectrum showed no significant change in the secondary structure of Hb after flavonoid addition, while molecular docking revealed different binding modes of quercetin and rutin with Hb. These results might provide new insights into the potential nutritional and physiological implications of rutin and quercetin with redox active heme proteins regarding their ani- and pro-oxidant effects. Copyright © 2016 Elsevier B.V. All rights reserved.

LAL (Lysosomal Acid Lipase) Promotes Reverse Cholesterol Transport In Vitro and In Vivo.

PubMed

Bowden, Kristin L; Dubland, Joshua A; Chan, Teddy; Xu, You-Hai; Grabowski, Gregory A; Du, Hong; Francis, Gordon A

2018-05-01

To explore the role of LAL (lysosomal acid lipase) in macrophage cholesterol efflux and whole-body reverse cholesterol transport. Immortalized peritoneal macrophages from lal -/- mice showed reduced expression of ABCA1 (ATP-binding cassette transporter A1) and ABCG1 (ATP-binding cassette transporter G1), reduced production of the regulatory oxysterol 27-hydroxycholesterol, and impaired suppression of cholesterol synthesis on exposure to acetylated low-density lipoprotein when compared with lal +/+ macrophages. LAL-deficient mice also showed reduced hepatic ABCG5 (ATP-binding cassette transporter G5) and ABCG8 (ATP-binding cassette transporter G8) expression compared with lal +/+ mice. LAL-deficient macrophages loaded with [ 3 H]-cholesteryl oleate-labeled acetylated low-density lipoprotein showed impaired efflux of released [ 3 H]-cholesterol to apoA-I (apolipoprotein A-I), with normalization of [ 3 H]-cholesteryl ester levels and partial correction of ABCA1 expression and cholesterol efflux to apoA-I when treated with exogenous rhLAL (recombinant human LAL protein). LAL-deficient mice injected intraperitoneally with lal -/- macrophages cholesterol loaded and labeled in the same way exhibited only 1.55±0.35% total injected [ 3 H]-cholesterol counts appearing in the feces for 48 h (n=30), compared with 5.38±0.92% in lal +/+ mice injected with labeled lal +/+ macrophages (n=27), P <0.001. To mimic the therapeutic condition of delivery of supplemental LAL in vivo, injection of labeled lal -/- macrophages into lal +/+ mice resulted in a significant increase in reverse cholesterol transport (2.60±0.46% of 3 H-cholesterol counts in feces at 48 hours [n=19]; P <0.001 when compared with injection into lal -/- mice). These results indicate a critical role for LAL in promoting both macrophage and whole-body reverse cholesterol transport and the ability of supplemental LAL to be taken up and correct reverse cholesterol transport in vivo. © 2018 American Heart Association, Inc.

Computational study of the inhibitory mechanism of the kinase CDK5 hyperactivity by peptide p5 and derivation of a pharmacophore

NASA Astrophysics Data System (ADS)

Cardone, A.; Brady, M.; Sriram, R.; Pant, H. C.; Hassan, S. A.

2016-06-01

The hyperactivity of the cyclic dependent kinase 5 (CDK5) induced by the activator protein p25 has been linked to a number of pathologies of the brain. The CDK5-p25 complex has thus emerged as a major therapeutic target for Alzheimer's disease (AD) and other neurodegenerative conditions. Experiments have shown that the peptide p5 reduces the CDK5-p25 activity without affecting the endogenous CDK5-p35 activity, whereas the peptide TFP5, obtained from p5, elicits similar inhibition, crosses the blood-brain barrier, and exhibits behavioral rescue of AD mice models with no toxic side effects. The molecular basis of the kinase inhibition is not currently known, and is here investigated by computer simulations. It is shown that p5 binds the kinase at the same CDK5/p25 and CDK5/p35 interfaces, and is thus a non-selective competitor of both activators, in agreement with available experimental data in vitro. Binding of p5 is enthalpically driven with an affinity estimated in the low µM range. A quantitative description of the binding site and pharmacophore is presented, and options are discussed to increase the binding affinity and selectivity in the design of drug-like compounds against AD.

Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage.

PubMed

Haendeler, Judith; Dröse, Stefan; Büchner, Nicole; Jakob, Sascha; Altschmied, Joachim; Goy, Christine; Spyridopoulos, Ioakim; Zeiher, Andreas M; Brandt, Ulrich; Dimmeler, Stefanie

2009-06-01

The enzyme telomerase and its catalytic subunit the telomerase reverse transcriptase (TERT) are important for maintenance of telomere length in the nucleus. Recent studies provided evidence for a mitochondrial localization of TERT. Therefore, we investigated the exact localization of TERT within the mitochondria and its function. Here, we demonstrate that TERT is localized in the matrix of the mitochondria. TERT binds to mitochondrial DNA at the coding regions for ND1 and ND2. Binding of TERT to mitochondrial DNA protects against ethidium bromide-induced damage. TERT increases overall respiratory chain activity, which is most pronounced at complex I and dependent on the reverse transcriptase activity of the enzyme. Moreover, mitochondrial reactive oxygen species are increased after genetic ablation of TERT by shRNA. Mitochondrially targeted TERT and not wild-type TERT revealed the most prominent protective effect on H(2)O(2)-induced apoptosis. Lung fibroblasts from 6-month-old TERT(-/-) mice (F2 generation) showed increased sensitivity toward UVB radiation and heart mitochondria exhibited significantly reduced respiratory chain activity already under basal conditions, demonstrating the protective function of TERT in vivo. Mitochondrial TERT exerts a novel protective function by binding to mitochondrial DNA, increasing respiratory chain activity and protecting against oxidative stress-induced damage.

Deciphering the kinetic mechanisms controlling selected plant ADP-glucose pyrophosphorylases.

PubMed

Boehlein, Susan K; Shaw, Janine R; Hwang, Seon K; Stewart, Jon D; Curtis Hannah, L

2013-07-15

ADP-Glc pyrophosphorylase (AGPase), a rate-limiting enzyme in starch biosynthesis, is controlled by thermostability and allosteric regulation. Previous studies suggested that redox affects turnover number and heat stability of AGPases. Here, we investigated how allostery and redox state affect kinetic mechanisms of the reduced, heat labile and the oxidized, heat stable potato tuber enzymes; the heat labile maize endosperm enzyme and a chimeric maize/potato heat stable enzyme that lacks the cysteine responsible for redox changes. With 3-PGA, all AGPases followed a Theorell-Chance Bi Bi mechanism with ATP binding first and ADP-Glc releasing last. 3-PGA increases the binding affinity for both substrates with little effect on velocity for the maize and MP isoforms. By contrast, 3-PGA increases the velocity and the affinity for G-1-P for the potato enzymes. Redox state does not affect kcat of the two potato isoforms. Without 3-PGA the oxidized potato enzyme exhibits a rapid equilibrium random Bi Bi mechanism with a dead end ternary complex. This fundamental change from rapid, ordered binding with little buildup of intermediates to a mechanism featuring relatively slow, random binding is unique to the oxidized potato tuber enzyme. Finally, ADP-Glc the physiologically relevant product of this enzyme has complex, isoform-specific effects on catalysis. Copyright © 2013 Elsevier Inc. All rights reserved.

Plant Flavone Apigenin Binds to Nucleic Acid Bases and Reduces Oxidative DNA Damage in Prostate Epithelial Cells

PubMed Central

Bhaskaran, Natarajan; Gupta, Sanjay

2014-01-01

Oxidative stress has been linked to prostate carcinogenesis as human prostate tissue is vulnerable to oxidative DNA damage. Apigenin, a dietary plant flavone, possesses anti-proliferative and anticancer effects; however, its antioxidant properties have not been fully elucidated. We investigated sub-cellular distribution of apigenin, it’s binding to DNA and protective effects against H2O2-induced DNA damage using transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells. Exposure of cells to apigenin exhibited higher accumulation in RWPE-1 and LNCaP cells, compared to PC-3 and DU145 cells. The kinetics of apigenin uptake in LNCaP cells was estimated with a Km value of 5 µmole/L and Vmax of 190 pmoles/million cells/h. Sub-cellular fractionation demonstrated that nuclear matrix retains the highest concentration of apigenin (45.3%), followed by cytosol (23.9%), nuclear membranes (17.9%) and microsomes (12.9%), respectively. Spectroscopic analysis of apigenin with calf-thymus DNA exhibited intercalation as the dominant binding mode to DNA duplex. Apigenin exposure resulted in significant genoprotective effects in H2O2-stressed RWPE-1 cells by reduction in reactive oxygen species levels. In addition, apigenin exposure suppressed the formation of 8-hydroxy-2′ deoxyguanosine and protected exposed cells from apoptosis. Our studies demonstrate that apigenin is readily taken up by normal prostatic epithelial cells and prostate cancer cells, and is incorporated into their nuclei, where its intercalation with nucleic acid bases may account for its antioxidant and chemopreventive activities. PMID:24614817

Plant flavone apigenin binds to nucleic acid bases and reduces oxidative DNA damage in prostate epithelial cells.

PubMed

Sharma, Haripaul; Kanwal, Rajnee; Bhaskaran, Natarajan; Gupta, Sanjay

2014-01-01

Oxidative stress has been linked to prostate carcinogenesis as human prostate tissue is vulnerable to oxidative DNA damage. Apigenin, a dietary plant flavone, possesses anti-proliferative and anticancer effects; however, its antioxidant properties have not been fully elucidated. We investigated sub-cellular distribution of apigenin, it's binding to DNA and protective effects against H2O2-induced DNA damage using transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells. Exposure of cells to apigenin exhibited higher accumulation in RWPE-1 and LNCaP cells, compared to PC-3 and DU145 cells. The kinetics of apigenin uptake in LNCaP cells was estimated with a Km value of 5 µmole/L and Vmax of 190 pmoles/million cells/h. Sub-cellular fractionation demonstrated that nuclear matrix retains the highest concentration of apigenin (45.3%), followed by cytosol (23.9%), nuclear membranes (17.9%) and microsomes (12.9%), respectively. Spectroscopic analysis of apigenin with calf-thymus DNA exhibited intercalation as the dominant binding mode to DNA duplex. Apigenin exposure resulted in significant genoprotective effects in H2O2-stressed RWPE-1 cells by reduction in reactive oxygen species levels. In addition, apigenin exposure suppressed the formation of 8-hydroxy-2' deoxyguanosine and protected exposed cells from apoptosis. Our studies demonstrate that apigenin is readily taken up by normal prostatic epithelial cells and prostate cancer cells, and is incorporated into their nuclei, where its intercalation with nucleic acid bases may account for its antioxidant and chemopreventive activities.

CDB-4124 and its putative monodemethylated metabolite, CDB-4453, are potent antiprogestins with reduced antiglucocorticoid activity: in vitro comparison to mifepristone and CDB-2914.

PubMed

Attardi, Barbara J; Burgenson, Janet; Hild, Sheri A; Reel, Jerry R; Blye, Richard P

2002-02-25

To obtain selective antiprogestins, we have examined the in vitro antiprogestational/antiglucocorticoid properties of two novel compounds, CDB-4124 and the putative monodemethylated metabolite, CDB-4453, in transcription and receptor binding assays and compared them to CDB-2914 and mifepristone. All four antiprogestins bound with high affinity to rabbit uterine progestin receptors (PR) and recombinant human PR-A and PR-B (rhPR-A, rhPR-B) and were potent inhibitors of R5020-induced transactivation of the PRE2-tk-luciferase (PRE2-tk-LUC) reporter plasmid and endogenous alkaline phosphatase production in T47D-CO human breast cancer cells. None of these compounds exhibited agonist activity in these cells. Induction of luciferase activity was potentiated about five-fold by 8-Br-cAMP under basal conditions and to the same extent in the presence of the PR antagonists. Mifepristone bound to rabbit thymic glucocorticoid receptors (GR) with approximately twice the avidity of the CDB antiprogestins. Inhibition of GR-mediated transcription of PRE2-tk-LUC was assessed in HepG2 human hepatoblastoma cells. Mifepristone exhibited greater antiglucocorticoid activity than CDB-2914, 4124, and 4453, about 12-, 22-, and 185-fold, respectively. Thus, while there was a good correlation between binding to PR and functional activity of these antiprogestins, GR binding was not predictive of their glucocorticoid antagonist activity. In agreement with our in vivo results, CDB-4124 and CDB-4453, as well as CDB-2914, are potent antiprogestins in vitro, but show considerably less antiglucocorticoid activity than mifepristone.

Mutations that abolish the ability of Ha-Ras to associate with Raf-1.

PubMed

Shirouzu, M; Koide, H; Fujita-Yoshigaki, J; Oshio, H; Toyama, Y; Yamasaki, K; Fuhrman, S A; Villafranca, E; Kaziro, Y; Yokoyama, S

1994-08-01

Recent studies have revealed that Ras can associate physically with Raf. In the present study, we tested 34 mutants of Ha-Ras carrying substitution(s) in the region of residues 23-71 for their ability to associate with Raf-1. Mouse Ba/F3 cell lysates were incubated with each mutant Ras protein, in either the guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)- or the guanosine 5'-[beta-thio]diphosphate (GDP beta S)-bound form, and the anti-Ras antibody Y13-238. The immunoprecipitates were analysed for the presence of Raf-1 by Western blotting with an anti-Raf-1 antibody. Six mutants of Ras, E31K, P34G, T35S, D38N, D57A and A59T, failed to bind Raf-1. Mutations N26G, V29A, S39A, Y40W, R41A, V44A, V45E, L56A and T58A partially reduced the ability to bind Raf-1. All the other mutants could associate with Raf-1 with nearly the same efficiency as that of wild-type Ras. Thus, the Raf-I-binding ability of Ras appears to be affected by mutations in the N-terminal region, and in particular, by those in and neighboring the effector region (residues 32-40) and in the region (residues 56-59) flanking the N-terminal of Switch II. The abilities to bind Raf-1 and to induce neurite outgrowth of pheochromocytoma (PC) 12 cells correlate to each other for 22 Ras mutants. However, mutation A59T, which does not reduce the neurite-inducing or transforming activities, abolishes the ability to bind Raf-1. In contrast, mutations Y32F, K42A and L53A, which impair the neurite-inducing activity of Ras, have no effect on the Ras.Raf-1 association. Partially reduced Raf-1-binding ability was observed for mutants V29A, S39A, Y40W, R41A, V44A, L56A and T58A, which exhibit full neurite-inducing activity, and also for mutant V45E, which has no activity of neurite induction.

Contribution of TyrB26 to the Function and Stability of Insulin

PubMed Central

Pandyarajan, Vijay; Phillips, Nelson B.; Rege, Nischay; Lawrence, Michael C.; Whittaker, Jonathan; Weiss, Michael A.

2016-01-01

Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface. We investigated the role of TyrB26, a conserved aromatic residue at this interface. To probe the evolutionary basis for such conservation, we constructed 18 variants at B26. Surprisingly, non-aromatic polar or charged side chains (such as Glu, Ser, or ornithine (Orn)) conferred high activity, whereas the weakest-binding analogs contained Val, Ile, and Leu substitutions. Modeling of variant complexes suggested that the B26 side chains pack within a shallow depression at the solvent-exposed periphery of the interface. This interface would disfavor large aliphatic side chains. The analogs with highest activity exhibited reduced thermodynamic stability and heightened susceptibility to fibrillation. Perturbed self-assembly was also demonstrated in studies of the charged variants (Orn and Glu); indeed, the GluB26 analog exhibited aberrant aggregation in either the presence or absence of zinc ions. Thus, although TyrB26 is part of insulin's receptor-binding surface, our results suggest that its conservation has been enjoined by the aromatic ring's contributions to native stability and self-assembly. We envisage that such classical structural relationships reflect the implicit threat of toxic misfolding (rather than hormonal function at the receptor level) as a general evolutionary determinant of extant protein sequences. PMID:27129279

Resistance of Echovirus 11 to ClO2 Is Associated with Enhanced Host Receptor Use, Altered Entry Routes, and High Fitness

PubMed Central

2017-01-01

Waterborne viruses can exhibit resistance to common water disinfectants, yet the mechanisms that allow them to tolerate disinfection are poorly understood. Here, we generated echovirus 11 (E11) with resistance to chlorine dioxide (ClO2) by experimental evolution, and we assessed the associated genotypic and phenotypic traits. ClO2 resistance emerged after E11 populations were repeatedly reduced (either by ClO2-exposure or by dilution) and then regrown in cell culture. The resistance was linked to an improved capacity of E11 to bind to its host cells, which was further attributed to two potential causes: first, the resistant E11 populations possessed mutations that caused amino acid substitutions from ClO2-labile to ClO2-stable residues in the viral proteins, which likely increased the chemical stability of the capsid toward ClO2. Second, resistant E11 mutants exhibited the capacity to utilize alternative cell receptors for host binding. Interestingly, the emergence of ClO2 resistance resulted in an enhanced replicative fitness compared to the less resistant starting population. Overall this study contributes to a better understanding of the mechanism underlying disinfection resistance in waterborne viruses, and processes that drive resistance development. PMID:28837336

Plant-produced anti-dengue virus monoclonal antibodies exhibit reduced antibody-dependent enhancement of infection activity.

PubMed

Dent, Matthew; Hurtado, Jonathan; Paul, Amber M; Sun, Haiyan; Lai, Huafang; Yang, Ming; Esqueda, Adrian; Bai, Fengwei; Steinkellner, Herta; Chen, Qiang

2016-12-01

The mAb E60 has the potential to be a desirable therapeutic molecule since it efficiently neutralizes all four serotypes of dengue virus (DENV). However, mammalian-cell-produced E60 exhibits antibody-dependent enhancement of infection (ADE) activity, rendering it inefficacious in vivo, and treated animals more susceptible to developing more severe diseases during secondary infection. In this study, we evaluated a plant-based expression system for the production of therapeutically suitable E60. The mAb was transiently expressed in Nicotiana benthamianaWT and a ∆XFT line, a glycosylation mutant lacking plant-specific N-glycan residues. The mAb was efficiently expressed and assembled in leaves and exhibited highly homogenous N-glycosylation profiles, i.e. GnGnXF3 or GnGn structures, depending on the expression host. Both E60 glycovariants demonstrated equivalent antigen-binding specificity and in vitro neutralization potency against DENV serotypes 2 and 4 compared with their mammalian-cell-produced counterpart. By contrast, plant-produced E60 exhibited reduced ADE activity in Fc gamma receptor expressing human cells. Our results suggest the ability of plant-produced antibodies to minimize ADE, which may lead to the development of safe and highly efficacious antibody-based therapeutics against DENV and other ADE-prone viral diseases. Our study provides so far unknown insight into the relationship between mAb N-glycosylation and ADE, which contributes to our understanding of how sugar moieties of antibodies modulate Fc-mediated functions and viral pathogenesis.

Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance

PubMed Central

Hall Sedlak, Ruth; Hnilova, Marketa; Grosh, Carolynn; Fong, Hanson; Baneyx, Francois; Schwartz, Dan; Sarikaya, Mehmet; Tamerler, Candan

2012-01-01

Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo. PMID:22286990

Contributions of the Histidine Side Chain and the N-terminal α-Amino Group to the Binding Thermodynamics of Oligopeptides to Nucleic Acids as a Function of pH

PubMed Central

Ballin, Jeff D.; Prevas, James P.; Ross, Christina R.; Toth, Eric A.; Wilson, Gerald M.; Record, M. Thomas

2010-01-01

Interactions of histidine with nucleic acid phosphates and histidine pKa shifts make important contributions to many protein-nucleic acid binding processes. To characterize these phenomena in simplified systems, we quantified binding of a histidine-containing model peptide HWKK (+NH3-His-Trp-Lys-Lys-NH2) and its lysine analog KWKK (+NH3-Lys-Trp-Lys-Lys-NH2) to a single-stranded RNA model, polyuridylate (polyU), by changes in tryptophan fluorescence as a function of salt concentration and pH. For both HWKK and KWKK, equilibrium binding constants, Kobs, and magnitudes of log-log salt derivatives SKobs ≡ (∂logKobs/∂log[Na+]), decreased with increasing pH in the manner expected for a titration curve model in which deprotonation of the histidine and α-amino groups weakens binding and reduces its salt-dependence. Fully protonated HWKK and KWKK exhibit the same Kobs and SKobs within uncertainty, and these SKobs values are consistent with limiting-law polyelectrolyte theory for +4 cationic oligopeptides binding to single-stranded nucleic acids. The pH-dependence of HWKK binding to polyU provides no evidence for pKa shifts nor any requirement for histidine protonation, in stark contrast to the thermodynamics of coupled protonation often seen for these cationic residues in the context of native protein structure where histidine protonation satisfies specific interactions (e.g., salt-bridge formation) within highly complementary binding interfaces. The absence of pKa shifts in our studies indicates that additional Coulombic interactions across the nonspecific-binding interface between RNA and protonated histidine or the α-amino group are not sufficient to promote proton uptake for these oligopeptides. We present our findings in the context of hydration models for specific versus nonspecific nucleic acid binding. PMID:20108951

Autoradiographic localization of sigma receptor binding sites in guinea pig and rat central nervous system with (+)3H-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine

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

Gundlach, A.L.; Largent, B.L.; Snyder, S.H.

1986-06-01

(+)3H-3-PPP ((+)3H-3-(3-Hydroxyphenyl)-N-(1-propyl)-piperidine) binds with high affinity to brain membranes with a pharmacological profile consistent with that of sigma receptors. The distribution of (+)3H-3-PPP binding sites in brain and spinal cord of both guinea pig and rat has been determined by in vitro autoradiography with binding densities quantitated by computer-assisted densitometry. (+)3H-3-PPP binding to slide-mounted brain sections is saturable and displays high affinity and a pharmacological specificity very similar to sites labeled in homogenates. (+)3H-3-PPP binding sites are heterogeneously distributed. Highest concentrations of binding sites occur in spinal cord, particularly the ventral horn and dorsal root ganglia; the pons-medulla, associated withmore » the cranial nerve and pontine nuclei and throughout the brain stem reticular formation; the cerebellum, over the Purkinje cell layer; the midbrain, particularly the central gray and red nucleus; and hippocampus, over the pyramidal cell layer. Lowest levels are seen in the basal ganglia and parts of the thalamus, while all other areas, including hypothalamus and cerebral cortex, exhibit moderate grain densities. Quinolinic acid-induced lesions of the hippocampus indicate that (+)3H-3-PPP labels hippocampal pyramidal cells and granule cells in the dentate gyrus. Intrastriatal injection of ibotenic acid dramatically reduces (+)3H-3-PPP binding in this area, while injection of 6-hydroxydopamine produces a relatively slight decrease. The distribution of (+)3H-3-PPP binding sites does not correlate with the receptor distribution of any recognized neurotransmitter or neuropeptide, including dopamine. However, there is a notable similarity between the distribution of (+)3H-3-PPP sites and high-affinity binding sites for psychotomimetic opioids, such as the benzomorphan (+)SKF 10,047.« less

Ibrutinib targets mutant-EGFR kinase with a distinct binding conformation.

PubMed

Wang, Aoli; Yan, Xiao-E; Wu, Hong; Wang, Wenchao; Hu, Chen; Chen, Cheng; Zhao, Zheng; Zhao, Peng; Li, Xixiang; Wang, Li; Wang, Beilei; Ye, Zi; Wang, Jinhua; Wang, Chu; Zhang, Wei; Gray, Nathanael S; Weisberg, Ellen L; Chen, Liang; Liu, Jing; Yun, Cai-Hong; Liu, Qingsong

2016-10-25

Ibrutinib, a clinically approved irreversible BTK kinase inhibitor for Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL) etc, has been reported to be potent against EGFR mutant kinase and currently being evaluated in clinic for Non Small Cell Lung Cancer (NSCLC). Through EGFR wt/mutant engineered isogenic BaF3 cell lines we confirmed the irreversible binding mode of Ibrutinib with EGFR wt/mutant kinase via Cys797. However, comparing to typical irreversible EGFR inhibitor, such as WZ4002, the washing-out experiments revealed a much less efficient covalent binding for Ibrutinib. The biochemical binding affinity examination in the EGFR L858R/T790M kinase revealed that, comparing to more efficient irreversible inhibitor WZ4002 (Kd: 0.074 μM), Ibrutinib exhibited less efficient binding (Kd: 0.18 μM). An X-ray crystal structure of EGFR (T790M) in complex with Ibrutinib exhibited a unique DFG-in/c-Helix-out inactive binding conformation, which partially explained the less efficiency of covalent binding and provided insight for further development of highly efficient irreversible binding inhibitor for the EGFR mutant kinase. These results also imply that, unlike the canonical irreversible inhibitor, sustained effective concentration might be required for Ibrutinib in order to achieve the maximal efficacy in the clinic application against EGFR driven NSCLC.

Proton and metal ion binding to natural organic polyelectrolytes-II. Preliminary investigation with a peat and a humic acid

USGS Publications Warehouse

Marinsky, J.A.; Reddy, M.M.

1984-01-01

We summarize here experimental studies of proton and metal ion binding to a peat and a humic acid. Data analysis is based on a unified physico-chemical model for reaction of simple ions with polyelectrolytes employing a modified Henderson-Hasselbalch equation. Peat exhibited an apparent intrinsic acid dissociation constant of 10-4.05, and an apparent intrinsic metal ion binding constant of: 400 for cadmium ion; 600 for zinc ion; 4000 for copper ion; 20000 for lead ion. A humic acid was found to have an apparent intrinsic proton binding constant of 10-2.6. Copper ion binding to this humic acid sample occurred at two types of sites. The first site exhibited reaction characteristics which were independent of solution pH and required the interaction of two ligands on the humic acid matrix to simultaneously complex with each copper ion. The second complex species is assumed to be a simple monodentate copper ion-carboxylate species with a stability constant of 18. ?? 1984.

Transfer of sulfur from IscS to IscU during Fe/S cluster assembly.

PubMed

Urbina, H D; Silberg, J J; Hoff, K G; Vickery, L E

2001-11-30

The cysteine desulfurase enzymes NifS and IscS provide sulfur for the biosynthesis of Fe/S proteins. NifU and IscU have been proposed to serve as template or scaffold proteins in the initial Fe/S cluster assembly events, but the mechanism of sulfur transfer from NifS or IscS to NifU or IscU has not been elucidated. We have employed [(35)S]cysteine radiotracer studies to monitor sulfur transfer between IscS and IscU from Escherichia coli and have used direct binding measurements to investigate interactions between the proteins. IscS catalyzed transfer of (35)S from [(35)S]cysteine to IscU in the absence of additional thiol reagents, suggesting that transfer can occur directly and without involvement of an intermediate carrier. Surface plasmon resonance studies and isothermal titration calorimetry measurements further revealed that IscU binds to IscS with high affinity (K(d) approximately 2 microm) in support of a direct transfer mechanism. Transfer was inhibited by treatment of IscU with iodoacetamide, and (35)S was released by reducing reagents, suggesting that transfer of persulfide sulfur occurs to cysteinyl groups of IscU. A deletion mutant of IscS lacking C-terminal residues 376-413 (IscSDelta376-413) displayed cysteine desulfurase activity similar to the full-length protein but exhibited lower binding affinity for IscU, decreased ability to transfer (35)S to IscU, and reduced activity in assays of Fe/S cluster assembly on IscU. The findings with IscSDelta376-413 provide additional support for a mechanism of sulfur transfer involving a direct interaction between IscS and IscU and suggest that the C-terminal region of IscS may be important for binding IscU.

Dectin-2 Is a C-Type Lectin Receptor that Recognizes Pneumocystis and Participates in Innate Immune Responses.

PubMed

Kottom, Theodore J; Hebrink, Deanne M; Jenson, Paige E; Marsolek, Paige L; Wüthrich, Marcel; Wang, Huafeng; Klein, Bruce; Yamasaki, Sho; Limper, Andrew H

2018-02-01

Pneumocystis is an important fungal pathogen that causes life-threatening pneumonia in patients with AIDS and malignancy. Lung fungal pathogens are recognized by C-type lectin receptors (CLRs), which bind specific ligands and stimulate innate immune responses. The CLR Dectin-1 was previously shown to mediate immune responses to Pneumocystis spp. For this reason, we investigated a potential role for Dectin-2. Rats with Pneumocystis pneumonia (PCP) exhibited elevated Dectin-2 mRNA levels. Soluble Dectin-2 carbohydrate-recognition domain fusion protein showed binding to intact Pneumocystis carinii (Pc) and to native Pneumocystis major surface glycoprotein/glycoprotein A (Msg/gpA). RAW macrophage cells expressing V5-tagged Dectin-2 displayed enhanced binding to Pc and increased protein tyrosine phosphorylation. Furthermore, the binding of Pc to Dectin-2 resulted in Fc receptor-γ-mediated intracellular signaling. Alveolar macrophages from Dectin-2-deficient mice (Dectin-2 -/- ) showed significant decreases in phospho-Syk activation after challenge with Pc cell wall components. Stimulation of Dectin-2 -/- alveolar macrophages with Pc components showed significant decreases in the proinflammatory cytokines IL-6 and TNF-α. Finally, during infection with Pneumocystis murina, Dectin-2 -/- mice displayed downregulated mRNA expression profiles of other CLRs implicated in fungal immunity. Although Dectin-2 -/- alveolar macrophages had reduced proinflammatory cytokine release in vitro, Dectin-2 -/- deficiency did not reduce the overall resistance of these mice in the PCP model, and organism burdens were statistically similar in the long-term immunocompromised and short-term immunocompetent PCP models. These results suggest that Dectin-2 participates in the initial innate immune signaling response to Pneumocystis, but its deficiency does not impair resistance to the organism.

Rice LGD1 containing RNA binding activity affects growth and development through alternative promoters.

PubMed

Thangasamy, Saminathan; Chen, Pei-Wei; Lai, Ming-Hsing; Chen, Jychian; Jauh, Guang-Yuh

2012-07-01

Tiller initiation and panicle development are important agronomical traits for grain production in Oryza sativa L. (rice), but their regulatory mechanisms are not yet fully understood. In this study, T-DNA mutant and RNAi transgenic approaches were used to functionally characterize a unique rice gene, LAGGING GROWTH AND DEVELOPMENT 1 (LGD1). The lgd1 mutant showed slow growth, reduced tiller number and plant height, altered panicle architecture and reduced grain yield. The fewer unelongated internodes and cells in lgd1 led to respective reductions in tiller number and to semi-dwarfism. Several independent LGD1-RNAi lines exhibited defective phenotypes similar to those observed in lgd1. Interestingly, LGD1 encodes multiple transcripts with different transcription start sites (TSSs), which were validated by RNA ligase-mediated rapid amplification of 5' and 3' cDNA ends (RLM-RACE). Additionally, GUS assays and a luciferase promoter assay confirmed the promoter activities of LGD1.1 and LGD1.5. LGD1 encoding a von Willebrand factor type A (vWA) domain containing protein is a single gene in rice that is seemingly specific to grasses. GFP-tagged LGD1 isoforms were predominantly detected in the nucleus, and weakly in the cytoplasm. In vitro northwestern analysis showed the RNA-binding activity of the recombinant C-terminal LGD1 protein. Our results demonstrated that LGD1 pleiotropically regulated rice vegetative growth and development through both the distinct spatiotemporal expression patterns of its multiple transcripts and RNA binding activity. Hence, the study of LGD1 will strengthen our understanding of the molecular basis of the multiple transcripts, and their corresponding polypeptides with RNA binding activity, that regulate pleiotropic effects in rice. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Contributions of a disulfide bond and a reduced cysteine side chain to the intrinsic activity of the HDL receptor SR-BI

PubMed Central

Yu, Miao; Lau, Thomas Y.; Carr, Steven A.; Krieger, Monty

2013-01-01

The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), binds HDL and mediates selective cholesteryl ester uptake. SR-BI's structure and mechanism are poorly understood. We used mass spectrometry to assign the two disulfide bonds in SR-BI that connect cysteines within the conserved Cys321-Pro322-Cys323 (CPC) motif and connect Cys280 to Cys334. We used site-specific mutagenesis to evaluate the contributions of the CPC motif and the side chain of extracellular Cys384 to HDL binding and lipid uptake. The effects of CPC mutations on activity were context dependent. Full wild-type (WT) activity required Pro322 and Cys323 only when Cys321 was present. Reduced intrinsic activities were observed for CXC and CPX, but not XXC, XPX or XXX mutants (X≠WT residue). Apparently, a free thiol side chain at position 321 that cannot form an intra-CPC disulfide bond with Cys323 is deleterious, perhaps because of aberrant disulfide bond formation. Pro322 may stabilize an otherwise strained CPC disulfide bond, thus supporting WT activity, but this disulfide bond is not absolutely required for activity. C384X (X=S,T,L,Y,G,A) mutants exhibited altered activities that varied with the side chain's size: larger side chains phenocopied WT SR-BI treated with its thiosemicarbazone inhibitor BLT-1 (increased binding, decreased uptake); smaller side chains produced almost inverse effects (increased uptake:binding ratio). C384X mutants were BLT-1 resistant, supporting the proposal that Cys384's thiol interacts with BLT-1. We discuss the implications of our findings on the functions of the extracellular loop cysteines in SR-BI and compare our results to those presented by other laboratories. PMID:23205738

VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability

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

Schwarz, Toni M.; Edwards, Megan R.; Diederichs, Audrey

ABSTRACT Zaire ebolavirus(EBOV),Bundibugyo ebolavirus(BDBV), andReston ebolavirus(RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA bindingmore » affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. IMPORTANCEThe interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of theEbolavirusgenus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA modulates inhibition of IFN signaling and VP24 stability. The effect of KPNA interaction on VP24 stability is a novel functional consequence of this virus-host interaction, and the differences identified between viral species may contribute to differences in pathogenesis.« less

Alterations in glucose and protein metabolism in animals subjected to simulated microgravity

NASA Technical Reports Server (NTRS)

Mondon, C. E.; Rodnick, K. J.; Azhar, S.; Reaven, G. M.; Dolkas, C. B.

1992-01-01

Reduction of physical activity due to disease or environmental restraints, such as total bed rest or exposure to spaceflight, leads to atrophy of skeletal muscle and is frequently accompanied by alterations in food intake and the concentration of metabolic regulatory hormones such as insulin. Hindlimb suspension of laboratory rats, as a model for microgravity, also shows marked atrophy of gravity-dependent muscles along with a reduced gain in body weight. Suspended rats exhibit enhanced sensitivity to insulin-induced glucose uptake when compared with normal control rats and resistance to insulin action when compared with control rats matched similarly for reduced body weight gain. These changes are accompanied by decreased insulin binding and tyrosine kinase activity in soleus but not plantaris muscle, unchanged glucose uptake by perfused hindlimb and decreased sensitivity but not responsiveness to insulin-induced suppression of net proteolysis in hindlimb skeletal muscle. These findings suggest that loss of insulin sensitivity during muscle atrophy is associated with decreased insulin binding and tyrosine kinase activity in atrophied soleus muscle along with decreased sensitivity to the effects of insulin on suppressing net protein breakdown but not on enhancing glucose uptake by perfused hindlimb.

Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis

PubMed Central

Lee, Hongsik; Jeong, Hyeyeun; Park, Sangeun; Yoo, Wonbaek; Choi, Soyoung; Choi, Kyungmin; Lee, Min-Goo; Lee, Mihwa; Cha, DaeRyong; Kim, Young-Sik; Han, Jeeyoung; Kim, Wonkon; Park, Sun-Hwa; Oh, Junseo

2015-01-01

Activated hepatic stellate cells (HSCs) play a key role in liver fibrosis, and inactivating HSCs has been considered a promising therapeutic approach. We previously showed that albumin and its derivative designed for stellate cell-targeting, retinol-binding protein–albumin domain III fusion protein (referred to as R-III), inactivate cultured HSCs. Here, we investigated the mechanism of action of albumin/R-III in HSCs and examined the anti-fibrotic potential of R-III in vivo. R-III treatment and albumin expression downregulated retinoic acid (RA) signaling which was involved in HSC activation. RA receptor agonist and retinaldehyde dehydrogenase overexpression abolished the anti-fibrotic effect of R-III and albumin, respectively. R-III uptake into cultured HSCs was significantly decreased by siRNA-STRA6, and injected R-III was localized predominantly in HSCs in liver. Importantly, R-III administration reduced CCl4- and bile duct ligation-induced liver fibrosis. R-III also exhibited a preventive effect against CCl4-inducd liver fibrosis. These findings suggest that the anti-fibrotic effect of albumin/R-III is, at least in part, mediated by downregulation of RA signaling and that R-III is a good candidate as a novel anti-fibrotic drug. PMID:25864124

NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING.

PubMed

Zhang, Lu; Mo, Jinyao; Swanson, Karen V; Wen, Haitao; Petrucelli, Alex; Gregory, Sean M; Zhang, Zhigang; Schneider, Monika; Jiang, Yan; Fitzgerald, Katherine A; Ouyang, Songying; Liu, Zhi-Jie; Damania, Blossom; Shu, Hong-Bing; Duncan, Joseph A; Ting, Jenny P-Y

2014-03-20

Stimulator of interferon genes (STING, also named MITA, MYPS, or ERIS) is an intracellular DNA sensor that induces type I interferon through its interaction with TANK-binding kinase 1 (TBK1). Here we found that the nucleotide-binding, leucine-rich-repeat-containing protein, NLRC3, reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP), and DNA viruses. NLRC3 associated with both STING and TBK1 and impeded STING-TBK1 interaction and downstream type I interferon production. By using purified recombinant proteins, we found NLRC3 to interact directly with STING. Furthermore, NLRC3 prevented proper trafficking of STING to perinuclear and punctated region, known to be important for its activation. In animals, herpes simplex virus 1 (HSV-1)-infected Nlrc3(-/-) mice exhibited enhanced innate immunity and reduced morbidity and viral load. This demonstrates the intersection of two key pathways of innate immune regulation, NLR and STING, to fine tune host response to intracellular DNA, DNA virus, and c-di-GMP. Copyright © 2014 Elsevier Inc. All rights reserved.

Alterations in glucose and protein metabolism in animals subjected to simulated microgravity

NASA Astrophysics Data System (ADS)

Mondon, C. E.; Rodnick, K. J.; Dolkas, C. B.; Azhar, S.; Reaven, G. M.

1992-09-01

Reduction of physical activity due to disease or environmental restraints, such as total bed rest or exposure to spaceflight, leads to atrophy of skeletal muscle and is frequently accompanied by alterations in food intake and the concentration of metabolic regulatory hormones such as insulin. Hindlimb suspension of laboratory rats, as a model for microgravity, also shows marked atrophy of gravity dependent muscles along with a reduced gain in body weight. Suspended rats exhibit enhanced sensitivity to insulin-induced glucose uptake when compared with normal control rats and resistance to insulin action when compared with control rats matched similarly for reduced body weight gain. These changes are accompanied by decreased insulin binding and tyrosine kinase activity in soleus but not plantaris muscle, unchanged glucose uptake by perfused hindlimb and decreased sensitivity but not responsiveness to insulin-induced suppression of net proteolysis in hindlimb skeletal muscle. These findings suggest that loss of insulin sensitivity during muscle atrophy is associated with decreased insulin binding and tyrosine kinase activity in atrophied soleus muscle along with decreased sensitivity to the effects of insulin on suppressing net protein breakdown but not on enhancing glucose uptake by perfused hindlimb.

Purification and kinetic characterization of recombinant human mitogen-activated protein kinase kinase kinase COT and the complexes with its cellular partner NF-kappa B1 p105.

PubMed

Jia, Yong; Quinn, Christopher M; Bump, Nancy J; Clark, Kevin M; Clabbers, Anca; Hardman, Jennifer; Gagnon, Andrew; Kamens, Joanne; Tomlinson, Medha J; Wishart, Neil; Allen, Hamish

2005-09-01

Cancer osaka thyroid (COT), a human MAP 3 K, is essential for lipopolysaccharide activation of the Erk MAPK cascade in macrophages. COT 30--467 is insoluble, whereas low levels of COT 30--397 can be expressed, but this protein is unstable. However, both COT 30--467 and COT 30--397 are expressed in a soluble and stable form when produced in complex with the C-terminal half of p105. The k(cat) of COT 30--397 is reduced approximately 47--fold in the COT 30--467/p105 Delta N complex. COT prefers Mn(2+) to Mg(2+) as the ATP metal cofactor, exhibiting an unusually high ATP K(m) in the presence of Mg(2+). When using Mn(2+) as the cofactor, the ATP K(m) is reduced to a level typical of most kinases. In contrast, the binding affinity of COT for its other substrate MEK is cofactor independent. Our results using purified proteins indicate that p105 binding improves COT solubility and stability while down-regulating kinase activity, consistent with cellular data showing that p105 functions as an inhibitor of COT.

The galactose-binding lectin isolated from Bauhinia bauhinioides Mart seeds inhibits neutrophil rolling and adhesion via primary cytokines.

PubMed

Girão, Deysen Kerlla Fernandes Bezerra; Cavada, Benildo Sousa; de Freitas Pires, Alana; Martins, Timna Varela; Franco, Álvaro Xavier; Morais, Cecília Mendes; Santiago do Nascimento, Kyria; Delatorre, Plinio; da Silva, Helton Colares; Nagano, Celso Shiniti; Assreuy, Ana Maria Sampaio; Soares, Pedro Marcos Gomes

2015-05-01

In this study, the amino acid sequence and anti-inflammatory effect of Bauhinia bauhinioides (BBL) lectin were evaluated. Tandem mass spectrometry revealed that BBL possesses 86 amino acid residues. BBL (1 mg/kg) intravenously injected in rats 30 min prior to inflammatory stimuli inhibited the cellular edema induced by carrageenan in only the second phase (21% - 3 h, 19% - 4 h) and did not alter the osmotic edema induced by dextran. BBL also inhibited carrageenan peritoneal neutrophil migration (51%), leukocyte rolling (58%) and adhesion (68%) and the neutrophil migration induced by TNF-α (64%). These effects were reversed by the association of BBL with galactose, demonstrating that the carbohydrate-binding domain is essential for lectin activity. In addition, BBL reduced myeloperoxidase activity (84%) and TNF-α (68%) and IL1-β (47%) levels. In conclusion, the present investigation demonstrated that BBL contains highly homologous isolectins, resulting in a total of 86 amino acid residues, and exhibits anti-inflammatory activity by inhibiting neutrophil migration by reducing TNF-α and IL1-β levels via the lectin domain. Copyright © 2015 John Wiley & Sons, Ltd.

Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

PubMed Central

Fago, Angela; Malte, Hans; Storz, Jay F.; Gorr, Thomas A.

2013-01-01

In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3−), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3− levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3− binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. PMID:23720132

Conformational Changes of Blood ACE in Chronic Uremia

PubMed Central

Petrov, Maxim N.; Shilo, Valery Y.; Tarasov, Alexandr V.; Schwartz, David E.; Garcia, Joe G. N.; Kost, Olga A.; Danilov, Sergei M.

2012-01-01

Background The pattern of binding of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) comprise a conformational ACE fingerprint and is a sensitive marker of subtle protein conformational changes. Hypothesis Toxic substances in the blood of patients with uremia due to End Stage Renal Disease (ESRD) can induce local conformational changes in the ACE protein globule and alter the efficacy of ACE inhibitors. Methodology/Principal Findings The recognition of ACE by 16 mAbs to the epitopes on the N and C domains of ACE was estimated using an immune-capture enzymatic plate precipitation assay. The precipitation pattern of blood ACE by a set of mAbs was substantially influenced by the presence of ACE inhibitors with the most dramatic local conformational change noted in the N-domain region recognized by mAb 1G12. The “short” ACE inhibitor enalaprilat (tripeptide analog) and “long” inhibitor teprotide (nonapeptide) produced strikingly different mAb 1G12 binding with enalaprilat strongly increasing mAb 1G12 binding and teprotide decreasing binding. Reduction in S-S bonds via glutathione and dithiothreitol treatment increased 1G12 binding to blood ACE in a manner comparable to enalaprilat. Some patients with uremia due to ESRD exhibited significantly increased mAb 1G12 binding to blood ACE and increased ACE activity towards angiotensin I accompanied by reduced ACE inhibition by inhibitory mAbs and ACE inhibitors. Conclusions/Significance The estimation of relative mAb 1G12 binding to blood ACE detects a subpopulation of ESRD patients with conformationally changed ACE, which activity is less suppressible by ACE inhibitors. This parameter may potentially serve as a biomarker for those patients who may need higher concentrations of ACE inhibitors upon anti-hypertensive therapy. PMID:23166630

Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor).

PubMed Central

Ishiai, M; Wada, C; Kawasaki, Y; Yura, T

1994-01-01

Replication of mini-F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherichia coli. The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaJ-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dimeric form. Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo. Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers. These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator. We propose that RepE is structurally and functionally differentiated and that monomerization of RepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication. Images PMID:8170998

Dpb11 may function with RPA and DNA to initiate DNA replication

PubMed Central

Bruck, Irina; Dhingra, Nalini; Martinez, Matthew P.

2017-01-01

Dpb11 is required for the initiation of DNA replication in budding yeast. We found that Dpb11 binds tightly to single-stranded DNA (ssDNA) or branched DNA structures, while its human homolog, TopBP1, binds tightly to branched-DNA structures. We also found that Dpb11 binds stably to CDK-phosphorylated RPA, the eukaryotic ssDNA binding protein, in the presence of branched DNA. A Dpb11 mutant specifically defective for DNA binding did not exhibit tight binding to RPA in the presence of DNA, suggesting that Dpb11-interaction with DNA may promote the recruitment of RPA to melted DNA. We then characterized a mutant of Dpb11 that is specifically defective in DNA binding in budding yeast cells. Expression of dpb11-m1,2,3,5,ΔC results in a substantial decrease in RPA recruitment to origins, suggesting that Dpb11 interaction with DNA may be required for RPA recruitment to origins. Expression of dpb11-m1,2,3,5,ΔC also results in diminished GINS interaction with Mcm2-7 during S phase, while Cdc45 interaction with Mcm2-7 is like wild-type. The reduced GINS interaction with Mcm2-7 may be an indirect consequence of diminished origin melting. We propose that the tight interaction between Dpb11, CDK-phosphorylated RPA, and branched-DNA may be required for the essential function of stabilizing melted origin DNA in vivo. We also propose an alternative model, wherein Dpb11-DNA interaction is required for some other function in DNA replication initiation, such as helicase activation. PMID:28467467

Structural characterization of β-catenin and RX-5902 binding to phospho-p68 RNA helicase by molecular dynamics simulation.

PubMed

Ali, Waqar; Shafique, Shagufta; Rashid, Sajid

2018-05-02

Emerging implications of probable ATP-dependent RNA helicase p68 in tumorigenesis and progression makes it a discerning target for cancer therapy. Recently it has been reported that tyrosyl-phosphorylation of p68 promotes β-catenin nuclear translocation and cancer metastasis through elevating the epithelial-mesenchymal transition. Despite recent advances, the structural characterization of this interaction, mode of action and induced conformational changes remain elusive. Here, through comparative structure analysis and molecular dynamics simulation assays, we explored comparative binding pattern of phospho-p68 against β-catenin. Conversely, due to the promising therapeutic potential of p68 in blocking the invasiveness and metastasis of cancer cells, we investigated the binding of heterocyclic N-substituted piperazine derivative-RX-5902 that inhibits the binding of phospho-p68 and β-catenin. Evidently, transactivation and C-terminal helicase domains of phospho-p68 exhibited dramatic conformational alterations to assist β-catenin and RX-5902 binding. As compared to unbound phospho-p68 (56.1 Å), the residual distances between transactivation domain-Ser79 and C-terminal helicase domain-Gln555 were reduced to 34.1 Å and 31 Å upon binding to β-catenin and RX-5902, respectively. In contrast, helicase ATP-binding domain remained conformationally stable throughout simulations. Clearly, the comparative docking-for-functional analysis of phospho-p68 against RX-5902 and β-catenin uncovered a spectrum of structural linkages associated with the molecular basis of β-catenin-dependent ATPase activity. Thus the outcomes of this study may provide a platform for the rational design of specific and potent inhibitors against phospho-p68 with a special emphasis on anticancer activity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Design and characterization of α-melanotropin peptide analogs cyclized through rhenium and technetium metal coordination

PubMed Central

Giblin, Michael F.; Wang, Nannan; Hoffman, Timothy J.; Jurisson, Silvia S.; Quinn, Thomas P.

1998-01-01

α-Melanocyte stimulating hormone (α-MSH) analogs, cyclized through site-specific rhenium (Re) and technetium (Tc) metal coordination, were structurally characterized and analyzed for their abilities to bind α-MSH receptors present on melanoma cells and in tumor-bearing mice. Results from receptor-binding assays conducted with B16 F1 murine melanoma cells indicated that receptor-binding affinity was reduced to approximately 1% of its original levels after Re incorporation into the cyclic Cys4,10, d-Phe7–α-MSH4-13 analog. Structural analysis of the Re–peptide complex showed that the disulfide bond of the original peptide was replaced by thiolate–metal–thiolate cyclization. A comparison of the metal-bound and metal-free structures indicated that metal complexation dramatically altered the structure of the receptor-binding core sequence. Redesign of the metal binding site resulted in a second-generation Re–peptide complex (ReCCMSH) that displayed a receptor-binding affinity of 2.9 nM, 25-fold higher than the initial Re–α-MSH analog. Characterization of the second-generation Re–peptide complex indicated that the peptide was still cyclized through Re coordination, but the structure of the receptor-binding sequence was no longer constrained. The corresponding 99mTc- and 188ReCCMSH complexes were synthesized and shown to be stable in phosphate-buffered saline and to challenges from diethylenetriaminepentaacetic acid (DTPA) and free cysteine. In vivo, the 99mTcCCMSH complex exhibited significant tumor uptake and retention and was effective in imaging melanoma in a murine-tumor model system. Cyclization of α-MSH analogs via 99mTc and 188Re yields chemically stable and biologically active molecules with potential melanoma-imaging and therapeutic properties. PMID:9788997

A chirality change in XPC- and Sfi1-derived peptides affects their affinity for centrin.

PubMed

Grecu, Dora; Irudayaraj, Victor Paul Raj; Martinez-Sanz, Juan; Mallet, Jean-Maurice; Assairi, Liliane

2016-04-01

The Ca(2+)-binding protein centrin binds to a hydrophobic motif (W(1)xxL(4)xxxL(8)) included in the sequence of several cellular targets: XPC (xeroderma pigmentosum group C protein), Sfi1 (suppressor of fermentation-induced loss of stress resistance protein1), and Sac3 [the central component of the transcription and mRNA export (TREX-2) complex]. However, centrin binding occurs in a reversed orientation (L(8)xxxL(4)xxW(1)) for Sfi1 and Sac3 compared with XPC. Because D-peptides have been investigated for future therapeutic use, we analyzed their centrin-binding properties. Their affinity for centrin was measured using isothermal titration calorimetry. The chirality change in the target-derived peptides affected their ability to bind centrin in a specific manner depending on the sequence orientation of the centrin-binding motif. In contrast to L-XPC-P10, D-XPC-P10 bound C-HsCen1 in a Ca(2+)-dependent manner and to a lesser extent. D-XPC-P10 exhibited a reduced affinity for C-HsCen1 (Ka=0.064 × 10(6) M(-1)) by a factor of 2000 compared with L-XPC-P10 (Ka=132 × 10(6) M(-1)). D-peptides have a lower affinity than L-peptides for centrin, and the strength of this affinity depends on the sequence orientation of the target-derived peptides. The residual affinity observed for D-XPC suggests that the use of d-peptides represents a promising strategy for inhibiting centrin binding to its targets. Copyright © 2016 Elsevier Inc. All rights reserved.

A role for anterior thalamic nuclei in affective cognition: interaction with environmental conditions.

PubMed

Dupire, Alexandra; Kant, Patricia; Mons, Nicole; Marchand, Alain R; Coutureau, Etienne; Dalrymple-Alford, John; Wolff, Mathieu

2013-05-01

Damage to anterior thalamic nuclei (ATN) is a well-known cause of diencephalic pathology that produces a range of cognitive deficits reminiscent of a hippocampal syndrome. Anatomical connections of the ATN also extend to cerebral areas that support affective cognition. Enriched environments promote recovery of declarative/relational memory after ATN lesions and are known to downregulate emotional behaviors. Hence, the performance of standard-housed and enriched ATN rats in a range of behavioral tasks engaging affective cognition was compared. ATN rats exhibited reduced anxiety responses in the elevated plus maze, increased activity and reduced corticosterone responses when exploring an open field, and delayed acquisition of a conditioned contextual fear response. ATN rats also exhibited reduced c-Fos and phosphorylated cAMP response element-binding protein (pCREB) immunoreactivity in the hippocampal formation and the amygdala after completion of the contextual fear test. Marked c-Fos hypoactivity and reduced pCREB levels were also evident in the granular retrosplenial cortex and, to a lesser extent, in the anterior cingulate cortex. Unlike standard-housed ATN rats, enriched ATN rats expressed virtually no fear of the conditioned context. These results show that the ATN regulate affective cognition and that damage to this region may produce markedly different behavioral effects as a function of environmental housing conditions. Copyright © 2013 Wiley Periodicals, Inc.

Involvement of cholinergic mechanisms in the behavioral effects of dietary fat consumption

PubMed Central

Morganstern, Irene; Ye, Zhiy; Liang, Sherry; Fagan, Shawn; Leibowitz, Sarah F.

2012-01-01

Clinical reports suggest a positive association between fat consumption and the incidence of hyperactivity, impulsivity and cognitive abnormalities. To investigate possible mechanisms underlying these disturbances under short-term conditions, we examined in Sprague-Dawley rats the influence of 7-day consumption of a high-fat diet (HFD) compared to chow on anxiety, novelty-seeking and exploratory behaviors and also on acetylcholine (ACh) neurotransmission that may mediate these behaviors. The HFD consumption, which elevated circulating fatty acids but produced no change in caloric intake or body weight, stimulated novelty-seeking and exploration in an open field, while reducing anxiety in an elevated plus maze. Using the Ellman assay to measure ACh esterase (AChE) activity that breaks down ACh, the second experiment showed HFD consumption to significantly reduce AChE activity in the frontal cortex, hypothalamus and midbrain. With measurements of [125I]-epibatidine or [125I]-bungarotoxin binding to nicotinic ACh receptors (nAChRs) containing β2 or α7 subunits, respectively, the results also showed HFD consumption to increase both β2-nAChR binding in the medial prefrontal cortex and substantia nigra and α7-nAChR binding in the lateral and ventromedial hypothalamus. When treated with an acute dose of the nicotinic antagonist, mecamylamine (0.5 mg/kg, sc), the HFD animals responded with significantly reduced exploratory and novelty-seeking behaviors, whereas the chow-consuming rats exhibited no response. These findings suggest that the exploratory and novelty-seeking behaviors induced by dietary fat may be mediated by enhanced nicotinic cholinergic activity, which is accompanied by increased density of β2-nAChRs in cortical and midbrain regions associated with impulsivity and locomotor activity and of α7-nAChRs in hypothalamic regions associated with arousal and energy balance. PMID:22765913

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia*

PubMed Central

Dong, Qian; Ernst, Sarah E.; Ostedgaard, Lynda S.; Shah, Viral S.; Ver Heul, Amanda R.; Welsh, Michael J.; Randak, Christoph O.

2015-01-01

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P1,P5-di(adenosine-5′) pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl− channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. PMID:25887396

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

PubMed

Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

2015-05-29

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Dual phosphorylation of Btk by Akt/protein kinase b provides docking for 14-3-3ζ, regulates shuttling, and attenuates both tonic and induced signaling in B cells.

PubMed

Mohammad, Dara K; Nore, Beston F; Hussain, Alamdar; Gustafsson, Manuela O; Mohamed, Abdalla J; Smith, C I Edvard

2013-08-01

Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in the Btk gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.

Dual Phosphorylation of Btk by Akt/Protein Kinase B Provides Docking for 14-3-3ζ, Regulates Shuttling, and Attenuates both Tonic and Induced Signaling in B Cells

PubMed Central

Nore, Beston F.; Hussain, Alamdar; Gustafsson, Manuela O.; Mohamed, Abdalla J.

2013-01-01

Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in the Btk gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation. PMID:23754751

Catalytic and spectroscopic analysis of blue copper-containing nitrite reductase mutants altered in the environment of the type 2 copper centre: implications for substrate interaction.

PubMed Central

Prudêncio, M; Eady, R R; Sawers, G

2001-01-01

The blue dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans is a trimer containing two types of Cu centre, three type 1 electron transfer centres and three type 2 centres. The latter have been implicated in the binding and reduction of nitrite. The Cu ion of the type 2 centre of the oxidized enzyme is ligated by three His residues, and additionally has a co-ordinated water molecule that is also hydrogen-bonded to the carboxyl of Asp(92) [Dodd, Van Beeumen, Eady and Hasnain (1998), J. Mol. Biol. 282, 369-382]. Two mutations of this residue have been made, one to a glutamic acid residue and a second to an asparagine residue; the effects of both mutations on the spectroscopic and catalytic properties of the enzyme have been analysed. EPR spectroscopy revealed that both mutants retained intact type 1 Cu centres with g( parallel)=2.12 (A( parallel)=0 mT) and g( perpendicular)=2.30 (A( perpendicular)=6.4 mT), which was consistent with their blue colour, but differed in their activities and in the spectroscopic properties of the type 2 centres. The D92E mutant had an altered geometry of its type 2 centre such that nitrite was no longer capable of binding to elicit changes in the EPR parameters of this centre. Accordingly, this mutation resulted in a form of NiR that had very low enzyme activity with the artificial electron donors reduced Methyl Viologen and sodium dithionite. As isolated, the EPR spectrum of the Asp(92)-->Asn (D92N) mutant showed no characteristic type 2 hyperfine lines. However, oxidation with iridium hexachloride partly restored a type 2 EPR signal, suggesting that type 2 copper is present in the enzyme but in a reduced, EPR-silent form. Like the Asp(92)-->Glu mutant, D92N had very low enzyme activities with either Methyl Viologen or dithionite. Remarkably, when the physiological electron donor reduced azurin I was used, both mutant proteins exhibited restoration of enzyme activity. The degree of restoration differed for the two mutants, with the D92N derivative exhibiting approx. 60% of the activity seen for the wild-type NiR. These findings suggest that on formation of an electron transfer complex with azurin, a conformational change in NiR occurs that returns the catalytic Cu centre to a functionally active state capable of binding and reducing nitrite. PMID:11139389

Analysis of myelomonocytic leukemic differentiation by a cell surface marker panel including a fucose-binding lectin from Lotus tetragonolobus.

PubMed

Elias, L; Van Epps, D E

1984-06-01

The fucose-binding lectin from Lotus tetragonolobus ( FBL -L) has been previously shown to bind specifically to normal cells of the myeloid and monocytic lineages. The purpose of this study was to explore the utility of fluoresceinated FBL -L as a leukemia differentiation marker in conjunction with a panel of other frequently used surface markers (Fc receptor, HLA-DR, OKM1, and antimonocyte antibody). FBL -L reacted with leukemic cells in 8/9 cases of clinically recognized acute myeloid leukemia, including myeloid blast crisis of chronic granulocytic leukemia, 3/3 cases of chronic phase chronic myelogenous leukemia, and in 2/7 cases of clinically undifferentiated acute leukemia. Correlations were noted between reactivity with FBL -L, and DR and Fc receptor expression. Among continuous cell lines, FBL -L bound with high intensity to a majority of HL-60 and U937 cells. The less well differentiated myeloblast cell lines, KG-1, KG1a , and HL-60 blast II, exhibited less FBL -L binding than HL-60 and U937. A moderate proportion of K562 cells exhibited low level binding of FBL -L. Several lymphoblastic cell lines exhibited a pattern of low intensity binding that was distinguishable from the high intensity binding pattern of the myeloblastic lines. FBL -L reactivity of U937 was enhanced by induction of differentiation with leukocyte conditioned medium, but not dimethylsulfoxide. Such treatments induced contrasting patterns of change of HL-60 and U937 when labeled with OKM1, alpha-Mono, and HLA-DR. These studies demonstrate the application of FBL -L to analysis and quantitation of myelomonocytic leukemic differentiation.

Uncoupling Lipid Metabolism from Inflammation through Fatty Acid Binding Protein-Dependent Expression of UCP2

PubMed Central

Xu, Hongliang; Hertzel, Ann V.; Steen, Kaylee A.; Wang, Qigui; Suttles, Jill

2015-01-01

Chronic inflammation in obese adipose tissue is linked to endoplasmic reticulum (ER) stress and systemic insulin resistance. Targeted deletion of the murine fatty acid binding protein (FABP4/aP2) uncouples obesity from inflammation although the mechanism underlying this finding has remained enigmatic. Here, we show that inhibition or deletion of FABP4/aP2 in macrophages results in increased intracellular free fatty acids (FFAs) and elevated expression of uncoupling protein 2 (UCP2) without concomitant increases in UCP1 or UCP3. Silencing of UCP2 mRNA in FABP4/aP2-deficient macrophages negated the protective effect of FABP loss and increased ER stress in response to palmitate or lipopolysaccharide (LPS). Pharmacologic inhibition of FABP4/aP2 with the FABP inhibitor HTS01037 also upregulated UCP2 and reduced expression of BiP, CHOP, and XBP-1s. Expression of native FABP4/aP2 (but not the non-fatty acid binding mutant R126Q) into FABP4/aP2 null cells reduced UCP2 expression, suggesting that the FABP-FFA equilibrium controls UCP2 expression. FABP4/aP2-deficient macrophages are resistant to LPS-induced mitochondrial dysfunction and exhibit decreased mitochondrial protein carbonylation and UCP2-dependent reduction in intracellular reactive oxygen species. These data demonstrate that FABP4/aP2 directly regulates intracellular FFA levels and indirectly controls macrophage inflammation and ER stress by regulating the expression of UCP2. PMID:25582199

Motor Deficits and Decreased Striatal Dopamine Receptor 2 Binding Activity in the Striatum-Specific Dyt1 Conditional Knockout Mice

PubMed Central

Yokoi, Fumiaki; Dang, Mai Tu; Li, Jianyong; Standaert, David G.; Li, Yuqing

2011-01-01

DYT1 early-onset generalized dystonia is a hyperkinetic movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Recently, significant progress has been made in studying pathophysiology of DYT1 dystonia using targeted mouse models. Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 knock-down (KD) mice exhibit motor deficits and alterations of striatal dopamine metabolisms, while Dyt1 knockout (KO) and Dyt1 ΔGAG homozygous KI mice show abnormal nuclear envelopes and neonatal lethality. However, it has not been clear whether motor deficits and striatal abnormality are caused by Dyt1 mutation in the striatum itself or the end results of abnormal signals from other brain regions. To identify the brain region that contributes to these phenotypes, we made a striatum-specific Dyt1 conditional knockout (Dyt1 sKO) mouse. Dyt1 sKO mice exhibited motor deficits and reduced striatal dopamine receptor 2 (D2R) binding activity, whereas they did not exhibit significant alteration of striatal monoamine contents. Furthermore, we also found normal nuclear envelope structure in striatal medium spiny neurons (MSNs) of an adult Dyt1 sKO mouse and cerebral cortical neurons in cerebral cortex-specific Dyt1 conditional knockout (Dyt1 cKO) mice. The results suggest that the loss of striatal torsinA alone is sufficient to produce motor deficits, and that this effect may be mediated, at least in part, through changes in D2R function in the basal ganglia circuit. PMID:21931745

Novel STAT3 phosphorylation inhibitors exhibit potent growth suppressive activity in pancreatic and breast cancer cells

PubMed Central

Lin, Li; Hutzen, Brian; Zuo, Mingxin; Ball, Sarah; Deangelis, Stephanie; Foust, Elizabeth; Pandit, Bulbul; Ihnat, Michael A.; Shenoy, Satyendra S.; Kulp, Samuel; Li, Pui-Kai; Li, Chenglong; Fuchs, James; Lin, Jiayuh

2010-01-01

The constitutive activation of Signal Transducer and Activator of Transcription 3 (STAT3) is frequently detected in most types of human cancer where it plays important roles in survival, drug-resistance, angiogenesis, and other functions. Targeting constitutive STAT3 signaling is thus an attractive therapeutic approach for these cancers. We have recently developed novel small molecule STAT3 inhibitors known as FLLL31 and FLLL32, which are derived from curcumin (the primary bioactive compound of turmeric). These compounds are designed to bind selectively to Janus Kinase 2 (JAK2) and the STAT3 SH2 domain, which serves crucial roles in STAT3 dimerization and signal transduction. Here we show that FLLL31 and FLLL32 are effective inhibitors of STAT3 phosphorylation, DNA binding activity, and transactivation in vitro, leading to the impediment of multiple oncogenic processes and the induction of apoptosis in pancreatic and breast cancer cell lines. FLLL31 and FLLL32 also inhibit colony formation in soft agar, cell invasion, and exhibit synergy with the anti-cancer drug doxorubicin against breast cancer cells. In addition, we show that FLLL32 can inhibit the induction of STAT3 phosphorylation by Interferon-α (IFNα) and Interleukin-6 (IL-6) in breast cancer cells. We also demonstrate that administration of FLLL32 can inhibit tumor growth and vascularity in chicken embryo xenografts as well as substantially reduce tumor volumes in mouse xenografts. Our findings highlight the potential of these new compounds and their efficacy in targeting pancreatic and breast cancers that exhibit constitutive STAT3 signaling. PMID:20215512

Conformational selection in protein binding and function

PubMed Central

Weikl, Thomas R; Paul, Fabian

2014-01-01

Protein binding and function often involves conformational changes. Advanced nuclear magnetic resonance (NMR) experiments indicate that these conformational changes can occur in the absence of ligand molecules (or with bound ligands), and that the ligands may “select” protein conformations for binding (or unbinding). In this review, we argue that this conformational selection requires transition times for ligand binding and unbinding that are small compared to the dwell times of proteins in different conformations, which is plausible for small ligand molecules. Such a separation of timescales leads to a decoupling and temporal ordering of binding/unbinding events and conformational changes. We propose that conformational-selection and induced-change processes (such as induced fit) are two sides of the same coin, because the temporal ordering is reversed in binding and unbinding direction. Conformational-selection processes can be characterized by a conformational excitation that occurs prior to a binding or unbinding event, while induced-change processes exhibit a characteristic conformational relaxation that occurs after a binding or unbinding event. We discuss how the ordering of events can be determined from relaxation rates and effective on- and off-rates determined in mixing experiments, and from the conformational exchange rates measured in advanced NMR or single-molecule fluorescence resonance energy transfer experiments. For larger ligand molecules such as peptides, conformational changes and binding events can be intricately coupled and exhibit aspects of conformational-selection and induced-change processes in both binding and unbinding direction. PMID:25155241

Contributions of arginines-43 and -94 of human choriogonadotropin. beta. to receptor binding and activation as determined by oligonucleotide-based mutagenesis

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

Fang Chen; Puett, D.

1991-10-22

Members of the glycoprotein hormone family contain a common {alpha} subunit and a hormone-specific {beta} subunit. Human choriogonadotropin (hCG) {beta} is a 145 amino acid residue protein glycosylated at 6 positions (2 N-linked and 4 O-linked oligosaccharides). In an effort to elucidate receptor determinants on hCG{beta}, the authors have used site-directed mutagenesis to prepare and express several mutant cDNAs with replacements at arginines-43 and -94. Arg-43 is invariant in all known mammalian CG/lutropin {beta} amino acid sequences, and Arg-94 is conserved in 10 of the 12 sequences. Moreover, various studies involving synthetic peptides and enzymatic digestions of intact {beta} chainsmore » suggest that these residues may be important in hCG receptor binding. Point mutants were made in which these two arginines were replaced with the corresponding residues in human follitropin {beta}, Leu-43 and Asp-94. The wild-type and mutant {beta} chains were expressed in CHO cells containing a stably integrated gene for bovine {alpha}, and heterodimer formation occurred. These heterologous gonadotropins were active in assays using transformed Leydig cells, competitive binding with standard {sup 125}I-hCG, and cAMP and progesterone production, but the potency was considerably less than that associated with the hCG{beta} wild-type-containing gonadotropin. The double-mutant protein Arg-43 to Leu/Arg-94 to Asp also associated with bovine {alpha}, but the resultant heterodimer exhibited only low activity. Replacement but the Lys-43-containing {beta} chain appeared to exhibit a low degree of subunit association or reduced stability relative to the expressed hCG{beta} wild type. These results demonstrate that arginines-43 and -94 contribute to receptor binding through a positive charge.« less

Impaired helix 12 dynamics due to proline 892 substitutions in the androgen receptor are associated with complete androgen insensitivity.

PubMed

Elhaji, Youssef A; Stoica, Ileana; Dennis, Sheldon; Purisima, Enrico O; Lumbroso, Rose; Beitel, Lenore K; Trifiro, Mark A

2006-03-15

Structural studies of the ligand-binding domain (LBD) of several steroid receptors have revealed that the dynamic properties of the C-terminal helix 12 (H12) are the major determinant of the activation mode of these receptors. H12 exhibits high mobility and different conformations in the absence of ligand. Upon ligand binding, H12 is stabilized in a precise position to seal the ligand-binding pocket and finalize the assembly of the activation function (AF-2) domain. In this study, we investigated the role of the conserved proline 892 of the androgen receptor (AR) in directing the dynamic location and orientation of the AR-H12. We used a combined approach including kinetic and biochemical assays with molecular dynamic simulations to analyze two substitutions (P892A and P892L) identified in individuals with complete androgen insensitivity syndrome. Our analyses revealed distinct mechanisms by which these substitutions impair H12 function resulting in severely defective receptors. The AR-P892A receptor exhibited reduced ligand binding and transactivational potential because of an increased flexibility in H12. The AR-P892L substitution renders the receptor inactive due to a distorted, unstructured and misplaced H12. To confirm the mutants' inability to stabilize H12 in an active position, we have developed a novel in vivo assay to evaluate the accessibility of the H12-docking site on the AR-LBD surface. An extrinsic AR-H12 peptide was able to interact with wild-type and mutant LBDs in the absence of ligand. Ligand-induced proper positioning of the intrinsic H12 of wild-type AR prevented these interactions, whereas the misplacement of the mutants' H12 did not. Proline at this position may be critical for H12 dynamics not only in the AR, but also in other nuclear receptors where this proline is conserved.

Investigation of the interaction between berberine and nucleosomes in solution: Spectroscopic and equilibrium dialysis approach

NASA Astrophysics Data System (ADS)

Rabbani-Chadegani, Azra; Mollaei, Hossein; Sargolzaei, Javad

2017-02-01

Berberine is a natural plant alkaloid with high pharmacological potential. Although its interaction with free DNA has been the subject of several reports, to date there is no work concerning the effect of berberine on nucleoprotein structure of DNA, the nucleosomes. The present study focuses on the binding affinity of berberine to nucleosomes and histone H1 employing various spectroscopic techniques, fluorescence, circular dichroism, thermal denaturation as well as equilibrium dialysis. The results showed that the binding of berberine to nucleosomes is positive cooperative with Ka = 5.57 × 103 M- 1. Berberine quenched with the chromophores of protein moiety of nucleosomes and reduced fluorescence emission intensity at 335 nm with Ksv value of 0.135. Binding of berberine to nucleosomes decreased the absorbance at 210 and 260 nm, produced hypochromicity in thermal denaturation profiles and its affinity to nucleoprotein structure of nucleosomes was much higher than to free DNA. Berberine also exhibited high affinity to histone H1 in solution and the binding was positive cooperative with. Ka = 3.61 × 103 M- 1. Moreover berberine decreased fluorescence emission intensity of H1 by quenching with tyrosine residue in its globular core domain. The circular dichroism profiles demonstrated that the binding of drug induced secondary structural changes in both DNA stacking and histone H1. It is concluded that berberine is genotoxic drug, interacts with nucleosomes and in this process histone H1 is involved to exert its anticancer activity.

Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain

PubMed Central

Müller, Mischa R.; Saunders, Kenneth; Grace, Christopher; Jin, Macy; Piche-Nicholas, Nicole; Steven, John; O’Dwyer, Ronan; Wu, Leeying; Khetemenee, Lam; Vugmeyster, Yulia; Hickling, Timothy P.; Tchistiakova, Lioudmila; Olland, Stephane; Gill, Davinder; Jensen, Allan; Barelle, Caroline J.

2012-01-01

Advances in recombinant antibody technology and protein engineering have provided the opportunity to reduce antibodies to their smallest binding domain components and have concomitantly driven the requirement for devising strategies to increase serum half-life to optimise drug exposure, thereby increasing therapeutic efficacy. In this study, we adopted an immunization route to raise picomolar affinity shark immunoglobulin new antigen receptors (IgNARs) to target human serum albumin (HSA). From our model shark species, Squalus acanthias, a phage display library encompassing the variable binding domain of IgNAR (VNAR) was constructed, screened against target, and positive clones were characterized for affinity and specificity. N-terminal and C-terminal molecular fusions of our lead hit in complex with a naïve VNAR domain were expressed, purified and exhibited the retention of high affinity binding to HSA, but also cross-selectivity to mouse, rat and monkey serum albumin both in vitro and in vivo. Furthermore, the naïve VNAR had enhanced pharmacokinetic (PK) characteristics in both N- and C-terminal orientations and when tested as a three domain construct with naïve VNAR flanking the HSA binding domain at both the N and C termini. Molecules derived from this platform technology also demonstrated the potential for clinical utility by being available via the subcutaneous route of delivery. This study thus demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies. PMID:23676205

Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain.

PubMed

Müller, Mischa R; Saunders, Kenneth; Grace, Christopher; Jin, Macy; Piche-Nicholas, Nicole; Steven, John; O'Dwyer, Ronan; Wu, Leeying; Khetemenee, Lam; Vugmeyster, Yulia; Hickling, Timothy P; Tchistiakova, Lioudmila; Olland, Stephane; Gill, Davinder; Jensen, Allan; Barelle, Caroline J

2012-01-01

Advances in recombinant antibody technology and protein engineering have provided the opportunity to reduce antibodies to their smallest binding domain components and have concomitantly driven the requirement for devising strategies to increase serum half-life to optimise drug exposure, thereby increasing therapeutic efficacy. In this study, we adopted an immunization route to raise picomolar affinity shark immunoglobulin new antigen receptors (IgNARs) to target human serum albumin (HSA). From our model shark species, Squalus acanthias, a phage display library encompassing the variable binding domain of IgNAR (VNAR) was constructed, screened against target, and positive clones were characterized for affinity and specificity. N-terminal and C-terminal molecular fusions of our lead hit in complex with a naïve VNAR domain were expressed, purified and exhibited the retention of high affinity binding to HSA, but also cross-selectivity to mouse, rat and monkey serum albumin both in vitro and in vivo. Furthermore, the naïve VNAR had enhanced pharmacokinetic (PK) characteristics in both N- and C-terminal orientations and when tested as a three domain construct with naïve VNAR flanking the HSA binding domain at both the N and C termini. Molecules derived from this platform technology also demonstrated the potential for clinical utility by being available via the subcutaneous route of delivery. This study thus demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies.

Crystal Structure and Computational Characterization of the Lytic Polysaccharide Monooxygenase GH61D from the Basidiomycota Fungus Phanerochaete chrysosporium*

PubMed Central

Wu, Miao; Beckham, Gregg T.; Larsson, Anna M.; Ishida, Takuya; Kim, Seonah; Payne, Christina M.; Himmel, Michael E.; Crowley, Michael F.; Horn, Svein J.; Westereng, Bjørge; Igarashi, Kiyohiko; Samejima, Masahiro; Ståhlberg, Jerry; Eijsink, Vincent G. H.; Sandgren, Mats

2013-01-01

Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain. PMID:23525113

Determining the Binding Sites of β-Cyclodextrin and Peptides by Electron-Capture Dissociation High Resolution Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Qi, Yulin; Geib, Timon; Volmer, Dietrich A.

2015-07-01

Cyclodextrins (CDs) are a group of cyclic oligosaccharides, which readily form inclusion complexes with hydrophobic compounds to increase bioavailability, thus making CDs ideal drug excipients. Recent studies have also shown that CDs exhibit a wide range of protective effects, preventing proteins from aggregation, degradation, and folding. These effects strongly depend on the binding sites on the protein surface. CDs only exhibit weak interactions with amino acids, however; conventional analytical techniques therefore usually fail to reveal the exact location of the binding sites. Moreover, some studies even suggest that CD inclusion complexes are merely electrostatic adducts. Here, electron capture dissociation (ECD) was applied in this proof-of-concept study to examine the exact nature of the CD/peptide complexes, and CD binding sites were unambiguously located for the first time via Fourier-transform ion cyclotron resonance (FTICR) tandem mass spectrometry.

High-oleic canola oil consumption enriches LDL particle cholesteryl oleate content and reduces LDL proteoglycan binding in humans.

PubMed

Jones, Peter J H; MacKay, Dylan S; Senanayake, Vijitha K; Pu, Shuaihua; Jenkins, David J A; Connelly, Philip W; Lamarche, Benoît; Couture, Patrick; Kris-Etherton, Penny M; West, Sheila G; Liu, Xiaoran; Fleming, Jennifer A; Hantgan, Roy R; Rudel, Lawrence L

2015-02-01

Oleic acid consumption is considered cardio-protective according to studies conducted examining effects of the Mediterranean diet. However, animal models have shown that oleic acid consumption increases LDL particle cholesteryl oleate content which is associated with increased LDL-proteoglycan binding and atherosclerosis. The objective was to examine effects of varying oleic, linoleic and docosahexaenoic acid consumption on human LDL-proteoglycan binding in a non-random subset of the Canola Oil Multi-center Intervention Trial (COMIT) participants. COMIT employed a randomized, double-blind, five-period, cross-over trial design. Three of the treatment oil diets: 1) a blend of corn/safflower oil (25:75); 2) high oleic canola oil; and 3) DHA-enriched high oleic canola oil were selected for analysis of LDL-proteoglycan binding in 50 participants exhibiting good compliance. LDL particles were isolated from frozen plasma by gel filtration chromatography and LDL cholesteryl esters quantified by mass-spectrometry. LDL-proteoglycan binding was assessed using surface plasmon resonance. LDL particle cholesterol ester fatty acid composition was sensitive to the treatment fatty acid compositions, with the main fatty acids in the treatments increasing in the LDL cholesterol esters. The corn/safflower oil and high-oleic canola oil diets lowered LDL-proteoglycan binding relative to their baseline values (p = 0.0005 and p = 0.0012, respectively). At endpoint, high-oleic canola oil feeding resulted in lower LDL-proteoglycan binding than corn/safflower oil (p = 0.0243) and DHA-enriched high oleic canola oil (p = 0.0249), although high-oleic canola oil had the lowest binding at baseline (p = 0.0344). Our findings suggest that high-oleic canola oil consumption in humans increases cholesteryl oleate percentage in LDL, but in a manner not associated with a rise in LDL-proteoglycan binding. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Botulinum neurotoxin G binds synaptotagmin-II in a mode similar to that of serotype B: tyrosine 1186 and lysine 1191 cause its lower affinity.

PubMed

Willjes, Gesche; Mahrhold, Stefan; Strotmeier, Jasmin; Eichner, Timo; Rummel, Andreas; Binz, Thomas

2013-06-04

Botulinum neurotoxins (BoNTs) block neurotransmitter release by proteolyzing SNARE proteins in peripheral nerve terminals. Entry into neurons occurs subsequent to interaction with gangliosides and a synaptic vesicle protein. Isoforms I and II of synaptotagmin were shown to act as protein receptors for two of the seven BoNT serotypes, BoNT/B and BoNT/G, and for mosaic-type BoNT/DC. BoNT/B and BoNT/G exhibit a homologous binding site for synaptotagmin whose interacting part adopts helical structure upon binding to BoNT/B. Whereas the BoNT/B-synaptotagmin-II interaction has been elucidated in molecular detail, corresponding information about BoNT/G is lacking. Here we systematically mutated the synaptotagmin binding site in BoNT/G and performed a comparative binding analysis with mutants of the cell binding subunit of BoNT/B. The results suggest that synaptotagmin takes the same overall orientation in BoNT/B and BoNT/G governed by the strictly conserved central parts of the toxins' binding site. The surrounding nonconserved areas differently contribute to receptor binding. Reciprocal mutations Y1186W and L1191Y increased the level of binding of BoNT/G approximately to the level of BoNT/B affinity, suggesting a similar synaptotagmin-bound state. The effects of the mutations were confirmed by studying the activity of correspondingly mutated full-length BoNTs. On the basis of these data, molecular modeling experiments were employed to reveal an atomistic model of BoNT/G-synaptotagmin recognition. These data suggest a reduced length and/or a bend in the C-terminal part of the synaptotagmin helix that forms upon contact with BoNT/G as compared with BoNT/B and are in agreement with the data of the mutational analyses.

Differential Regulation of Angiogenesis using Degradable VEGF-Binding Microspheres

PubMed Central

Belair, David G.; Miller, Michael J.; Wang, Shoujian; Darjatmokon, Soesiawati R.; Binder, Bernard Y.K.; Sheibani, Nader; Murphy, William L.

2016-01-01

Vascular endothelial growth factor (VEGF) spatial and temporal activity must be tightly controlled during angiogenesis to form perfusable vasculature in a healing wound. The native extracellular matrix (ECM) regulates growth factor activity locally via sequestering, and researchers have used ECM-mimicking approaches to regulate the activity of VEGF in cell culture and in vivo. However, the impact of dynamic, affinity-mediated growth factor sequestering has not been explored in detail with biomaterials. Here, we sought to modulate VEGF activity dynamically over time using poly(ethylene glycol) microspheres containing VEGF-binding peptides (VBPs) and exhibiting varying degradation rates. The degradation rate of VBP microspheres conferred a differential ability to up- or down-regulate VEGF activity in culture with primary human endothelial cells. VBP microspheres with fast-degrading crosslinks reduced VEGF activity and signaling, while VBP microspheres with no inherent degradability sequestered and promoted VEGF activity in culture with endothelial cells. VBP microspheres with degradable crosslinks significantly reduced neovascularization in vivo, but neither non-degradable VBP microspheres nor bolus delivery of soluble VBP reduced neovascularization. The covalent incorporation of VBP to degradable microspheres was required to reduce neovascularization in a mouse model of choroidal neovascularization in vivo, which demonstrates a potential clinical application of degradable VBP microspheres to reduce pathological angiogenesis. The results herein highlight the ability to modulate the activity of a sequestered growth factor by changing the crosslinker identity within PEG hydrogel microspheres. The insights gained here may instruct the design and translation of affinity-based growth factor sequestering biomaterials for regenerative medicine applications. PMID:27061268

Negatively Cooperative Binding of High Density Lipoprotein to the HDL Receptor SR-BI†

PubMed Central

Nieland, Thomas J.F.; Xu, Shangzhe; Penman, Marsha; Krieger, Monty

2011-01-01

Scavenger receptor class B, type I (SR-BI) is a high-density lipoprotein (HDL) receptor, which also binds low density lipoprotein (LDL), and mediates the cellular selective uptake of cholesteryl esters from lipoproteins. SR-BI also is a co-receptor for hepatitis C virus and a signaling receptor that regulates cell metabolism. Many investigators have reported that lipoproteins bind to SR-BI via a single class of independent (not interacting), high affinity binding sites (one site model). We have re-investigated the ligand concentration dependence of 125I-HDL binding to SR-BI and SR-BI-mediated specific uptake of [3H]CE from [3H]CE-HDL using an expanded range of ligand concentrations (<1 µg protein/ml, lower than previously reported). Scatchard and non-linear least squares model fitting analyses of the binding and uptake data were both inconsistent with a single class of independent binding sites binding univalent lipoprotein ligands. The data are best fit by models in which SR-BI has either two independent classes of binding sites, or one class of sites exhibiting negative cooperativity due to either classic allostery or ensemble effects (‘ lattice model’). Similar results were observed for LDL. Application of the ‘infinite dilution’ dissociation rate method established that the binding of 125I-HDL to SR-BI at 4 °C exhibits negative cooperativity. The unexpected complexity of the interactions of lipoproteins with SR-BI should be taken into account when interpreting the results of experiments that explore the mechanism(s) by which SR-BI mediates ligand binding, lipid transport and cell signaling. PMID:21254782

orbital selective correlation reduce in collapse tetragonal phase of CaFe2(As0.935P0.065)2 and electronic structure reconstruction studied by angel resolved photoemission spectroscopy

NASA Astrophysics Data System (ADS)

Zeng, Lingkun

We performed an angle-resolved photoemission spectroscopy (ARPES) study of the CaFe2(As0.935P0.065)2 in the collapse tetragonal(CT) phase and uncollapse tetragonal(UCT) phase. We find in the CT phase the electronic correlation dramatically reduces respective to UCT phase. Meanwhile, the reduction of correlation in CT phase show an orbital selective effect: correlation in dxy reduces the most, and then dxz/yz, while the one in dz2-r2 almost keeps the same. In CT phase, almost all bands sink downwards to higher binding energy, leading to the hole like bands around Brillouin zone(BZ) center sink below EF compared with UCT phase. However, the electron pocket around Brillouin Zone(BZ) corner(M) in UCT phase, forms a hole pocket around BZ center(Z point) in CT phase. Moreover, the dxy exhibits larger movement down to higher binding energy, resulting in farther away from dyz/xz and closer to dxy.We propose the electron filling ,namely high spin state in UCT phase to low spin state in CT phase(due to competing between crystal structure field and Hund's coupling), other than the Fermi surface nesting might be responsible for the absent of magnetic ordering.

Perfusion alterations converge with patterns of pathological spread in transactive response DNA-binding protein 43 proteinopathies.

PubMed

Ferraro, Pilar M; Jester, Charles; Olm, Christopher A; Placek, Katerina; Agosta, Federica; Elman, Lauren; McCluskey, Leo; Irwin, David J; Detre, John A; Filippi, Massimo; Grossman, Murray; McMillan, Corey T

2018-04-17

Amyotrophic lateral sclerosis (ALS) and the behavioral variant of frontotemporal dementia (bvFTD) commonly share the presence of transactive response DNA-binding protein 43 (TDP-43) inclusions. Structural magnetic resonance imaging studies demonstrated evidence for TDP-43 pathology spread, but while structural imaging usually reveals overt neuronal loss, perfusion imaging may detect more subtle neural activity alterations. We evaluated perfusion as an early marker for incipient pathology-associated brain alterations in TDP-43 proteinopathies. Cortical thickness (CT) and perfusion measurements were obtained in ALS (N = 18), pathologically and/or genetically confirmed bvFTD-TDP (N = 12), and healthy controls (N = 33). bvFTD showed reduced frontotemporal CT, hypoperfusion encompassing orbitofrontal and temporal cortices, and hyperperfusion in motor and occipital regions. ALS did not show reduced CT, but exhibited hypoperfusion in motor and temporal regions, and hyperperfusion in frontal and occipital cortices. Frontotemporal hypoperfusion and reduced CT correlated with cognitive and behavioral impairments as investigated using Mini-Mental State Examination and Philadelphia Brief Assessment of Cognition in bvFTD, and hypoperfusion in motor regions correlated with motor disability as measured by the ALS Functional Rating Scale-Revised in ALS. Hypoperfusion marked early pathologically involved regions, while hyperperfusion characterized regions of late pathological involvement. Distinct perfusion patterns may provide early markers of pathology distribution in TDP-43 proteinopathies. Copyright © 2018 Elsevier Inc. All rights reserved.

Constitutive activity of the δ-opioid receptor expressed in C6 glioma cells: identification of non-peptide δ-inverse agonists

PubMed Central

Neilan, Claire L; Akil, Huda; Woods, James H; Traynor, John R

1999-01-01

G-protein coupled receptors can exhibit constitutive activity resulting in the formation of active ternary complexes in the absence of an agonist. In this study we have investigated constitutive activity in C6 glioma cells expressing either the cloned δ-(OP1) receptor (C6δ), or the cloned μ-(OP3) opioid receptor (C6μ).Constitutive activity was measured in the absence of Na+ ions to provide an increased signal. The degree of constitutive activity was defined as the level of [35S]-GTPγS binding that could be inhibited by pre-treatment with pertussis toxin (PTX). In C6δ cells the level of basal [35S]-GTPγS binding was reduced by 51.9±6.1 fmols mg−1 protein, whereas in C6μ and C6 wild-type cells treatment with PTX reduced basal [35S]-GTPγS binding by only 10.0±3.5 and 8.6±3.1 fmols mg−1 protein respectively.The δ-antagonists N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864), 7-benzylidenenaltrexone (BNTX) and naltriben (NTB), in addition to clocinnamox (C-CAM), acted as δ-opioid receptor inverse agonists. Naloxone, buprenorphine, and naltrindole were neutral antagonists. Furthermore, naltrindole blocked the reduction in [35S]-GTPγS binding caused by the inverse agonists. The inverse agonists did not inhibit basal [35S]-GTPγS binding in C6μ or C6 wild-type cell membranes.Competition binding assays in C6δ cell membranes revealed a leftward shift in the displacement curve of [3H]-naltrindole by ICI 174,864 and C-CAM in the presence of NaCl and the GTP analogue, GppNHp. There was no change in the displacement curve for BNTX or NTB under these conditions.These data confirm the presence of constitutive activity associated with the δ-opioid receptor and identify three novel, non-peptide, δ-opioid inverse agonists. PMID:10516632

Fetal programming: prenatal testosterone excess leads to fetal growth retardation and postnatal catch-up growth in sheep.

PubMed

Manikkam, Mohan; Crespi, Erica J; Doop, Douglas D; Herkimer, Carol; Lee, James S; Yu, Sunkyung; Brown, Morton B; Foster, Douglas L; Padmanabhan, Vasantha

2004-02-01

Alterations in the maternal endocrine, nutritional, and metabolic environment disrupt the developmental trajectory of the fetus, leading to adult diseases. Female offspring of rats, subhuman primates, and sheep treated prenatally with testosterone (T) develop reproductive/metabolic defects during adult life similar to those that occur after intrauterine growth retardation. In the present study we determined whether prenatal T treatment produces growth-retarded offspring. Cottonseed oil or T propionate (100 mg, im) was administered twice weekly to pregnant sheep between 30-90 d gestation (term = 147 d; cottonseed oil, n = 16; prenatal T, n = 32). Newborn weight and body dimensions were measured the day after birth, and postnatal weight gain was monitored for 4 months in all females and in a subset of males. Consistent with its action, prenatal T treatment produced females and males with greater anogenital distances relative to controls. Prenatal T treatment reduced body weights and heights of newborns from both sexes and chest circumference of females. Prenatally T-treated females, but not males, exhibited catch-up growth during 2-4 months of postnatal life. Plasma IGF-binding protein-1 and IGF-binding protein-2, but not IGF-I, levels of prenatally T-treated females were elevated in the first month of life, a period when the prenatally T-treated females were not exhibiting catch-up growth. This is suggestive of reduced IGF availability and potential contribution to growth retardation. These findings support the concept that fetal growth retardation and postnatal catch-up growth, early markers of future adult diseases, can also be programmed by prenatal exposure to excess sex steroids.

Dysregulation of hepatic fatty acid metabolism in chronic kidney disease.

PubMed

Jin, Kyubok; Norris, Keith; Vaziri, Nosratola D

2013-02-01

Chronic kidney disease (CKD) results in hypertriglyceridemia which is largely due to impaired clearance of triglyceride-rich lipoproteins occasioned by downregulation of lipoprotein lipase and very low-density lipoprotein (LDL) receptor in the skeletal muscle and adipose tissue and of hepatic lipase and LDL receptor-related protein in the liver. However, data on the effect of CKD on fatty acid metabolism in the liver is limited and was investigated here. Male Sprague-Dawley rats were randomized to undergo 5/6 nephrectomy (CRF) or sham operation (control) and observed for 12 weeks. The animals were then euthanized and their liver tissue tested for nuclear translocation (activation) of carbohydrate-responsive element binding protein (ChREBP) and sterol-responsive element binding protein-1 (SREBP-1) which independently regulate the expression of key enzyme in fatty acid synthesis, i.e. fatty acid synthase (FAS) and acyl-CoA carboxylase (ACC) as well as nuclear Peroxisome proliferator-activated receptor alpha (PPARα) which regulates the expression of enzymes involved in fatty acid oxidation and transport, i.e. L-FABP and CPT1A. In addition, the expression of ATP synthase α, ATP synthase β, glycogen synthase and diglyceride acyltransferase 1 (DGAT1) and DGAT2 were determined. Compared with controls, the CKD rats exhibited hypertriglyceridemia, elevated plasma and liver tissue free fatty acids, increased nuclear ChREBP and reduced nuclear SREBP-1 and PPARα, upregulation of ACC and FAS and downregulation of L-FABP, CPT1A, ATP synthase α, glycogen synthase and DGAT in the liver tissue. Liver in animals with advanced CKD exhibits ChREBP-mediated upregulation of enzymes involved in fatty acid synthesis, downregulation of PPARα-regulated fatty acid oxidation system and reduction of DGAT resulting in reduced fatty acid incorporation in triglyceride.

Rate and extent of protein localization is controlled by peptide-binding domain association kinetics and morphology.

PubMed

Mills, Evan; Truong, Kevin

2009-06-01

Protein localization is an important regulatory mechanism in many cell signaling pathways such as cytoskeletal organization and genetic regulation. The specific mechanism of protein localization determines the kinetics and morphological constraints of protein translocation, and thus affects the rate and extent of localization. To investigate the affect of localization kinetics and morphology on protein localization, we designed a protein localization system based on Ca(2+)-calmodulin and Src homology 3 domain binding peptides that can translocate between specific localizations in response to a Ca(2+) signal. We used a stochastic biomolecular simulator to predict that such a protein localization system will exhibit slower and less complete translocations when the association kinetics of a binding domain and peptide are reduced. As well, we predicted that increasing the diffusion resistance by manipulating the morphology of the system would similarly impair translocation speed and completeness. We then constructed a network of synthetic fusion proteins and showed that these predictions could be qualitatively confirmed in vitro. This work provides a basis for explaining the different characteristics (rate and extent) of protein transport and localization in cells as a consequence of the kinetics and morphology of the transport mechanism.

The Abl-related gene (Arg) requires its F-actin-microtubule cross-linking activity to regulate lamellipodial dynamics during fibroblast adhesion.

PubMed

Miller, Ann L; Wang, Yinxiang; Mooseker, Mark S; Koleske, Anthony J

2004-05-10

Microtubules (MTs) help establish and maintain cell polarity by promoting actin-dependent membrane protrusion at the leading edge of the cell, but the molecular mechanisms that mediate cross-talk between actin and MTs during this process are unclear. We demonstrate that the Abl-related gene (Arg) nonreceptor tyrosine kinase is required for dynamic lamellipodial protrusions after adhesion to fibronectin. arg-/- fibroblasts exhibit reduced lamellipodial dynamics as compared with wild-type fibroblasts, and this defect can be rescued by reexpression of an Arg-yellow fluorescent protein fusion. We show that Arg can bind MTs with high affinity and cross-link filamentous actin (F-actin) bundles and MTs in vitro. MTs concentrate and insert into Arg-induced F-actin-rich cell protrusions. Arg requires both its F-actin-binding domains and its MT-binding domain to rescue the defects in lamellipodial dynamics of arg-/- fibroblasts. These findings demonstrate that Arg can mediate physical contact between F-actin and MTs at the cell periphery and that this cross-linking activity is required for Arg to regulate lamellipodial dynamics in fibroblasts. Copyright the Rockefeller University Press

The cell-penetrating peptide domain from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) has anti-inflammatory activity in vitro and in vivo.

PubMed

Lee, Jue-Yeon; Seo, Yoo-Na; Park, Hyun-Jung; Park, Yoon-Jeong; Chung, Chong-Pyoung

2012-03-23

A heparin-binding peptide (HBP) sequence from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified and was shown to exhibit cell penetration activity. This cell penetration induced an anti-inflammatory reaction in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. HBP penetrated the cell membrane during the 10 min treatment and reduced the LPS-induced production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cytokines (TNF-α and IL-6) in a concentration-dependent manner. Additionally, HBP inhibited the LPS-induced upregulation of cytokines, including TNF-α and IL-6, and decreased the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. HBP inhibited NF-κB-dependent inflammatory responses by directly blocking the phosphorylation and degradation of IκBα and by subsequently inhibiting the nuclear translocation of the p65 subunit of NF-κB. Taken together, this novel HBP may be potentially useful candidate for anti-inflammatory treatments and can be combined with other drugs of interest to transport attached molecules into cells. Copyright © 2012 Elsevier Inc. All rights reserved.

A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin

NASA Technical Reports Server (NTRS)

Lu, C.; Fedoroff, N.

2000-01-01

Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant's responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N-(1-naph-thyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1, encodes a 419-amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein-protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level.

Fomiroid A, a Novel Compound from the Mushroom Fomitopsis nigra, Inhibits NPC1L1-Mediated Cholesterol Uptake via a Mode of Action Distinct from That of Ezetimibe

PubMed Central

Chiba, Tomohiro; Sakurada, Tsuyoshi; Watanabe, Rie; Yamaguchi, Kohji; Kimura, Yasuhisa; Kioka, Noriyuki; Kawagishi, Hirokazu; Matsuo, Michinori; Ueda, Kazumitsu

2014-01-01

Hypercholesterolemia is one of the key risk factors for coronary heart disease, a major cause of death in developed countries. Suppression of NPC1L1-mediated dietary and biliary cholesterol absorption is predicted to be one of the most effective ways to reduce the risk of hypercholesterolemia. In a screen for natural products that inhibit ezetimibe glucuronide binding to NPC1L1, we found a novel compound, fomiroid A, in extracts of the mushroom Fomitopsis nigra. Fomiroid A is a lanosterone derivative with molecular formula C30H48O3. Fomiroid A inhibited ezetimibe glucuronide binding to NPC1L1, and dose-dependently prevented NPC1L1-mediated cholesterol uptake and formation of esterified cholesterol in NPC1L1-expressing Caco2 cells. Fomiroid A exhibited a pharmacological chaperone activity that corrected trafficking defects of the L1072T/L1168I mutant of NPC1L1. Because ezetimibe does not have such an activity, the binding site and mode of action of fomiroid A are likely to be distinct from those of ezetimibe. PMID:25551765

The “gating” residues Ile199 and Tyr326 in human monoamine oxidase B function in substrate and inhibitor recognition

PubMed Central

Milczek, Erika M.; Binda, Claudia; Rovida, Stefano; Mattevi, Andrea; Edmondson, Dale E.

2011-01-01

Summary The major structural difference between human monoamine oxidases A (MAO A) and B (MAO B) is that MAO A has a monopartite substrate cavity of ~550 Å3 volume and MAO B contains a dipartite cavity structure with volumes of ~290 Å3 (entrance cavity) and ~400 Å3 (substrate cavity). Ile199 and Tyr326 side chains separate these two cavities in MAO B. To probe the function of these gating residues, Ile199Ala and Ile199Ala Tyr326Ala mutant forms of MAO B were investigated. Structural data on the Ile199Ala MAO B mutant show no alterations in active site geometries compared to WT enzyme while the Ile199Ala-Tyr326Ala MAO B mutant exhibits alterations in residues 100–103 which are part of the loop gating the entrance to the active site. Both mutant enzymes exhibit catalytic properties with increased amine KM but unaltered kcat values. The altered KM values on mutation are attributed to the influence of the cavity structure in the binding and subsequent deprotonation of the amine substrate. Both mutant enzymes exhibit weaker binding affinities relative to WT enzyme for small reversible inhibitors. Ile199Ala MAO B exhibits an increase in binding affinity for reversible MAO B specific inhibitors which bridge both cavities. The Ile199Ala-Tyr326Ala double mutant exhibits inhibitor binding properties more similar to those of MAO A than to MAO B. These results demonstrate the bipartite cavity structure in MAO B plays an important role in substrate and inhibitor recognition to distinguish its specificities from those of MAO A and provides insights into specific reversible inhibitor design for these membrane-bound enzymes. PMID:21978362

Characterization of Transcription Factor Gene OsDRAP1 Conferring Drought Tolerance in Rice

PubMed Central

Huang, Liyu; Wang, Yinxiao; Wang, Wensheng; Zhao, Xiuqin; Qin, Qiao; Sun, Fan; Hu, Fengyi; Zhao, Yan; Li, Zichao; Fu, Binying; Li, Zhikang

2018-01-01

HIGHLIGHTS Overexpressing and RNA interfering OsDRAP1 transgenic rice plants exhibited significantly improved and reduced drought tolerance, but accompanied with negative effects on development and yield. The dehydration responsive element binding (DREBs) genes are important transcription factors which play a crucial role in plant abiotic stress tolerances. In this study, we functionally characterized a DREB2-like gene, OsDRAP1 conferring drought tolerance (DT) in rice. OsDRAP1, containing many cis-elements in its promoter region, was expressed in all organs (mainly expressed in vascular tissues) of rice, and induced by a variety of environmental stresses and plant hormones. Overexpressing OsDRAP1 transgenic plants exhibited significantly improved DT; while OsDRAP1 RNA interfering plants exhibited significantly reduced DT which also accompanied with significant negative effects on development and yield. Overexpression of OsDRAP1 has a positive impact on maintaining water balance, redox homeostasis and vascular development in transgenic rice plants under drought stress. OsDRAP1 interacted with many genes/proteins and could activate many downstream DT related genes, including important transcription factors such as OsCBSX3 to response drought stress, indicating the OsDRAP1-mediated pathways for DT involve complex genes networks. All these results provide a basis for further complete understanding of the OsDRAP1 mediated gene networks and their related phenotypic effects. PMID:29449862

Cell wall reactivity of acidophilic and alkaliphilic bacteria determined by potentiometric titrations and Cd adsorption experiments.

PubMed

Kenney, Janice P L; Fein, Jeremy B

2011-05-15

In this study, we used potentiometric titrations and Cd adsorption experiments to determine the binding capacities of two acidophilic (A. cryptum and A. acidophilum) and two alkaliphilic (B. pseudofirmus and B. circulans) bacterial species in order to determine if any consistent trends could be observed relating bacterial growth environment to proton and Cd binding properties and to compare those binding behaviors to those of neutrophilic bacteria. All of the bacterial species studied exhibited significant proton buffering over the pH range in this study, with the alkaliphiles exhibiting significantly higher acidity constants than the acidophiles as well as the neutrophilic bacterial consortia. The calculated average site concentrations for each of the bacteria in this study are within 2σ experimental error of each other, with the exception of A. cryptum, which has a significantly higher Site 2 concentration than the other species. Despite differing acidity constants between the acidophiles and alkaliphiles, all bacteria except A. cryptum exhibited remarkably similar Cd adsorption behavior to each other, and the observed extent of adsorption was also similar to that predicted from a generalized model derived using neutrophilic bacterial consortia. This study demonstrates that bacteria that grow under extreme conditions exhibit similar proton and metal adsorption behavior to that of previously studied neutrophilic species and that a single set of proton and metal binding constants can be used to model the behavior of bacterial adsorption under a wide range of environmental conditions.

A novel dissociative steroid VBP15 reduces MUC5AC gene expression in airway epithelial cells but lacks the GRE mediated transcriptional properties of dexamethasone.

PubMed

Garvin, Lindsay M; Chen, Yajun; Damsker, Jesse M; Rose, Mary C

2016-06-01

Overproduction of secretory mucins contributes to morbidity/mortality in inflammatory lung diseases. Inflammatory mediators directly increase expression of mucin genes, but few drugs have been shown to directly repress mucin gene expression. IL-1β upregulates the MUC5AC mucin gene in part via the transcription factors NFκB while the glucocorticoid Dexamethasone (Dex) transcriptionally represses MUC5AC expression by Dex-activated GR binding to two GRE cis-sites in the MUC5AC promoter in lung epithelial cells. VBP compounds (ReveraGen BioPharma) maintain anti-inflammatory activity through inhibition of NFκB but exhibit reduced GRE-mediated transcriptional properties associated with adverse side-effects and thus have potential to minimize harmful side effects of long-term steroid therapy in inflammatory lung diseases. We investigated VBP15 efficacy as an anti-mucin agent in two types of airway epithelial cells and analyzed the transcription factor activity and promoter binding associated with VBP15-induced MUC5AC repression. VBP15 reduced MUC5AC mRNA abundance in a dose- and time-dependent manner similar to Dex in the presence or absence of IL-1β in A549 and differentiated human bronchial epithelial cells. Repression was abrogated in the presence of RU486, demonstrating a requirement for GR in the VBP15-induced repression of MUC5AC. Inhibition of NFκB activity resulted in reduced baseline expression of MUC5AC indicating that constitutive activity maintains MUC5AC production. Chromatin immunoprecipitation analysis demonstrated lack of GR and of p65 (NFκB) binding to composite GRE domains in the MUC5AC promoter following VBP15 exposure of cells, in contrast to Dex. These data demonstrate that VBP15 is a novel anti-mucin agent that mediates the reduction of MUC5AC gene expression differently than the classical glucocorticoid, Dex. Copyright © 2016 Elsevier Ltd. All rights reserved.

Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity.

PubMed

Srinivasan, Sundaramoorthy; Fernández-Sampedro, Miguel A; Morillo, Margarita; Ramon, Eva; Jiménez-Rosés, Mireia; Cordomí, Arnau; Garriga, Pere

2018-03-27

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Novel interactions of CAPS (Ca2+-dependent activator protein for secretion) with the three neuronal SNARE proteins required for vesicle fusion.

PubMed

Daily, Neil J; Boswell, Kristin L; James, Declan J; Martin, Thomas F J

2010-11-12

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca(2+)-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion*

PubMed Central

Daily, Neil J.; Boswell, Kristin L.; James, Declan J.; Martin, Thomas F. J.

2010-01-01

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca2+-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis. PMID:20826818

A systematic review of the evidence of reduced allergenicity and clinical benefit of food hydrolysates in dogs with cutaneous adverse food reactions.

PubMed

Olivry, Thierry; Bizikova, Petra

2010-02-01

Several hydrolysate-based diets have been commercialized for helping diagnose or treat dogs with cutaneous adverse food reactions (CAFR). This systematic review was performed to examine the evidence in favour of reduced immunological and clinical allergenicity of hydrolysates in dogs with CAFR. Citation databases, meeting abstracts and article bibliographies were scanned for relevant citations, and companies were contacted to provide unpublished reports. Eleven studies relevant to this study were identified. Some evidence of reduced serum IgE binding to a soy hydrolysate (1 study) and decreased intradermal test reactivity to hydrolysed proteins (three studies) was found. In four reports, the feeding of dogs suspected of having CAFR with hydrolysate-based diets reduced or eliminated clinical signs in a variable proportion of subjects. The percentage of dogs with CAFR that still reacted to these hydrolysate-based diets could not be assessed, however. Importantly, up to 50% of dogs with CAFR enrolled in three controlled studies exhibited increases in clinical signs after ingesting partial hydrolysates derived from foods to which they were hypersensitive. In conclusion, the limited number of studies undertaken point to reduced - but not eliminated - immunological and clinical allergenicity of hydrolysate-based commercial diets. A variable proportion of dogs with CAFR will exhibit a worsening of clinical signs when fed partial hydrolysates. Clinicians must weigh the clinical benefit of these diets versus their high cost and low risk of reduced appetence or gastrointestinal sign development. At this time, hydrolysate-containing diets are probably best used in dogs suspected not to be hypersensitive to their individual components.

Enhanced stability of monomer fold correlates with extreme drug resistance of HIV-1 protease.

PubMed

Louis, John M; Tözsér, József; Roche, Julien; Matúz, Krisztina; Aniana, Annie; Sayer, Jane M

2013-10-29

During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20(T26A) and dimeric PR20 exhibit Tm values 6-7.5 °C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PR(T26A) by ~8 °C, similar to PR20(T26A). However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR(D25N) in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ΔTm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.

Allosteric communication between the pyridoxal 5'-phosphate (PLP) and heme sites in the H2S generator human cystathionine β-synthase.

PubMed

Yadav, Pramod Kumar; Xie, Peter; Banerjee, Ruma

2012-11-02

Human cystathionine β-synthase (CBS) is a unique pyridoxal 5'-phosphate (PLP)-dependent enzyme that has a regulatory heme cofactor. Previous studies have demonstrated the importance of Arg-266, a residue at the heme pocket end of α-helix 8, for communication between the heme and PLP sites. In this study, we have examined the role of the conserved Thr-257 and Thr-260 residues, located at the other end of α-helix 8 on the heme electronic environment and on activity. The mutations at the two positions destabilize PLP binding, leading to lower PLP content and ~2- to ~500-fold lower activity compared with the wild-type enzyme. Activity is unresponsive to PLP supplementation, consistent with the pyridoxine-nonresponsive phenotype of the T257M mutation in a homocystinuric patient. The H(2)S-producing activities, also impacted by the mutations, show a different pattern of inhibition compared with the canonical transsulfuration reaction. Interestingly, the mutants exhibit contrasting sensitivities to the allosteric effector, S-adenosylmethionine (AdoMet); whereas T257M and T257I are inhibited, the other mutants are hyperactivated by AdoMet. All mutants showed an increased propensity of the ferrous heme to form an inactive species with a 424 nm Soret peak and exhibited significantly reduced enzyme activity in the ferrous and ferrous-CO states. Our results provide the first evidence for bidirectional transmission of information between the cofactor binding sites, suggest the additional involvement of this region in allosteric communication with the regulatory AdoMet-binding domain, and reveal the potential for independent modulation of the canonical transsulfuration versus H(2)S-generating reactions catalyzed by CBS.

Ebola Virus Binding to Tim-1 on T Lymphocytes Induces a Cytokine Storm

PubMed Central

Younan, Patrick; Iampietro, Mathieu; Nishida, Andrew; Ramanathan, Palaniappan; Santos, Rodrigo I.; Dutta, Mukta; Lubaki, Ndongala Michel; Koup, Richard A.; Katze, Michael G.

2017-01-01

ABSTRACT Ebola virus (EBOV) disease (EVD) results from an exacerbated immunological response that is highlighted by a burst in the production of inflammatory mediators known as a “cytokine storm.” Previous reports have suggested that nonspecific activation of T lymphocytes may play a central role in this phenomenon. T-cell immunoglobulin and mucin domain-containing protein 1 (Tim-1) has recently been shown to interact with virion-associated phosphatidylserine to promote infection. Here, we demonstrate the central role of Tim-1 in EBOV pathogenesis, as Tim-1−/− mice exhibited increased survival rates and reduced disease severity; surprisingly, only a limited decrease in viremia was detected. Tim-1−/− mice exhibited a modified inflammatory response as evidenced by changes in serum cytokines and activation of T helper subsets. A series of in vitro assays based on the Tim-1 expression profile on T cells demonstrated that despite the apparent absence of detectable viral replication in T lymphocytes, EBOV directly binds to isolated T lymphocytes in a phosphatidylserine–Tim-1-dependent manner. Exposure to EBOV resulted in the rapid development of a CD4Hi CD3Low population, non-antigen-specific activation, and cytokine production. Transcriptome and Western blot analysis of EBOV-stimulated CD4+ T cells confirmed the induction of the Tim-1 signaling pathway. Furthermore, comparative analysis of transcriptome data and cytokine/chemokine analysis of supernatants highlight the similarities associated with EBOV-stimulated T cells and the onset of a cytokine storm. Flow cytometry revealed virtually exclusive binding and activation of central memory CD4+ T cells. These findings provide evidence for the role of Tim-1 in the induction of a cytokine storm phenomenon and the pathogenesis of EVD. PMID:28951472

Adsorption-Coupled Diffusion of Gold Nanoclusters within a Large-Pore Protein Crystal Scaffold.

PubMed

Hartje, Luke F; Munsky, Brian; Ni, Thomas W; Ackerson, Christopher J; Snow, Christopher D

2017-08-17

Large-pore protein crystals (LPCs) are ordered biologically derived nanoporous materials exhibiting pore diameters greater than 8 nm. These substantial pores distinguish LPCs from typical nanoporous scaffolds, enabling engineered LPC materials to readily uptake, immobilize, and release macromolecular guests. In this study, macromolecular transport within an LPC environment was experimentally and computationally investigated by studying adsorption-coupled diffusion of Au 25 (glutathione) 18 nanoclusters within a cross-linked LPC scaffold via time-lapse confocal microscopy, bulk equilibrium adsorption, and hindered diffusion simulation. Equilibrium adsorption data is congruent with a Langmuir adsorption model, exhibiting strong binding behavior between nanoclusters and the scaffold. The standard Gibbs free energy of binding is equivalent to -37.2 kJ/mol, and the maximum binding capacity of 1.25 × 10 3 mg/g corresponds to approximately 29 nanoclusters per LPC unit cell. The hindered diffusion model showed good agreement with experimental data, revealing a pore diffusion coefficient of 3.7 × 10 -7 cm 2 /s under low nanocluster concentration. Furthermore, the model was sufficient to determine adsorption and desorption kinetic values for k a and k d equal to 13 cm 3 /mol·s and 1.7 × 10 -7 s -1 , respectively. At higher nanocluster concentrations, the simulated pore diffusion coefficient could be reduced by 3 orders of magnitude to 3.4 × 10 -10 cm 2 /s due to the effects of pore occlusion. This study demonstrates a strategy to analyze adsorption-coupled diffusion data to better understand complex transport of fluorescent macromolecules into LPCs. This approach fits the observable fluorescence data to the key molecular details and will benefit downstream efforts to engineer LPC-based nanoporous materials.

The importance of being kinked: role of Pro residues in the selectivity of the helical antimicrobial peptide P5.

PubMed

Bobone, Sara; Bocchinfuso, Gianfranco; Park, Yoonkyung; Palleschi, Antonio; Hahm, Kyung-Soo; Stella, Lorenzo

2013-12-01

Antimicrobial peptides (AMPs) are promising compounds for developing new antibiotic drugs against drug-resistant bacteria. Many of them kill bacteria by perturbing their membranes but exhibit no significant toxicity towards eukaryotic cells. The identification of the features responsible for this selectivity is essential for their pharmacological development. AMPs exhibit few conserved features, but a statistical analysis of an AMP sequence database indicated that many α-helical AMPs surprisingly have a helix-breaking Pro residue in the middle of their sequence. To discriminate among the different possible hypotheses for the functional role of this feature, we designed an analogue of the antimicrobial peptide P5, in which the central Pro was deleted (analogue P5Del). Pro removal resulted in a dramatic increase of toxicity. This was explained by the observation that P5Del binds both charged and neutral membranes, whereas P5 has no appreciable affinity towards neutral bilayers. CD and simulative data provided a rationalization of this behavior. In solution P5, due to the presence of Pro, attains compact conformations, in which its apolar residues are partially shielded from the solvent, whereas P5Del is more helical. These structural differences reduce the hydrophobic driving force for association of P5 to neutral membranes, whereas its binding to anionic bilayers can still take place because of electrostatic attraction. After membrane binding, the Pro residue does not preclude the attainment of a membrane-active amphiphilic helical conformation. These findings shed light on the role of Pro residues in the selectivity of AMPs and provide hints for the design of new, highly selective compounds. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

V-1 regulates capping protein activity in vivo

PubMed Central

Jung, Goeh; Wu, Xufeng S.; Piszczek, Grzegorz; Chen, Bi-Chang; Betzig, Eric; Hammer, John A.

2016-01-01

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1–null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1’s ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their “actin phenotype.” PMID:27791032

V-1 regulates capping protein activity in vivo.

PubMed

Jung, Goeh; Alexander, Christopher J; Wu, Xufeng S; Piszczek, Grzegorz; Chen, Bi-Chang; Betzig, Eric; Hammer, John A

2016-10-25

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."

Cytochrome P450 binding studies of novel tacrine derivatives: Predicting the risk of hepatotoxicity.

PubMed

McEneny-King, Alanna; Osman, Wesseem; Edginton, Andrea N; Rao, Praveen P N

2017-06-01

The 1,2,3,4-tetrahydroacridine derivative tacrine was the first drug approved to treat Alzheimer's disease (AD). It is known to act as a potent cholinesterase inhibitor. However, tacrine was removed from the market due to its hepatotoxicity concerns as it undergoes metabolism to toxic quinonemethide species through the cytochrome P450 enzyme CYP1A2. Despite these challenges, tacrine serves as a useful template in the development of novel multi-targeting anti-AD agents. In this regard, we sought to evaluate the risk of hepatotoxicity in a series of C9 substituted tacrine derivatives that exhibit cholinesterase inhibition properties. The hepatotoxic potential of tacrine derivatives was evaluated using recombinant cytochrome (CYP) P450 CYP1A2 and CYP3A4 enzymes. Molecular docking studies were conducted to predict their binding modes and potential risk of forming hepatotoxic metabolites. Tacrine derivatives compound 1 (N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) and 2 (6-chloro-N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) which possess a C9 3,4-dimethoxybenzylamino substituent exhibited weak binding to CYP1A2 enzyme (1, IC 50 =33.0µM; 2, IC 50 =8.5µM) compared to tacrine (CYP1A2 IC 50 =1.5µM). Modeling studies show that the presence of a bulky 3,4-dimethoxybenzylamino C9 substituent prevents the orientation of the 1,2,3,4-tetrahydroacridine ring close to the heme-iron center of CYP1A2 thereby reducing the risk of forming hepatotoxic species. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Gedunin and Azadiradione: Human Pancreatic Alpha-Amylase Inhibiting Limonoids from Neem (Azadirachta indica) as Anti-Diabetic Agents.

PubMed

Ponnusamy, Sudha; Haldar, Saikat; Mulani, Fayaj; Zinjarde, Smita; Thulasiram, Hirekodathakallu; RaviKumar, Ameeta

2015-01-01

Human pancreatic α-amylase (HPA) inhibitors offer an effective strategy to lower postprandial hyperglycemia via control of starch breakdown. Limonoids from Azadirachta indica known for their therapeutic potential were screened for pancreatic α-amylase inhibition, a known anti-diabetic target. Studies were carried out to reveal their mode of action so as to justify their hypoglycemic potential. Of the nine limonoids isolated/semi-synthesized from A.indica and screened for α-amylase inhibition, azadiradione and exhibited potential inhibition with an IC50 value of 74.17 and 68.38 μM, respectively against HPA under in vitro conditions. Further screening on AR42J α-amylase secretory cell line for cytotoxicity and bioactivity revealed that azadiradione and gedunin exhibited cytotoxicity with IC50 of 11.1 and 13.4μM. Maximal secreted α-amylase inhibition of 41.8% and 53.4% was seen at 3.5 and 3.3μM, respectively. Michaelis-Menten kinetics suggested a mixed mode of inhibition with maltopentaose (Ki 42.2, 18.6 μM) and starch (Ki' 75.8, 37.4 μM) as substrate with a stiochiometry of 1:1 for both azadiradione and gedunin, respectively. The molecular docking simulation indicated plausible π-alkyl and alkyl-alkyl interactions between the aromatic amino acids and inhibitors. Fluorescence and CD confirmed the involvement of tryptophan and tyrosine in ligand binding to HPA. Thermodynamic parameters suggested that binding is enthalpically and entropically driven with ΔG° of -21.25 kJ mol-1 and -21.16 kJ mol-1 for azadiradione and gedunin, respectively. Thus, the limonoids azadiradione and gedunin could bind and inactivate HPA (anti-diabetic target) and may prove to be lead drug candidates to reduce/control post-prandial hyperglycemia.

Molecular Docking Studies of Catechin and Its Derivatives as Anti-bacterial Inhibitor for Glucosamine-6-Phosphate Synthase

NASA Astrophysics Data System (ADS)

Fikrika, H.; Ambarsari, L.; Sumaryada, T.

2016-01-01

Molecular docking simulation of catechin and its derivatives on Glucosamine-6- Phosphate Synthase (GlmS) has been performed in this research. GlmS inhibition by a particular ligand will suppress the production of bacterial cell wall and significantly reduce the population of invading bacteria. In this study, catechin derivatives i.e epicatechin, galloatechin and epigalloatechin were found to have stronger binding affinities as compared to natural ligand of GlmS, Fructose-6-Phosphate (F6P). Those three ligands were docked on the same pocket in GlmS target as F6P, with 70% binding sites similarity. Based on the docking results, gallocatechin turns out to be the most potent ligand for anti-bacterial agent with ΔG= -8.00 kcal/mol. The docking between GlmS and catechin derivatives are characterized by a constant present of a strong hydrogen bond between functional group O3 and Ser-349. This hydrogen bond most likely plays a significant role in the docking mechanism and binding modes selection. The surprising result is catechin itself exhibited a quite strong binding with GlmS (ΔG= -7.80 kcal.mol), but docked on a completely different pocket compared to other ligands. This results suggest that catechin might still have a curing effect but with a completely different pathway and mechanism as compared to its derivatives.

The RNA-binding protein CsrA plays a central role in positively regulating virulence factors in Erwinia amylovora

PubMed Central

Ancona, Veronica; Lee, Jae Hoon; Zhao, Youfu

2016-01-01

The GacS/GacA two-component system (also called GrrS/GrrA) is a global regulatory system which is highly conserved among gamma-proteobacteria. This system positively regulates non-coding small regulatory RNA csrB, which in turn binds to the RNA-binding protein CsrA. However, how GacS/GacA-Csr system regulates virulence traits in E. amylovora remains unknown. Results from mutant characterization showed that the csrB mutant was hypermotile, produced higher amount of exopolysaccharide amylovoran, and had increased expression of type III secretion (T3SS) genes in vitro. In contrast, the csrA mutant exhibited complete opposite phenotypes, including non-motile, reduced amylovoran production and expression of T3SS genes. Furthermore, the csrA mutant did not induce hypersensitive response on tobacco or cause disease on immature pear fruits, indicating that CsrA is a positive regulator of virulence factors. These findings demonstrated that CsrA plays a critical role in E. amylovora virulence and suggested that negative regulation of virulence by GacS/GacA acts through csrB sRNA, which binds to CsrA and neutralizes its positive effect on T3SS gene expression, flagellar formation and amylovoran production. Future research will be focused on determining the molecular mechanism underlying the positive regulation of virulence traits by CsrA. PMID:27845410

The RNA-binding protein CsrA plays a central role in positively regulating virulence factors in Erwinia amylovora.

PubMed

Ancona, Veronica; Lee, Jae Hoon; Zhao, Youfu

2016-11-15

The GacS/GacA two-component system (also called GrrS/GrrA) is a global regulatory system which is highly conserved among gamma-proteobacteria. This system positively regulates non-coding small regulatory RNA csrB, which in turn binds to the RNA-binding protein CsrA. However, how GacS/GacA-Csr system regulates virulence traits in E. amylovora remains unknown. Results from mutant characterization showed that the csrB mutant was hypermotile, produced higher amount of exopolysaccharide amylovoran, and had increased expression of type III secretion (T3SS) genes in vitro. In contrast, the csrA mutant exhibited complete opposite phenotypes, including non-motile, reduced amylovoran production and expression of T3SS genes. Furthermore, the csrA mutant did not induce hypersensitive response on tobacco or cause disease on immature pear fruits, indicating that CsrA is a positive regulator of virulence factors. These findings demonstrated that CsrA plays a critical role in E. amylovora virulence and suggested that negative regulation of virulence by GacS/GacA acts through csrB sRNA, which binds to CsrA and neutralizes its positive effect on T3SS gene expression, flagellar formation and amylovoran production. Future research will be focused on determining the molecular mechanism underlying the positive regulation of virulence traits by CsrA.

Important role of N108 residue in binding of bovine foamy virus transactivator Tas to viral promoters.

PubMed

Bing, Tiejun; Zhang, Suzhen; Liu, Xiaojuan; Liang, Zhibin; Shao, Peng; Zhang, Song; Qiao, Wentao; Tan, Juan

2016-06-30

Bovine foamy virus (BFV) encodes the transactivator BTas, which enhances viral gene transcription by binding to the long terminal repeat promoter and the internal promoter. In this study, we investigated the different replication capacities of two similar BFV full-length DNA clones, pBS-BFV-Y and pBS-BFV-B. Here, functional analysis of several chimeric clones revealed a major role for the C-terminal region of the viral genome in causing this difference. Furthermore, BTas-B, which is located in this C-terminal region, exhibited a 20-fold higher transactivation activity than BTas-Y. Sequence alignment showed that these two sequences differ only at amino acid 108, with BTas-B containing N108 and BTas-Y containing D108 at this position. Results of mutagenesis studies demonstrated that residue N108 is important for BTas binding to viral promoters. In addition, the N108D mutation in pBS-BFV-B reduced the viral replication capacity by about 1.5-fold. Our results suggest that residue N108 is important for BTas binding to BFV promoters and has a major role in BFV replication. These findings not only advances our understanding of the transactivation mechanism of BTas, but they also highlight the importance of certain sequence polymorphisms in modulating the replication capacity of isolated BFV clones.

Characterization of the primary interaction between the mating pheromone, alpha-factor, and its receptor in Saccharomyces cerevisiae

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

Raths, S.K.

1987-01-01

Alpha-factor is a peptide of thirteen amino acids which is required for mating between the haploid mating types, a and ..cap alpha.., in Saccharomyces cerevisiae. An analogue of alpha-factor, DHP/sup 8/ DHP/sup 11/ Nle/sup 12/ tridecapeptide, was catalytically reduced in the presence of /sup 3/H gas for production of a radiolabeled pheromone suitable for use in binding studies. Incorporation of tritium resulted in /sup 3/H-alpha-factor with high specific activity, purity, biological activity and long shelf-life. Binding studies revealed that alpha-factor interacts with its receptor via a simple, reversible process which obeys the law of mass action. Association and dissociation kineticsmore » indicate values of 2.92 x 10/sup 6/ M/sup /minus/1/ min/sup -1/ for k/sub 1/ and between 4 and 7 x 10/sup /minus/2/ min/sup /minus/1/ for k/sub /minus/1/. Saturation binding studies reveal an equilibrium dissociation constant equal to 2.32 x 10/sup /minus/8/ M which approximate the kinetically-derived K/sub D/ of 2.12 x 10/sup /minus/8/ M. Scatchard and Hill analyses as well as dissociation behavior in the presence of excess unlabeled ligand indicate alpha-factor interacts with a homogeneous population of binding sites which do not interact and exhibit one affinity for the alpha-factor pheromone.« less

Insights into the binding behavior of bovine serum albumin to black carbon nanoparticles and induced cytotoxicity

NASA Astrophysics Data System (ADS)

Wu, Hai; Chen, Miaomiao; Shang, Mengting; Li, Xiang; Mu, Kui; Fan, Suhua; Jiang, Shuanglin; Li, Wenyong

2018-07-01

Black carbon (BC) is a main component of particulate matter (PM2.5). Due to their small size (<100 nm), inhaled ultrafine BC nanoparticles may penetrate the lung alveoli, where they interact with surfactant proteins and lipids, causing more serious damage to human health. Here, BC was analyzed to investigate the binding mechanism of its interaction with protein and induction of cytotoxicity changes. The binding process and protein conformation between BC and a serum protein (bovine serum albumin, BSA) were monitored by using a fluorescence quenching technique and UV-vis absorption, Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies. The experimental results revealed that the fluorescence quenching of BSA induced by BC was a static quenching process and the hydrophobic force played the critical role in the interaction. The native conformation of BSA on the BC surface was slightly disturbed but obvious structural unfolding of the secondary structure did not occur. In the cytotoxicity study, BC nanoparticles with low concentrations exhibited strong toxicity towards BEAS-2B cells. However, the toxicity of BC nanoparticles could be mitigated by the presence of BSA. Therefore, proteins in biological fluids likely reduce the toxic effect of BC on human health. These findings delineated the binding mechanism and the toxicity between BC and the BSA-BC system, contributing to the understanding of the biological effects of BC exposure on human health in polluted atmospheres.

Characterization of glucagon-like peptide-1 receptor-binding determinants.

PubMed

Xiao, Q; Jeng, W; Wheeler, M B

2000-12-01

Glucagon-like peptide 1 (GLP-1) is a potent insulinotropic hormone currently under study as a therapeutic agent for type 2 diabetes. Since an understanding of the molecular mechanisms leading to high-affinity receptor (R) binding and activation may facilitate the development of more potent GLP-1R agonists, we have localized specific regions of GLP-1R required for binding. The purified N-terminal fragment (hereafter referred to as NT) of the GLP-1R produced in either insect (Sf9) or mammalian (COS-7) cells was shown to bind GLP-1. The physical interaction of NT with GLP-1 was first demonstrated by cross-linking ((125)I-GLP-1/NT complex band at approximately 28 kDa) and secondly by attachment to Ni(2+)-NTA beads. The GLP-1R NT protein attached to beads bound GLP-1, but with lower affinity (inhibitory concentration (IC(50)): 4.5 x 10(-7) M) than wild-type (WT) GLP-1R (IC(50): 5.2 x 10(-9)M). The low affinity of GLP-1R NT suggested that other receptor domains may contribute to GLP-1 binding. This was supported by studies using chimeric glucose-dependent insulinotropic polypeptide (GIP)/GLP-1 receptors. GIP(1-151)/GLP-1R, but not GIP(1-222)/GLP-1R, exhibited specific GLP-1 binding and GLP-1-induced cAMP production, suggesting that the region encompassing transmembrane (TM) domain 1 through to TM3 was required for binding. Since it was hypothesized that certain charged or polar amino acids in this region might be involved in binding, these residues (TM2-TM3) were analyzed by substitution mutagenesis. Five mutants (K197A, D198A, K202A, D215A, R227A) displayed remarkably reduced binding affinity. These studies indicate that the NT domain of the GLP-1R is able to bind GLP-1, but charged residues concentrated at the distal TM2/extracellular loop-1 (EC1) interface (K197, D198, K202) and in EC1 (D215 and R227) probably contribute to the binding determinants of the GLP-1R.

Identification of the bioactive and consensus peptide motif from Momordica charantia insulin receptor-binding protein.

PubMed

Lo, Hsin-Yi; Li, Chia-Cheng; Ho, Tin-Yun; Hsiang, Chien-Yun

2016-08-01

Many food bioactive peptides with diverse functions have been discovered by studying plant proteins. We have previously identified a 68-residue insulin receptor (IR)-binding protein (mcIRBP) from Momordica charantia that exhibits hypoglycemic effects in mice via interaction with IR. By in vitro digestion, we found that mcIRBP-19, spanning residues 50-68 of mcIRBP, enhanced the binding of insulin to IR, stimulated the phosphorylation of PDK1 and Akt, induced the expression of glucose transporter 4, and stimulated both the uptake of glucose in cells and the clearance of glucose in diabetic mice. Furthermore, mcIRBP-19 homologs were present in various plants and shared similar β-hairpin structures and IR kinase-activating abilities to mcIRBP-19. In conclusion, our findings suggested that mcIRBP-19 is a blood glucose-lowering bioactive peptide that exhibits IR-binding potentials. Moreover, we newly identified novel IR-binding bioactive peptides in various plants which belonged to different taxonomic families. Copyright © 2016 Elsevier Ltd. All rights reserved.

Force fields for describing the solution-phase synthesis of shape-selective metal nanoparticles

NASA Astrophysics Data System (ADS)

Zhou, Ya; Al-Saidi, Wissam; Fichthorn, Kristen

2013-03-01

Polyvinylpyrrolidone (PVP) and polyethylene oxide (PEO) are structure-directing agents that exhibit different performance in the polyol synthesis of Ag nanostructures. The success of these structure-directing agents in selective nanostructure synthesis is often attributed to their selective binding to Ag(100) facets. We use first-principles, density-functional theory (DFT) calculations in a vacuum environment to show that PVP has a stronger preference to bind to Ag(100) than to Ag(111), whereas PEO exhibits much weaker selectivity. To understand the role of solvent in the surface-sensitive binding, we develop classical force fields to describe the interactions of the structure-directing (PVP and PEO) and solvent (ethylene glycol) molecules with various Ag substrates. We parameterize the force fields through force-and-energy matching to DFT results using simulated annealing. We validate the force fields by comparisons to DFT and experimental binding energies. Our force fields reproduce the surface-sensitive binding predicted by DFT calculations. Molecular dynamics simulations based on these force fields can be used to reveal the role of solvent, polymer chain length, and polymer concentration in the selective synthesis of Ag nanostructures.

Molecular coevolution of mammalian ribosomal gene terminator sequences and the transcription termination factor TTF-I.

PubMed Central

Evers, R; Grummt, I

1995-01-01

Both the DNA elements and the nuclear factors that direct termination of ribosomal gene transcription exhibit species-specific differences. Even between mammals--e.g., human and mouse--the termination signals are not identical and the respective transcription termination factors (TTFs) which bind to the terminator sequence are not fully interchangeable. To elucidate the molecular basis for this species-specificity, we have cloned TTF-I from human and mouse cells and compared their structural and functional properties. Recombinant TTF-I exhibits species-specific DNA binding and terminates transcription both in cell-free transcription assays and in transfection experiments. Chimeric constructs of mouse TTF-I and human TTF-I reveal that the major determinant for species-specific DNA binding resides within the C terminus of TTF-I. Replacing 31 C-terminal amino acids of mouse TTF-I with the homologous human sequences relaxes the DNA-binding specificity and, as a consequence, allows the chimeric factor to bind the human terminator sequence and to specifically stop rDNA transcription. Images Fig. 2 Fig. 3 Fig. 4 PMID:7597036

Social insect genomes exhibit dramatic evolution in gene composition and regulation while preserving regulatory features linked to sociality

PubMed Central

Simola, Daniel F.; Wissler, Lothar; Donahue, Greg; Waterhouse, Robert M.; Helmkampf, Martin; Roux, Julien; Nygaard, Sanne; Glastad, Karl M.; Hagen, Darren E.; Viljakainen, Lumi; Reese, Justin T.; Hunt, Brendan G.; Graur, Dan; Elhaik, Eran; Kriventseva, Evgenia V.; Wen, Jiayu; Parker, Brian J.; Cash, Elizabeth; Privman, Eyal; Childers, Christopher P.; Muñoz-Torres, Monica C.; Boomsma, Jacobus J.; Bornberg-Bauer, Erich; Currie, Cameron R.; Elsik, Christine G.; Suen, Garret; Goodisman, Michael A.D.; Keller, Laurent; Liebig, Jürgen; Rawls, Alan; Reinberg, Danny; Smith, Chris D.; Smith, Chris R.; Tsutsui, Neil; Wurm, Yannick; Zdobnov, Evgeny M.; Berger, Shelley L.; Gadau, Jürgen

2013-01-01

Genomes of eusocial insects code for dramatic examples of phenotypic plasticity and social organization. We compared the genomes of seven ants, the honeybee, and various solitary insects to examine whether eusocial lineages share distinct features of genomic organization. Each ant lineage contains ∼4000 novel genes, but only 64 of these genes are conserved among all seven ants. Many gene families have been expanded in ants, notably those involved in chemical communication (e.g., desaturases and odorant receptors). Alignment of the ant genomes revealed reduced purifying selection compared with Drosophila without significantly reduced synteny. Correspondingly, ant genomes exhibit dramatic divergence of noncoding regulatory elements; however, extant conserved regions are enriched for novel noncoding RNAs and transcription factor–binding sites. Comparison of orthologous gene promoters between eusocial and solitary species revealed significant regulatory evolution in both cis (e.g., Creb) and trans (e.g., fork head) for nearly 2000 genes, many of which exhibit phenotypic plasticity. Our results emphasize that genomic changes can occur remarkably fast in ants, because two recently diverged leaf-cutter ant species exhibit faster accumulation of species-specific genes and greater divergence in regulatory elements compared with other ants or Drosophila. Thus, while the “socio-genomes” of ants and the honeybee are broadly characterized by a pervasive pattern of divergence in gene composition and regulation, they preserve lineage-specific regulatory features linked to eusociality. We propose that changes in gene regulation played a key role in the origins of insect eusociality, whereas changes in gene composition were more relevant for lineage-specific eusocial adaptations. PMID:23636946

Weakly Hydrated Surfaces and the Binding Interactions of Small Biological Solutes

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

Brady, J. W.; Tavagnacco, L.; Ehrlich, L.

2012-04-01

Extended planar hydrophobic surfaces, such as are found in the side chains of the amino acids histidine, phenylalanine, tyrosine, and tryptophan, exhibit an affinity for the weakly hydrated faces of glucopyranose. In addition, molecular species such as these, including indole, caffeine, and imidazole, exhibit a weak tendency to pair together by hydrophobic stacking in aqueous solution. These interactions can be partially understood in terms of recent models for the hydration of extended hydrophobic faces and should provide insight into the architecture of sugar-binding sites in proteins.

Weakly hydrated surfaces and the binding interactions of small biological solutes.

PubMed

Brady, John W; Tavagnacco, Letizia; Ehrlich, Laurent; Chen, Mo; Schnupf, Udo; Himmel, Michael E; Saboungi, Marie-Louise; Cesàro, Attilio

2012-04-01

Extended planar hydrophobic surfaces, such as are found in the side chains of the amino acids histidine, phenylalanine, tyrosine, and tryptophan, exhibit an affinity for the weakly hydrated faces of glucopyranose. In addition, molecular species such as these, including indole, caffeine, and imidazole, exhibit a weak tendency to pair together by hydrophobic stacking in aqueous solution. These interactions can be partially understood in terms of recent models for the hydration of extended hydrophobic faces and should provide insight into the architecture of sugar-binding sites in proteins.

Diurnal rhythm of melatonin binding in the rat suprachiasmatic nucleus

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

Laitinen, J.T.; Castren, E.; Vakkuri, O.

1989-03-01

We used quantitative in vitro autoradiography to localize and characterize 2-/sup 125/I-melatonin binding sites in the rat suprachiasmatic nuclei in relation to pineal melatonin production. In a light:dark cycle of 12:12 h, binding density exhibited significant diurnal variation with a peak at the dark-light transition and a trough 12 hours later. Saturation studies suggested that the decreased binding at light-dark transition might be due to a shift of the putative melatonin receptor to a low affinity state.

The Interaction of Integrin αIIbβ3 with Fibrin Occurs through Multiple Binding Sites in the αIIb β-Propeller Domain*

PubMed Central

Podolnikova, Nataly P.; Yakovlev, Sergiy; Yakubenko, Valentin P.; Wang, Xu; Gorkun, Oleg V.; Ugarova, Tatiana P.

2014-01-01

The currently available antithrombotic agents target the interaction of platelet integrin αIIbβ3 (GPIIb-IIIa) with fibrinogen during platelet aggregation. Platelets also bind fibrin formed early during thrombus growth. It was proposed that inhibition of platelet-fibrin interactions may be a necessary and important property of αIIbβ3 antagonists; however, the mechanisms by which αIIbβ3 binds fibrin are uncertain. We have previously identified the γ370–381 sequence (P3) in the γC domain of fibrinogen as the fibrin-specific binding site for αIIbβ3 involved in platelet adhesion and platelet-mediated fibrin clot retraction. In the present study, we have demonstrated that P3 can bind to several discontinuous segments within the αIIb β-propeller domain of αIIbβ3 enriched with negatively charged and aromatic residues. By screening peptide libraries spanning the sequence of the αIIb β-propeller, several sequences were identified as candidate contact sites for P3. Synthetic peptides duplicating these segments inhibited platelet adhesion and clot retraction but not platelet aggregation, supporting the role of these regions in fibrin recognition. Mutant αIIbβ3 receptors in which residues identified as critical for P3 binding were substituted for homologous residues in the I-less integrin αMβ2 exhibited reduced cell adhesion and clot retraction. These residues are different from those that are involved in the coordination of the fibrinogen γ404–411 sequence and from auxiliary sites implicated in binding of soluble fibrinogen. These results map the binding of fibrin to multiple sites in the αIIb β-propeller and further indicate that recognition specificity of αIIbβ3 for fibrin differs from that for soluble fibrinogen. PMID:24338009

The PHD Finger Protein MMD1/DUET Ensures the Progression of Male Meiotic Chromosome Condensation and Directly Regulates the Expression of the Condensin Gene CAP-D3.

PubMed

Wang, Jun; Niu, Baixiao; Huang, Jiyue; Wang, Hongkuan; Yang, Xiaohui; Dong, Aiwu; Makaroff, Christopher; Ma, Hong; Wang, Yingxiang

2016-08-01

Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation. © 2016 American Society of Plant Biologists. All rights reserved.

The PHD Finger Protein MMD1/DUET Ensures the Progression of Male Meiotic Chromosome Condensation and Directly Regulates the Expression of the Condensin Gene CAP-D3[OPEN

PubMed Central

Wang, Jun; Niu, Baixiao; Huang, Jiyue; Wang, Hongkuan; Yang, Xiaohui; Dong, Aiwu

2016-01-01

Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation. PMID:27385818

Suppression of innate inflammation and immunity by interleukin-37.

PubMed

Dinarello, Charles A; Nold-Petry, Claudia; Nold, Marcel; Fujita, Mayumi; Li, Suzhao; Kim, Soohyun; Bufler, Philip

2016-05-01

IL-37 is unique in the IL-1 family in that unlike other members of the family, IL-37 broadly suppresses innate immunity. IL-37 can be elevated in humans with inflammatory and autoimmune diseases where it likely functions to limit inflammation. Transgenic mice expressing human IL-37 (IL37-tg) exhibit less severe inflammation in models of endotoxin shock, colitis, myocardial infarction, lung, and spinal cord injury. IL37-tg mice have reduced antigen-specific responses and dendritic cells (DCs) from these mice exhibit characteristics of tolerogenic DCs. Compared to aging wild-type (WT) mice, aging IL37-tg mice are protected against B-cell leukemogenesis and heart failure. Treatment of WT mice with recombinant human IL-37 has been shown to be protective in several models of inflammation and injury. IL-37 binds to the IL-18 receptor but then recruits the orphan IL-1R8 (formerly TIR8 or SIGIRR) in order to function as an inhibitor. Here, we review the discovery of IL-37, its production, release, and mechanisms by which IL-37 reduces inflammation and suppresses immune responses. The data reviewed here suggest a therapeutic potential for IL-37. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comprehensive behavioral analysis of RNG105 (Caprin1) heterozygous mice: Reduced social interaction and attenuated response to novelty

PubMed Central

Ohashi, Rie; Takao, Keizo; Miyakawa, Tsuyoshi; Shiina, Nobuyuki

2016-01-01

RNG105 (also known as Caprin1) is a major RNA-binding protein in neuronal RNA granules, and is responsible for mRNA transport to dendrites and neuronal network formation. A recent study reported that a heterozygous mutation in the Rng105 gene was found in an autism spectrum disorder (ASD) patient, but it remains unclear whether there is a causal relation between RNG105 deficiency and ASD. Here, we subjected Rng105+/− mice to a comprehensive behavioral test battery, and revealed the influence of RNG105 deficiency on mouse behavior. Rng105+/− mice exhibited a reduced sociality in a home cage and a weak preference for social novelty. Consistently, the Rng105+/− mice also showed a weak preference for novel objects and novel place patterns. Furthermore, although the Rng105+/− mice exhibited normal memory acquisition, they tended to have relative difficulty in reversal learning in the spatial reference tasks. These findings suggest that the RNG105 heterozygous knockout leads to a reduction in sociality, response to novelty and flexibility in learning, which are implicated in ASD-like behavior. PMID:26865403

Expression of E-selectin ligand-1 (CFR/ESL-1) on hepatic stellate cells: implications for leukocyte extravasation and liver metastasis.

PubMed

Antoine, Marianne; Tag, Carmen G; Gressner, Axel M; Hellerbrand, Claus; Kiefer, Paul

2009-02-01

Leukocytes and tumor cells use E-selectin binding ligands to attach to activated endothelial cells expressing E-selectin during inflammation or metastasis. The cysteine-rich fibroblast growth factor receptor (CFR) represents the main E-selectin ligand (ESL-1) on granulocytes and its expression is exclusively modified by alpha(1,3)-fucosyltransferases IV or VII (FucT4 and FucT7). Hepatic stellate cells (HSC) are pericytes of liver sinusoidal endothelial cells. The activation of HSC and transdifferentiation into a myofibroblastic phenotype is involved in the repair of liver tissue injury, liver regeneration and angiogenesis of liver metastases. In the present study, we demonstrated that HSC expressed CFR together with FucT7 and exhibited a functional E-selectin binding activity on their cell surface. Since HSC appear to be oxygen-sensing cells, the expression of E-selectin binding activity was analyzed in HSC under a hypoxic atmosphere. While the expression of the glycoprotein CFR was unaffected by hypoxia, the cell-associated E-selectin binding activity decreased. However, under the same conditions, mRNA expression of the modifying enzyme FucT7 increased. The loss of E-selectin binding activity, therefore, appears to be neither the result of a reduced expression of the modifying transferase nor the expression of the backbone glycoprotein. After the transient transfection of HSC with CFR cDNA, the E-selectin binding activity (ESL-1) was efficiently released into the supernatant. Therefore, we hypothesize that under hypoxia, ESL-1 is shed from activated HSC. Our findings provide a novel perspective on the function of HSC in liver metastasis and inflammatory liver diseases.

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter.

PubMed

Hohl, Michael; Hürlimann, Lea M; Böhm, Simon; Schöppe, Jendrik; Grütter, Markus G; Bordignon, Enrica; Seeger, Markus A

2014-07-29

ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5'-(β,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.

Hypotonic activation of short ClC3 isoform is modulated by direct interaction between its cytosolic C-terminal tail and subcortical actin filaments.

PubMed

McCloskey, Diana T; Doherty, Lynda; Dai, Yan-Ping; Miller, Lisa; Hume, Joseph R; Yamboliev, Ilia A

2007-06-08

Short ClC3 isoform (sClC3) functions as a volume-sensitive outwardly rectifying anion channel (VSOAC) in some cell types. In previous studies, we have shown that the hypotonic activation of sClC3 is linked to cell swelling-mediated remodeling of the actin cytoskeleton. In the present study, we have tested the hypothesis that the cytosolic tails of sClC3 bind to actin directly and that binding modulates the hypotonic activation of the channel. Co-sedimentation assays in vitro demonstrated a strong binding between the glutathione S-transferase-fused cytosolic C terminus of sClC3 (GST-sClC3-CT) to filamentous actin (F-actin) but not to globular monomeric actin (G-actin). The GST-fused N terminus (GST-sClC3-NT) exhibited low binding affinity to both G- and F-actin. Co-sedimentation experiments with progressively truncated GST-sClC3-CT indicated that the F-actin binding region is located between amino acids 690 and 760 of sClC3. Two synthetic peptides mapping basic clusters of the cytosolic sClC3-CT (CTP2, isoleucine 716 to leucine 734; and CTP3, proline 688 to proline 709) prevented binding of GST-sClC3-CT to F-actin in vitro. Dialysis into NIH/3T3 cells of these two peptides (but not of synthetic peptide CTP1 (isoleucine 737 to glutamine 748)) reduced the maximal current density by 60 and 38%, respectively. Based on these results, we have concluded that, by direct interaction with subcortical actin filaments, sClC3 contributes to the hypotonic stress-induced VSOACs in NIH/3T3 cells.

Mutations in the Putative Zinc-Binding Motif of UL52 Demonstrate a Complex Interdependence between the UL5 and UL52 Subunits of the Human Herpes Simplex Virus Type 1 Helicase/Primase Complex

PubMed Central

Chen, Yan; Carrington-Lawrence, Stacy D.; Bai, Ping; Weller, Sandra K.

2005-01-01

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface. PMID:15994803

Mutations in the putative zinc-binding motif of UL52 demonstrate a complex interdependence between the UL5 and UL52 subunits of the human herpes simplex virus type 1 helicase/primase complex.

PubMed

Chen, Yan; Carrington-Lawrence, Stacy D; Bai, Ping; Weller, Sandra K

2005-07-01

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface.

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody.

PubMed

Monnet, Céline; Jorieux, Sylvie; Souyris, Nathalie; Zaki, Ouafa; Jacquet, Alexandra; Fournier, Nathalie; Crozet, Fabien; de Romeuf, Christophe; Bouayadi, Khalil; Urbain, Rémi; Behrens, Christian K; Mondon, Philippe; Fontayne, Alexandre

2014-01-01

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGen™) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling(®) platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.

Stereoselective binding of agonists to the β2-adrenergic receptor: insights into molecular details and thermodynamics from molecular dynamics simulations.

PubMed

Plazinska, Anita; Plazinski, Wojciech

2017-05-02

The β 2 -adrenergic receptor (β 2 -AR) is one of the most studied G-protein-coupled receptors. When interacting with ligand molecules, it exhibits a binding characteristic that is strongly dependent on ligand stereoconfiguration. In particular, many experimental and theoretical studies confirmed that stereoisomers of an important β 2 -AR agonist, fenoterol, are associated with diverse mechanisms of binding and activation of β 2 -AR. The objective of the present study was to explore the stereoselective binding of fenoterol to β 2 -AR through the application of an advanced computational methodology based on enhanced-sampling molecular dynamics simulations and potentials of interactions tailored to investigate the stereorecognition effects. The results remain in very good, quantitative agreement with the experimental data (measured in the context of ligand-receptor affinities and their dependence on the temperature), which provides an additional validation for the applied computational protocols. Additionally, our results contribute to the understanding of stereoselective agonist binding by β 2 -AR. Although the significant role of the N293 6.55 residue is confirmed, we additionally show that stereorecognition does not depend solely on the N293-ligand interactions; the stereoselective effects rely on the co-operation of several residues located on both the 6th and 7th transmembrane domains and on extracellular loops. The magnitude and character of the contributions of these residues may be very diverse and result in either enhancing or reducing the stereoselective effects. The same is true when considering the enthalpic and entropic contributions to the binding free energies, which also are dependent on the ligand stereoconfiguration.

Dual Regulation of a Chimeric Plant Serine/Threonine Kinase by Calcium and Calcium/Calmodulin

NASA Technical Reports Server (NTRS)

Takezawa, D.; Ramachandiran, S.; Paranjape, V.; Poovaiah, B. W.

1996-01-01

A chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain was recently cloned from plants. The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca(2+)/calmodulin-dependent manner. The calmodulin-binding region of CCAMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca(2+)/calmodulin-dependent protein kinase (CaMKII). CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of P-32/mol) that is stimulated 3.4-fold by Ca(2+) (0.339 mol of P-32/mol), while calmodulin inhibits Ca(2+)-stimulated autophosphorylation to the basal level. A deletion mutant lacking the visinin-like domain did not show Ca(2+)-simulated autophosphorylation activity but retained Ca(2+)/calmodulin-dependent protein kinase activity at a reduced level. Ca(2+)-dependent mobility shift assays using E.coli-expressed protein from residues 358-520 revealed that Ca(2+) binds to the visinin-like domain. Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca(2+)-induced conformational changes in the visinin-like domain. Autophosphorylation of CCaMK increases Ca(2+)/calmodulin-dependent protein kinase activity by about 5-fold, whereas it did not affect its C(2+)-independent activity. This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca(2+) and Ca(2+)/calmodulin. The presence of a visinin-like Ca(2+)-binding domain in CCaMK adds an additional Ca(2+)-sensing mechanism not previously known to exist in the Ca(2+)/calmodulin-mediated signaling cascade in plants.

Prefoldin 6 is required for normal microtubule dynamics and organization in Arabidopsis

PubMed Central

Gu, Ying; Deng, Zhiping; Paredez, Alexander R.; DeBolt, Seth; Wang, Zhi-Yong; Somerville, Chris

2008-01-01

Newly translated tubulin molecules undergo a series of complex interactions with nascent chain-binding chaperones, including prefoldin (PFD) and chaperonin-containing TCP-1 (CCT). By screening for oryzalin hypersensitivity, we identified several mutants of Arabidopsis that have lesions in PFD subunits. The pfd6–1 mutant exhibits a range of microtubule defects, including hypersensitivity to oryzalin, defects in cell division, cortical array organization, and microtubule dynamicity. Consistent with phenotypic analysis, proteomic analysis indicates several isoforms of tubulins were reduced in pfd6–1. These results support the concept that the function of microtubules is critically dependent on the absolute amount of tubulins. PMID:19004800

Restless legs syndrome-associated intronic common variant in Meis1 alters enhancer function in the developing telencephalon.

PubMed

Spieler, Derek; Kaffe, Maria; Knauf, Franziska; Bessa, José; Tena, Juan J; Giesert, Florian; Schormair, Barbara; Tilch, Erik; Lee, Heekyoung; Horsch, Marion; Czamara, Darina; Karbalai, Nazanin; von Toerne, Christine; Waldenberger, Melanie; Gieger, Christian; Lichtner, Peter; Claussnitzer, Melina; Naumann, Ronald; Müller-Myhsok, Bertram; Torres, Miguel; Garrett, Lillian; Rozman, Jan; Klingenspor, Martin; Gailus-Durner, Valérie; Fuchs, Helmut; Hrabě de Angelis, Martin; Beckers, Johannes; Hölter, Sabine M; Meitinger, Thomas; Hauck, Stefanie M; Laumen, Helmut; Wurst, Wolfgang; Casares, Fernando; Gómez-Skarmeta, Jose Luis; Winkelmann, Juliane

2014-04-01

Genome-wide association studies (GWAS) identified the MEIS1 locus for Restless Legs Syndrome (RLS), but causal single nucleotide polymorphisms (SNPs) and their functional relevance remain unknown. This locus contains a large number of highly conserved noncoding regions (HCNRs) potentially functioning as cis-regulatory modules. We analyzed these HCNRs for allele-dependent enhancer activity in zebrafish and mice and found that the risk allele of the lead SNP rs12469063 reduces enhancer activity in the Meis1 expression domain of the murine embryonic ganglionic eminences (GE). CREB1 binds this enhancer and rs12469063 affects its binding in vitro. In addition, MEIS1 target genes suggest a role in the specification of neuronal progenitors in the GE, and heterozygous Meis1-deficient mice exhibit hyperactivity, resembling the RLS phenotype. Thus, in vivo and in vitro analysis of a common SNP with small effect size showed allele-dependent function in the prospective basal ganglia representing the first neurodevelopmental region implicated in RLS.

Tackling CO Poisoning with Single Atom Alloy Catalysts

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

Liu, Jilei; Lucci, Felicia R.; Yang, Ming

2016-05-01

Platinum (Pt) catalysts are extensively used in the chemical industry and as electrocatalysts in fuel cells. Pt is notorious for its sensitivity to poisoning by strong CO adsorption. Here we demonstrate that the single atom alloy (SAA) strat-egy applied to Pt reduces the binding strength of CO while maintaining catalytic performance. By using surface sensi-tive studies, we accurately determined the binding strength of CO to different Pt ensembles, and this in turn guided the preparation of PtCu alloy nanoparticles. The atomic ratio Pt:Cu = 1:120 yielded a SAA which exhibited excellent CO tolerance in H2 activation, the key elementary stepmore » for hy-drogenation and hydrogen electro-oxidation. As a probe reaction, the selective hydrogenation of acetylene to ethene was performed under flow conditions on the SAA nanopar-ticles supported on alumina without activity loss in the pres-ence of CO. The ability to maintain reactivity in the presence of CO is vital to other industrial reactions including fuel reforming and methanol/ethanol fuel cells.« less

In silico identification of potent pancreatic triacylglycerol lipase inhibitors from traditional Chinese medicine.

PubMed

Chen, Kuan-Yu; Chang, Su-Sen; Chen, Calvin Yu-Chian

2012-01-01

Pancreatic triacylglycerol lipase (PNLIP) are primary lipases that are critical for triacylglyceride digestion in human. Since reduced metabolism of triacylglyceride might be a plausible concept for weight loss, we screened for potential PNLIP inhibitors from traditional Chinese medicine (TCM) with the aim to identify weight loss candidate compounds. TCM candidates Aurantiamide, Cnidiadin, and 2-hexadecenoic acid exhibited higher Dock Scores than the commercial drug Orlistat, and were also predicted to have inhibitory characteristics against PNLIP using constructed MLR (R(2) = 0.8664) and SVM (R(2) = 0.9030) models. Molecular dynamics indicated that the TCM-PNLIP complexes formed were stable. We identified that the PNLIP binding site has several residues that can serve as anchors, and a hydrophobic corridor that provides additional stability to the complex. Aurantiamide, Cnidiadin, and 2-hexadecenoic acid all have features that correspond to these binding site features, indicating their potential as candidates for PNLIP inhibitors. The information presented in this study may provide helpful insights to designing novel weight-control drugs.

An HMGA2-IGF2BP2 axis regulates myoblast proliferation and myogenesis.

PubMed

Li, Zhizhong; Gilbert, Jason A; Zhang, Yunyu; Zhang, Minsi; Qiu, Qiong; Ramanujan, Krishnan; Shavlakadze, Tea; Eash, John K; Scaramozza, Annarita; Goddeeris, Matthew M; Kirsch, David G; Campbell, Kevin P; Brack, Andrew S; Glass, David J

2012-12-11

A group of genes that are highly and specifically expressed in proliferating skeletal myoblasts during myogenesis was identified. Expression of one of these genes, Hmga2, increases coincident with satellite cell activation, and later its expression significantly declines correlating with fusion of myoblasts into myotubes. Hmga2 knockout mice exhibit impaired muscle development and reduced myoblast proliferation, while overexpression of HMGA2 promotes myoblast growth. This perturbation in proliferation can be explained by the finding that HMGA2 directly regulates the RNA-binding protein IGF2BP2. Add-back of IGF2BP2 rescues the phenotype. IGF2BP2 in turn binds to and controls the translation of a set of mRNAs, including c-myc, Sp1, and Igf1r. These data demonstrate that the HMGA2-IGF2BP2 axis functions as a key regulator of satellite cell activation and therefore skeletal muscle development. Copyright © 2012 Elsevier Inc. All rights reserved.

Group B Streptococcal Serine-Rich Repeat Proteins Promote Interaction With Fibrinogen and Vaginal Colonization

PubMed Central

Wang, Nai-Yu; Patras, Kathryn A.; Seo, Ho Seong; Cavaco, Courtney K.; Rösler, Berenice; Neely, Melody N.; Sullam, Paul M.; Doran, Kelly S.

2014-01-01

Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 “latching” domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr–fibrinogen interactions in the female reproductive tract. PMID:24620021

Characterization of WY 14,643 and its Complex with Aldose Reductase

PubMed Central

Sawaya, Michael R.; Verma, Malkhey; Balendiran, Vaishnavi; Rath, Nigam P.; Cascio, Duilio; Balendiran, Ganesaratnam K.

2016-01-01

The peroxisome proliferator, WY 14,643 exhibits a pure non-competitive inhibition pattern in the aldehyde reduction and in alcohol oxidation activities of human Aldose reductase (hAR). Fluorescence emission measurements of the equilibrium dissociation constants, Kd, of oxidized (hAR•NADP+) and reduced (hAR•NADPH) holoenzyme complexes display a 2-fold difference between them. Kd values for the dissociation of WY 14,643 from the oxidized (hAR•NADP+•WY 14,643) and reduced (hAR•NADPH•WY 14,643) ternary complexes are comparable to each other. The ternary complex structure of hAR•NADP+•WY 14,643 reveals the first structural evidence of a fibrate class drug binding to hAR. These observations demonstrate how fibrate molecules such as WY 14,643, besides being valued as agonists for PPAR, also inhibit hAR. PMID:27721416

Water-soluble Manganese and Iron Mesotetrakis(carboxyl)porphyrin: DNA Binding, Oxidative Cleavage, and Cytotoxic Activities.

PubMed

Shi, Lei; Jiang, Yi-Yu; Jiang, Tao; Yin, Wei; Yang, Jian-Ping; Cao, Man-Li; Fang, Yu-Qi; Liu, Hai-Yang

2017-06-29

Two new water-soluble metal carboxyl porphyrins, manganese (III) meso -tetrakis (carboxyl) porphyrin and iron (III) meso -tetrakis (carboxyl) porphyrin, were synthesized and characterized. Their interactions with ct-DNA were investigated by UV-Vis titration, fluorescence spectra, viscosity measurement and CD spectra. The results showed they can strongly bind to ct-DNA via outside binding mode. Electrophoresis experiments revealed that both complexes can cleave pBR322 DNA efficiently in the presence of hydrogen peroxide, albeit 2-Mn exhibited a little higher efficiency. The inhibitor tests suggest the oxidative DNA cleavage by these two complexes may involve hydroxyl radical active intermediates. Notably, 2-Mn exhibited considerable photocytotoxicity against Hep G2 cell via triggering a significant generation of ROS and causing disruption of MMP after irradiation.

A BPTTF-based self-assembled electron-donating triangle capable of C60 binding.

PubMed

Goeb, Sébastien; Bivaud, Sébastien; Dron, Paul Ionut; Balandier, Jean-Yves; Chas, Marcos; Sallé, Marc

2012-03-25

A kinetically stable self-assembled redox-active triangle is isolated. The resulting electron-donating cavity, which incorporates three BPTTF units, exhibits a remarkable binding ability for electron-deficient C(60), supported by a favorable combination of structural and electronic features.

17 CFR 230.402 - Number of copies; binding; signatures.

Code of Federal Regulations, 2010 CFR

2010-04-01

...; signatures. 230.402 Section 230.402 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION...; binding; signatures. (a) Three copies of the complete registration statement, including exhibits and all... bound and may contain facsimile versions of manual signatures in accordance with paragraph (e) of this...

The cytotoxic effect of spiroflavanone derivatives, their binding ability to human serum albumin (HSA) and a DFT study on the mechanism of their synthesis

NASA Astrophysics Data System (ADS)

Budzisz, Elzbieta; Paneth, Piotr; Geromino, Inacrist; Muzioł, Tadeusz; Rozalski, Marek; Krajewska, Urszula; Pipiak, Paulina; Ponczek, Michał B.; Małecka, Magdalena; Kupcewicz, Bogumiła

2017-06-01

This paper examines the cytotoxic effect of nine compounds with spiropyrazoline structures, and determines the reaction mechanism between diazomethane and selected benzylideneflavanones, their lipophilicity, and their binding ability to human serum albumin. The cytotoxic effect was determined on two human leukaemia cell lines (HL-60 and NALM-6) and melanoma WM-115 cells, as well as on normal human umbilical vein endothelial cells (HUVEC). The highest cytotoxicity was exhibited by compound B7: it was found to have an IC50 of less than 10 μM for all three cancer cell lines, with five to 12-fold lower sensitivity against normal cells (HUVEC). All the compounds exhibit comparable affinity energy in human serum albumin binding (from -8.1 to -8.6 kcal mol-1) but vary in their binding sites depending on the substituent. X-ray crystallography of two derivatives confirmed their synthetic pathway, and their structures were carefully examined.

Cooperative binding of Annexin A5 to phosphatidylserine on apoptotic cell membranes

NASA Astrophysics Data System (ADS)

Janko, Christina; Jeremic, Ivica; Biermann, Mona; Chaurio, Ricardo; Schorn, Christine; Muñoz, Luis E.; Herrmann, Martin

2013-12-01

Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major ‘eat-me’ signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.

A cationic amphiphilic peptide ABP-CM4 exhibits selective cytotoxicity against leukemia cells.

PubMed

Chen, Yu Qing; Min, Cui; Sang, Ming; Han, Yang Yang; Ma, Xiao; Xue, Xiao Qing; Zhang, Shuang Quan

2010-08-01

Some cationic antibacterial peptides exhibit a broad spectrum of cytotoxic activity against cancer cells, which could provide a new class of anticancer drugs. In the present study, the anticancer activity of ABP-CM4, an antibacterial peptide from Bombyx mori, against leukemic cell lines THP-1, K562 and U937 was evaluated, and the cytotoxicity compared with the effects on non-cancerous mammalian cells, including peripheral blood mononuclear cells (PBMCs), HEK-293 and erythrocytes. ABP-CM4 reduced the number of viable cells of the leukemic cell lines after exposure for 24h. The reduction was concentration dependent, and the IC50 values ranged from 14 to 18 microM. Conversely, ABP-CM4, even at 120 microM, exhibited no cytotoxicity toward HEK-293 or PBMCs, indicating that there was no significant effect on these two types of non-cancer cells. ABP-CM4 at a concentration of 200 microM had no hemolytic activity on mammalian erythrocytes. Together, these results suggested a selective cytotoxicity in leukemia cells. Flow cytometry demonstrated that the binding activity of ABP-CM4 to leukemia cells was much higher than that to HEK-293 or PBMCs, and there was almost no binding to erythrocytes. FITC-labeled ABP-CM4 molecules were examined under a confocal microscope and found to be concentrated at the surface of leukemia cells and changes of the cell membrane were determined by a cell permeability assay, which led us to the conclusion that ABP-CM4 could act at the cell membrane for its anticancer activity on leukemia cells. Collectively, our results indicated that ABP-CM4 has the potential for development as a novel antileukemic agent. Copyright 2010 Elsevier Inc. All rights reserved.

Subunit Dissociation and Metal Binding by Escherichia coli apo-Manganese Superoxide Dismutase

PubMed Central

Whittaker, Mei M.; Lerch, Thomas F.; Kirillova, Olga; Chapman, Michael S.; Whittaker, James W.

2010-01-01

Metal binding by apo-manganese superoxide dismutase (apo-MnSOD) is essential for functional maturation of the enzyme. Previous studies have demonstrated that metal binding by apo-MnSOD is conformationally gated, requiring protein reorganization for the metal to bind. We have now solved the X-ray crystal structure of apo-MnSOD at 1.9 Å resolution. The organization of active site residues is independent of the presence of the metal cofactor, demonstrating that protein itself templates the unusual metal coordination geometry. Electrophoretic analysis of mixtures of apo- and (Mn2)-MnSOD, dye-conjugated protein, or C-terminal Strep-tag II fusion protein reveals a dynamic subunit exchange process associated with cooperative metal binding by the two subunits of the dimeric protein. In contrast, (S126C) (SS) apo-MnSOD, which contains an inter-subunit covalent disulfide crosslink, exhibits anticooperative metal binding. The protein concentration dependence of metal uptake kinetics implies that protein dissociation is involved in metal binding by the wild type apo-protein, although other processes may also contribute to gating metal uptake. Protein concentration dependent small-zone size exclusion chromatography is consistent with apo-MnSOD dimer dissociation at low protein concentration (KD = 1×10−6 M). Studies on metal uptake by apo-MnSOD in Escherichia coli cells show that the protein exhibits similar behavior in vivo and in vitro. PMID:21044611

Stimulation of iodine organification in porcine thyroid cells by thyroid stimulators.

PubMed

Ginsberg, J; Shewring, G; Howells, R; Smith, B R; Hall, R

Several Graves' sera were simultaneously assessed in a bioassay based on the ability of porcine thyroid cells to organify 125I and in a radioreceptor assay for TSH receptor binding activity. Both assay systems were sensitive to 1 mcU/ml (final concentration) of unlabelled bovine TSH. Six Graves' sera were studied in detail over a wide (0-1.0 mcl sera) dose response range in repeat determinations. Two sera exhibited parallel binding and stimulating. However, two sera revealed significant inhibition of 125I-TSH binding prior to the demonstration of stimulation and the other two sera showed stimulatory capabilities before significant binding was evident. IgG was prepared from one serum by ammonium sulphate precipitation and chromatography on Sepharose 6B and then subjected to preparative isoelectric focusing. The isoelectric distribution of the two activities were found to be identical with major peaks of activity at pl=9.5 and pl=8.5. In summary: 1) each Graves' sera exhibits different dose-response curves with respect to binding and stimulation, 2) at certain concentrations of sera, only binding or stimulation were evident, 3) neither assay was consistently more sensitive for the presence of Graves' immunoglobulins, 4) for one Graves' sera, binding and stimulation could not be separated by isoelectric focusing. These studies would suggest each Graves' immunoglobulin has inherently different characteristics in its interaction with the TSH receptor.

Comparison of Genome-Wide Binding of MyoD in Normal Human Myogenic Cells and Rhabdomyosarcomas Identifies Regional and Local Suppression of Promyogenic Transcription Factors

PubMed Central

MacQuarrie, Kyle L.; Yao, Zizhen; Fong, Abraham P.; Diede, Scott J.; Rudzinski, Erin R.; Hawkins, Douglas S.

2013-01-01

Rhabdomyosarcoma is a pediatric tumor of skeletal muscle that expresses the myogenic basic helix-loop-helix protein MyoD but fails to undergo terminal differentiation. Prior work has determined that DNA binding by MyoD occurs in the tumor cells, but myogenic targets fail to activate. Using MyoD chromatin immunoprecipitation coupled to high-throughput sequencing and gene expression analysis in both primary human muscle cells and RD rhabdomyosarcoma cells, we demonstrate that MyoD binds in a similar genome-wide pattern in both tumor and normal cells but binds poorly at a subset of myogenic genes that fail to activate in the tumor cells. Binding differences are found both across genomic regions and locally at specific sites that are associated with binding motifs for RUNX1, MEF2C, JDP2, and NFIC. These factors are expressed at lower levels in RD cells than muscle cells and rescue myogenesis when expressed in RD cells. MEF2C is located in a genomic region that exhibits poor MyoD binding in RD cells, whereas JDP2 exhibits local DNA hypermethylation in its promoter in both RD cells and primary tumor samples. These results demonstrate that regional and local silencing of differentiation factors contributes to the differentiation defect in rhabdomyosarcomas. PMID:23230269

Studies on the chitin/chitosan binding properties of six cuticular proteins analogous to peritrophin 3 from Bombyx mori.

PubMed

Qu, M; Ren, Y; Liu, Y; Yang, Q

2017-08-01

Chitin deacetylation is required to make the cuticle rigid and compact through chitin chain crosslinking. Thus it is presumed that specialized proteins are required to bind deacetylated chitin chains together. However, deacetylated-chitin binding proteins have not ever been reported. In a previous work, six cuticular proteins analogous to peritrophin 3 (CPAP3s) were found to be abundant in the moulting fluid of Bombyx mori. In this study, these BmCPAP3s (BmCPAP3-A1, BmCPAP3-A2, BmCPAP3-B, BmCPAP3-C, BmCPAP3-D1 and BmCPAP3-D2) were cloned and expressed in Escherichia coli and purified using metal-chelating affinity chromatography. Their binding activities demonstrated that although all of the BmCPAP3s showed similar binding abilities toward crystalline chitin and colloidal chitin, they differed in their affinities toward partially and fully deacetylated chitin. Amongst them, BmCPAP3-D1 exhibited the highest binding activity toward deacetylated chitin. The gene expression pattern of BmCPAP3-D1 was similar to BmCPAP3-A1 and BmCPAP3-C at most stages except that it was dramatically upregulated at the beginning of the pupa to adult transition stage. This work is the first report of a chitin-binding protein, BmCPAP3-D1, which exhibits high binding affinity to deacetylated chitin. © 2017 The Royal Entomological Society.

Computer-aided rational design of novel EBF analogues with an aromatic ring.

PubMed

Wang, Shanshan; Sun, Yufeng; Du, Shaoqing; Qin, Yaoguo; Duan, Hongxia; Yang, Xinling

2016-06-01

Odorant binding proteins (OBPs) are important in insect olfactory recognition. These proteins bind specifically to insect semiochemicals and induce their seeking, mating, and alarm behaviors. Molecular docking and molecular dynamics simulations were performed to provide computational insight into the interaction mode between AgamOBP7 and novel (E)-β-farnesene (EBF) analogues with an aromatic ring. The ligand-binding cavity in OBP7 was found to be mostly hydrophobic due to the presence of several nonpolar residues. The interactions between the EBF analogues and the hydrophobic residues in the binding cavity increased in strength as the distance between them decreased. The EBF analogues with an N-methyl formamide or ester linkage had higher docking scores than those with an amide linkage. Moreover, delocalized π-π and electrostatic interactions were found to contribute significantly to the binding between the ligand benzene ring and nearby protein residues. To design new compounds with higher activity, four EBF analogues D1-D4 with a benzene ring were synthesized and evaluated based on their docking scores and binding affinities. D2, which had an N-methyl formamide group linkage, exhibited stronger binding than D1, which had an amide linkage. D4 exhibited particularly strong binding due to multiple hydrophobic interactions with the protein. This study provides crucial foundations for designing novel EBF analogues based on the OBP structure. Graphical abstract The design strategy of new EBF analogues based on the OBP7 structure.

Chemical Proteomics Reveals Ferrochelatase as a Common Off-target of Kinase Inhibitors.

PubMed

Klaeger, Susan; Gohlke, Bjoern; Perrin, Jessica; Gupta, Vipul; Heinzlmeir, Stephanie; Helm, Dominic; Qiao, Huichao; Bergamini, Giovanna; Handa, Hiroshi; Savitski, Mikhail M; Bantscheff, Marcus; Médard, Guillaume; Preissner, Robert; Kuster, Bernhard

2016-05-20

Many protein kinases are valid drug targets in oncology because they are key components of signal transduction pathways. The number of clinical kinase inhibitors is on the rise, but these molecules often exhibit polypharmacology, potentially eliciting desired and toxic effects. Therefore, a comprehensive assessment of a compound's target space is desirable for a better understanding of its biological effects. The enzyme ferrochelatase (FECH) catalyzes the conversion of protoporphyrin IX into heme and was recently found to be an off-target of the BRAF inhibitor Vemurafenib, likely explaining the phototoxicity associated with this drug in melanoma patients. This raises the question of whether FECH binding is a more general feature of kinase inhibitors. To address this, we applied a chemical proteomics approach using kinobeads to evaluate 226 clinical kinase inhibitors for their ability to bind FECH. Surprisingly, low or submicromolar FECH binding was detected for 29 of all compounds tested and isothermal dose response measurements confirmed target engagement in cells. We also show that Vemurafenib, Linsitinib, Neratinib, and MK-2461 reduce heme levels in K562 cells, verifying that drug binding leads to a loss of FECH activity. Further biochemical and docking experiments identified the protoporphyrin pocket in FECH as one major drug binding site. Since the genetic loss of FECH activity leads to photosensitivity in humans, our data strongly suggest that FECH inhibition by kinase inhibitors is the molecular mechanism triggering photosensitivity in patients. We therefore suggest that a FECH assay should generally be part of the preclinical molecular toxicology package for the development of kinase inhibitors.

Suicidal inactivation of methemoglobin by generation of thiyl radical: insight into NAC mediated protection in RBC.

PubMed

Balaji, S N; Trivedi, V

2013-07-01

N-acetyl-L-cysteine (NAC) improves antioxidant potentials of RBCs to provide protection against oxidative stress induced hemolysis. The antioxidant mechanism of NAC to reduce oxidative stress in RBC, studied through inactivation of pro-oxidant MetHb. NAC causes irreversible inactivation of the MetHb in an H2O2 dependent manner, and the inactivation follows the pseudo- first- order kinetics. The kinetic constants are ki = 8.5μM, kinact = 0.706 min(-1) and t1/2 = 0.9 min. Spectroscopic studies indicate that MetHb accepts NAC as a substrate and oxidizes through a single electron transfer mechanism to the NACox. The single e- oxidation product of NAC has been identified as the 5, 5'- dimethyl-1- pyrroline N- oxide (DMPO) adduct of the sulfur centered radical (a(N) = 15.2 G and a(H)=16.78 G). Binding studies indicate that NACox interacts at the heme moiety and NAC oxidation through MetHb is essential for NAC binding. Heme-NAC adduct dissociated from MetHb and identified (m/z 1011.19) as 2:1 ratio of NAC/heme in the adduct. TEMPO and PBN treatment reduces NAC binding to MetHb and protects against inactivation confirms the role of thiyl radical in the inactivation process. Furthermore, scavenging thiyl radicals by TEMPO abolish the protective effect of NAC in hemolysis. Current work highlights antioxidant mechanism of NAC through NAC thiyl radical generation, and MetHb inactivation to exhibit protection in RBC against oxidative stress induced hemolysis.

How many atoms are required to characterize accurately trajectory fluctuations of a protein?

NASA Astrophysics Data System (ADS)

Cukier, Robert I.

2010-06-01

Large molecules, whose thermal fluctuations sample a complex energy landscape, exhibit motions on an extended range of space and time scales. Principal component analysis (PCA) is often used to extract dominant motions that in proteins are typically domain motions. These motions are captured in the large eigenvalue (leading) principal components. There is also information in the small eigenvalues, arising from approximate linear dependencies among the coordinates. These linear dependencies suggest that instead of using all the atom coordinates to represent a trajectory, it should be possible to use a reduced set of coordinates with little loss in the information captured by the large eigenvalue principal components. In this work, methods that can monitor the correlation (overlap) between a reduced set of atoms and any number of retained principal components are introduced. For application to trajectory data generated by simulations, where the overall translational and rotational motion needs to be eliminated before PCA is carried out, some difficulties with the overlap measures arise and methods are developed to overcome them. The overlap measures are evaluated for a trajectory generated by molecular dynamics for the protein adenylate kinase, which consists of a stable, core domain, and two more mobile domains, referred to as the LID domain and the AMP-binding domain. The use of reduced sets corresponding, for the smallest set, to one-eighth of the alpha carbon (CA) atoms relative to using all the CA atoms is shown to predict the dominant motions of adenylate kinase. The overlap between using all the CA atoms and all the backbone atoms is essentially unity for a sum over PCA modes that effectively capture the exact trajectory. A reduction to a few atoms (three in the LID and three in the AMP-binding domain) shows that at least the first principal component, characterizing a large part of the LID-binding and AMP-binding motion, is well described. Based on these results, the overlap criterion should be applicable as a guide to postulating and validating coarse-grained descriptions of generic biomolecular assemblies.

Genome-wide identification of novel expression signatures reveal distinct patterns and prevalence of binding motifs for p53, nuclear factor-κB and other signal transcription factors in head and neck squamous cell carcinoma

PubMed Central

Yan, Bin; Yang, Xinping; Lee, Tin-Lap; Friedman, Jay; Tang, Jun; Van Waes, Carter; Chen, Zhong

2007-01-01

Background Differentially expressed gene profiles have previously been observed among pathologically defined cancers by microarray technologies, including head and neck squamous cell carcinomas (HNSCCs). However, the molecular expression signatures and transcriptional regulatory controls that underlie the heterogeneity in HNSCCs are not well defined. Results Genome-wide cDNA microarray profiling of ten HNSCC cell lines revealed novel gene expression signatures that distinguished cancer cell subsets associated with p53 status. Three major clusters of over-expressed genes (A to C) were defined through hierarchical clustering, Gene Ontology, and statistical modeling. The promoters of genes in these clusters exhibited different patterns and prevalence of transcription factor binding sites for p53, nuclear factor-κB (NF-κB), activator protein (AP)-1, signal transducer and activator of transcription (STAT)3 and early growth response (EGR)1, as compared with the frequency in vertebrate promoters. Cluster A genes involved in chromatin structure and function exhibited enrichment for p53 and decreased AP-1 binding sites, whereas clusters B and C, containing cytokine and antiapoptotic genes, exhibited a significant increase in prevalence of NF-κB binding sites. An increase in STAT3 and EGR1 binding sites was distributed among the over-expressed clusters. Novel regulatory modules containing p53 or NF-κB concomitant with other transcription factor binding motifs were identified, and experimental data supported the predicted transcriptional regulation and binding activity. Conclusion The transcription factors p53, NF-κB, and AP-1 may be important determinants of the heterogeneous pattern of gene expression, whereas STAT3 and EGR1 may broadly enhance gene expression in HNSCCs. Defining these novel gene signatures and regulatory mechanisms will be important for establishing new molecular classifications and subtyping, which in turn will promote development of targeted therapeutics for HNSCC. PMID:17498291

Electronic-property dependent interactions between tetracycline and graphene nanomaterials in aqueous solution.

PubMed

He, Lin; Liu, Fei-Fei; Zhao, Mengyao; Qi, Zhen; Sun, Xuefei; Afzal, Muhammad Zaheer; Sun, Xiaomin; Li, Yanhui; Hao, Jingcheng; Wang, Shuguang

2018-04-01

Understanding the interactions between graphene nanomaterials (GNMs) and antibiotics in aqueous solution is critical to both the engineering applications of GNMs and the assessment of their potential impact on the fate and transport of antibiotics in the aquatic environment. In this study, adsorption of one common antibiotic, tetracycline, by graphene oxide (GO) and reduced graphene oxide (RGO) was examined with multi-walled carbon nanotubes (MWCNTs) and graphite as comparison. The results showed that the tetracycline adsorption capacity by the four selected carbonaceous materials on the unit mass basis followed an order of GO>RGO>MWCNTs>graphite. Upon normalization by surface area, graphite, RGO and MWCNTs had almost the same high tetracycline adsorption affinity while GO exhibited the lowest. We proposed π-electron-property dependent interaction mechanisms to explain the observed different adsorption behaviors. Density functional theory (DFT) calculations suggested that the oxygen-containing functional groups on GO surface reduced its π-electron-donating ability, and thus decreased the π-based interactions between tetracycline and GO surface. Comparison of adsorption efficiency at different pH indicated that electrostatic interaction also played an important role in tetracycline-GO interactions. Site energy analysis confirmed a highly heterogeneous distribution of the binding sites and strong tetracycline binding affinity of GO surface. Copyright © 2017. Published by Elsevier B.V.

Sitagliptin down-regulates retinol-binding protein 4 and reduces insulin resistance in gestational diabetes mellitus: a randomized and double-blind trial.

PubMed

Sun, Xia; Zhang, Zhendong; Ning, Hui; Sun, Hong; Ji, Xianghong

2017-06-01

Gestational diabetes mellitus (GDM) is a condition that affects increasing number of pregnant women worldwide. Sitagliptin was reported to alleviate symptoms of type 2 diabetes mellitus by reducing serum levels of retinol-binding protein 4 (RBP-4). We investigated the effectiveness of sitagliptin on insulin sensitivity parameters in GDM patients. Pregnant GDM women in the 2nd trimester were recruited for this study. Participants were then assigned randomly to sitagliptin treatment group or placebo treatment group, and administered sitagliptin or placebo daily for 16 weeks. Glucose and insulin profiles, as well as serum RBP-4 level, were measured at both baseline and end of the study. After 16 weeks of treatment, participants in the STL group exhibited significantly improved levels of fasting plasma glucose and serum insulin, homeostasis model of assessment of β cell function (HOMA-β) and insulin resistance (HOMA-IR), compared with those in the placebo group. Serum levels of RBP-4 were also markedly decreased in the sitagliptin treatment group, and more importantly it was positively correlated with improved insulin resistance parameters. Our study supports a potentially promising role of sitagliptin in improving insulin resistance by decreasing RBP-4 in GDM-affected women.

Physiological and transcriptome response to cadmium in cosmos (Cosmos bipinnatus Cav.) seedlings.

PubMed

Liu, Yujing; Yu, Xiaofang; Feng, Yimei; Zhang, Chao; Wang, Chao; Zeng, Jian; Huang, Zhuo; Kang, Houyang; Fan, Xing; Sha, Lina; Zhang, Haiqin; Zhou, Yonghong; Gao, Suping; Chen, Qibing

2017-10-31

To date, several species of Asteraceae have been considered as Cd-accumulators. However, little information on the Cd tolerance and associated mechanisms of Asteraceae species Cosmos bipinnatus, is known. Presently, several physiological indexes and transcriptome profiling under Cd stress were investigated. C. bipinnatus exhibited strong Cd tolerance and recommended as a Cd-accumulator, although the biomasses were reduced by Cd. Meanwhile, Cd stresses reduced Zn and Ca uptake, but increased Fe uptake. Subcellular distribution indicated that the vacuole sequestration in root mainly detoxified Cd under lower Cd stress. Whilst, cell wall binding and vacuole sequestration in root co-detoxified Cd under high Cd exposure. Meanwhile, 66,407 unigenes were assembled and 41,674 (62.75%) unigenes were annotated in at least one database. 2,658 DEGs including 1,292 up-regulated unigenes and 1,366 down-regulated unigenes were identified under 40 μmol/L Cd stress. Among of these DEGs, ZIPs, HMAs, NRAMPs and ABC transporters might participate in Cd uptake, translocation and accumulation. Many DEGs participating in several processes such as cell wall biosynthesis, GSH metabolism, TCA cycle and antioxidant system probably play critical roles in cell wall binding, vacuole sequestration and detoxification. These results provided a novel insight into the physiological and transcriptome response to Cd in C. bipinnatus seedlings.

Functional and Selective Bacterial Interfaces Using Cross-Scaffold Gold Binding Peptides

NASA Astrophysics Data System (ADS)

Adams, Bryn L.; Hurley, Margaret M.; Jahnke, Justin P.; Stratis-Cullum, Dimitra N.

2015-11-01

We investigated the functional and selective activity of three phage-derived gold-binding peptides on the Escherichia coli ( E. coli) bacterial cell surface display scaffold (eCPX) for the first time. Gold-binding peptides, p3-Au12 (LKAHLPPSRLPS), p8#9 (VSGSSPDS), and Midas-2 (TGTSVLIATPYV), were compared side-by-side through experiment and simulation. All exhibited strong binding to an evaporated gold film, with approximately a 4-log difference in binding between each peptide and the control sample. The increased affinity for gold was also confirmed by direct visualization of samples using Scanning Electron Microscopy (SEM). Peptide dynamics in solution were performed to analyze innate structure, and all three were found to have a high degree of flexibility. Preferential binding to gold over silicon for all three peptides was demonstrated, with up to four orders of magnitude selectivity exhibited by p3-Au12. The selectivity was also clearly evident through SEM analysis of the boundary between the gold film and silicon substrate. Functional activity of bound E. coli cells was further demonstrated by stimulating filamentation and all three peptides were characterized as prolific relative to control samples. This work shows great promise towards functional and active bacterial-hybrid gold surfaces and the potential to enable the next generation living material interfaces.

Substitutional impact on biological activity of new water soluble Ni(II) complexes: Preparation, spectral characterization, X-ray crystallography, DNA/protein binding, antibacterial activity and in vitro cytotoxicity.

PubMed

Umadevi, C; Kalaivani, P; Puschmann, H; Murugan, S; Mohan, P S; Prabhakaran, R

2017-02-01

A series of new water soluble nickel(II) complexes containing triphenylphosphine and 4-methoxysalicylaldehyde-4(N)-substituted thiosemicarbazones were synthesized and characterized. Crystallographic investigations confirmed the structure of the complexes (1-4) having the general structure [Ni(4-Msal-Rtsc)(PPh 3 )] (Where R=H (1); CH 3 (2); C 2 H 5 (3); C 6 H 5 (4)) which showed that thiosemicarbazone ligands coordinated to nickel(II) ion as ONS tridentate bibasic donor. DNA/BSA protein binding ability of the ligands and their new complexes were studied by taking calf-thymus DNA (CT-DNA) and Bovine serum albumin (BSA) through absorption and emission titrations. Ethidium bromide (EB) displacement study showed the intercalative binding trend of the complexes to DNA. From the albumin binding studies, the mechanism of quenching was found as static and the alterations in the secondary structure of BSA by the compounds were confirmed with synchronous spectral studies. The binding affinity of the complexes to CT-DNA and BSA has the order of [Ni(4-Msal-etsc)(PPh 3 )] (3) >[Ni(4-Msal-mtsc)(PPh 3 )] (2) >[Ni(4-Msal-tsc)(PPh 3 )] (1) >[Ni(4-Msal-ptsc)(PPh 3 )] (4). In vitro cytotoxicity of the complexes was tested on human lung cancer cells (A549), human cervical cancer cells (HeLa), human liver carcinoma cells (Hep G2). All the complexes exhibited significant activity against three cancer cells. Among them, complex 4 exhibited almost 2.5 fold activity than cisplatin in A549 and HepG2 cell lines. In HeLa cell line, the complexes exhibited significant activity which is less than cisplatin. While comparing the activity of the complexes in A549 and HepG2 cell lines it falls in the order 4>1>2>3>cisplatin. The results obtained from DNA, protein binding and cytotoxicity studies, it is concluded that the cytotoxicity of the complexes as determined by MTT assay were not unduly influenced by the complexes having different binding efficiency with DNA and protein. The complexes exhibited good spectrum of antibacterial activity against four pathogenic bacteria such as E. faecalis (gram +ve), S. aureus (gram +ve), E. coli (gram -ve) and P. aeruginosa (gram -ve). Copyright © 2016 Elsevier B.V. All rights reserved.

Binding and degradation of 125I-gastrin by plasma membranes from homogenized rat gastric mucosa.

PubMed

Kleveland, P M; Waldum, H L

1986-06-01

Binding of 125I-gastrin to the 270-30,000 g fraction from homogenized rat oxyntic mucosa was studied. 'Specific' binding was calculated by subtracting the binding at excess cold gastrin from the binding with labelled gastrin (250 pM) only. At 30 degrees C specific binding rose rapidly to a short-lived maximum before falling gradually, whereas at 15 degrees C and 0 degree C specific binding rose gradually to a higher plateau level. The reduced binding at 30 degrees C could be caused by degradation of either the tracer or the binding site or by a combination of these two events. Degradation of 125I-gastrin was evaluated by trichloroacetic acid (TCA) precipitation, fast protein liquid chromatography, and binding to a gastrin antibody (immunoreactivity). The effect of incubation on the binding site was evaluated by preincubation of the homogenate fraction before adding gastrin. In separate studies, the proteolytic activity of the homogenate fraction was studied by TCA precipitation of radioactive casein. Different enzyme inhibitors tested were virtually ineffective in preventing gastrin and casein degradation. Only lowering the incubation temperature to 15 degrees C or lower could prevent this degradation. The reduced and transient binding of 125I-gastrin at 30 degrees C most probably reflects tracer degradation. Accordingly, the gastrin binding experiments were performed at 15 degrees C. Only homogenates from the oxyntic area of the stomach bound 125I-gastrin specifically and with a Kd of 0.8 nM (Scatchard analysis). However, micromolar concentrations of unlabelled gastrin were required to inhibit half maximal binding of the tracer. The tracer binding was unaffected by secretin, slightly reduced by a CCK-9 analogue, and more markedly reduced by pentagastrin.

Plasma sex steroid binding in Chiroptera.

PubMed

Kwiecinski, G G; Damassa, D A; Gustafson, A W; Armao, M E

1987-04-01

Plasma steroid binding was examined in samples obtained from seven species of bats representing four different families. A specific sex steroid-binding protein (SBP) was identified by steady-state polyacrylamide gel electrophoresis in representatives of two families, the phyllostomids and the vespertilionids. In these species, as in primates, SBP not only exhibited high affinity for the androgens testosterone and dihydrotestosterone (DHT), but also for estradiol. A specific SBP was not identified in the tropical American vampire bat or in the two species of pteropodids examined. In all species examined, except for the vampire bat, a specific corticosteroid-binding globulin (CBG) was also identified. In addition to binding glucocorticoids, CBG in these species appeared to bind androgens as well.

Sex Differences in Serotonin 1 Receptor Binding in Rat Brain

NASA Astrophysics Data System (ADS)

Fischette, Christine T.; Biegon, Anat; McEwen, Bruce S.

1983-10-01

Male and female rats exhibit sex differences in binding by serotonin 1 receptors in discrete areas of the brain, some of which have been implicated in the control of ovulation and of gonadotropin release. The sex-specific changes in binding, which occur in response to the same hormonal (estrogenic) stimulus, are due to changes in the number of binding sites. Castration alone also affects the number of binding sites in certain areas. The results lead to the conclusion that peripheral hormones modulate binding by serotonin 1 receptors. The status of the serotonin receptor system may affect the reproductive capacity of an organism and may be related to sex-linked emotional disturbances in humans.

The influence of surface integrin binding patterns on specific biomaterial-cell interactions

NASA Astrophysics Data System (ADS)

Beranek, Maggi Marie

As the future of biomaterials progresses toward bioactivity, the biomaterial surface must control non-specific protein adsorption and encourage selective protein and cell adsorption. Integrins alphavbeta3, alpha 1beta1, alpha5beta1 and alpha Mbeta2 are expressed on cells involved in endothelialization, inflammation, and intimal hyperplasia. These cellular events play a vital role in biomaterial biocompatibility, especially in the vascular environment. The overall hypothesis of these studies is that biomaterial surfaces exhibit selective integrin binding, which then specifies differential cell binding. To test this hypothesis, four specific aims were developed. The first aim was designed to determine whether metal and polymeric biomaterials exhibit selective integrin binding. The tested materials included 316L stainless steel, nitinol, gold, Elgiloy RTM, poly(D, L-lactide-co-glycolide), polycarbonate urethane and expanded polytetrafluoroethylene. Discrete integrin binding patterns were detected microscopically using integrin specific fluorescent antibodies. Stainless steel exhibited high level integrin alpha1beta 1 and low level integrin alphaMbeta2 binding pattern. This suggests that this metal surface should selectively encourage endothelial cell to inflammatory cell binding. In contrast, gold bound ten times the amount of integrin alphaMbeta2 compared to integrin alpha1beta1, which should encourage inflammatory cell adhesion. The 65/35 poly(D, L-lactide-co-glycolide) was the only polymeric biomaterial tested that had integrin binding levels comparable to metal biomaterials. Based on these observations, a combinational biomaterial with a surface pattern of 65/35 poly(D, L-lactide-co-glycolide) dots on a 316L stainless steel background was created. A pattern of high level integrin alpha1beta1 binding and low level integrin alpha Mbeta2 binding on this combinational surface indicates that this surface should selectively favor endothelial cell binding. In the second aim, the response of surface-bound integrins to flow-related shear stress was examined. Based on fluorescent analysis, total alphavbeta 3, alpha1beta1, and alpha5beta 1 appeared to increase on stainless steel after 90-minute low shear stress exposure, whereas only alpha5beta1 appeared to increase when exposed to high shear. 65/35 poly(D, L-lactide-co-glycolide) exhibited increased total binding of alpha5beta1 and alphaMbeta2, when exposed to either shear stress level. Exposure to either shear stress regimen appeared to increase binding of all integrins on the combinational surface. These responses to shear stress suggest differential integrin binding affinity compared to stainless steel. Using antibodies specific to the integrin subunits, the apparent increase in surface-bound integrins was found to be related to a surface disassociation of alpha and beta subunits. The third aim evaluated human aortic endothelial cells and acute monocytic leukemia cells (THP-1) cell binding to the tested biomaterial surfaces under both static and flow conditions. Both stainless steel and the combinational surface had increased endothelial cell binding compared to monocyte attachment. Pre-incubation of the surface with the specific integrins significantly inhibited human aortic endothelial cell binding. Aim four was designed to investigate the influence of surface bound integrins on human aortic endothelial cell migration under shear stress. If biomaterial surface integrin binding patterns are specific, then pre-bound surface integrins should competitively inhibit binding of cellular integrins to the surface. Cell migration distance on to alphavbeta3, alpha 1beta1, and alpha5beta1 pre-incubated stainless steel was decreased ten-fold, and decreased by three-fold on both 65/35 poly(D, L-lactide-coglycolide) and combinational surfaces compared to the respective bare surfaces. In contrast, migration distance on to alphaMbeta2 pre-coated stainless steel and combinational surface was decreased by only sixty percent and only fifty percent on alphaMbeta2 precoated 65/35 poly(D, L -lactide-co-glycolide). These results suggested that surface binding sites are selective and critical in governing endothelial cell migration. In conclusion, these results support the hypothesis that a surface that encourages specific integrin binding would promote differential cell binding. The novel integrin binding model used in this investigation may be a methodology that can be employed to evaluate potential vascular biomaterials.

The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2

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

Jha, Jyoti K.; Li, Mi; Ghirlando, Rodolfo

Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations inrctBthat reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in amore » dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding. IMPORTANCE The capacity of proteins to undergo remodeling provides opportunities to control their function. However, remodeling remains a poorly understood aspect of the structure-function paradigm due to its dynamic nature. Here we have studied remodeling of the initiator of replication ofVibrio choleraeChr2 by the molecular chaperone, DnaK. We show that DnaK binds to a site on the Chr2 initiator (RctB) that promotes initiation by reducing the initiator’s propensity to dimerize. Dimerization of the initiator of the putative plasmid progenitor of Chr2 is also reduced by DnaK, which promotes initiation. Paradoxically, the DnaK binding also promotes replication inhibition by reducing an autoinhibitory activity of RctB. In the plasmid-to-chromosome transition, it appears that the initiator has acquired an autoinhibitory activity and along with it a new chaperone activity that apparently helps to control replication inhibition independently of replication promotion.« less

Overexpression of the essential Sis1 chaperone reduces TDP-43 effects on toxicity and proteolysis

PubMed Central

Park, Sei-Kyoung; Hong, Joo Y.; Arslan, Fatih; Tietsort, Alex; Tank, Elizabeth M. H.; Li, Xingli

2017-01-01

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by selective loss of motor neurons with inclusions frequently containing the RNA/DNA binding protein TDP-43. Using a yeast model of ALS exhibiting TDP-43 dependent toxicity, we now show that TDP-43 overexpression dramatically alters cell shape and reduces ubiquitin dependent proteolysis of a reporter construct. Furthermore, we show that an excess of the Hsp40 chaperone, Sis1, reduced TDP-43’s effect on toxicity, cell shape and proteolysis. The strength of these effects was influenced by the presence of the endogenous yeast prion, [PIN+]. Although overexpression of Sis1 altered the TDP-43 aggregation pattern, we did not detect physical association of Sis1 with TDP-43, suggesting the possibility of indirect effects on TDP-43 aggregation. Furthermore, overexpression of the mammalian Sis1 homologue, DNAJB1, relieves TDP-43 mediated toxicity in primary rodent cortical neurons, suggesting that Sis1 and its homologues may have neuroprotective effects in ALS. PMID:28531192

Near-infrared fluorophores as biomolecular probes

NASA Astrophysics Data System (ADS)

Patonay, Gabor; Beckford, Garfield; Strekowski, Lucjan; Henary, Maged; Merid, Yonathan

2010-02-01

Near-Infrared (NIR) fluorescence has been valuable in analytical and bioanalytical chemistry. NIR probes and labels have been used for several applications, including hydrophobicity of protein binding sites, DNA sequencing, immunoassays, CE separations, etc. The NIR region (700-1100 nm) has advantages for the spectroscopist due to the inherently lower background interference from the biological matrix and the high molar absorptivities of NIR chromophores. During the studies we report here several NIR dyes were prepared to determine the role of the hydrophobicity of NIR dyes and their charge in binding to amino acids and proteins, e.g., serum albumins. We synthesized NIR dye homologs containing the same chromophore but substituents of varying hydrophobicity. Hydrophobic moieties were represented by alkyl and aryl groups. These NIR dyes of varying hydrophobicity exhibited varying degrees of H-aggregation in aqueous solution indicating that the degree of H-aggregation could be used as an indicator to predict binding characteristics to serum albumins. In order to understand what factors may be important in the binding process, spectral behavior of these varying hydrophobicity dyes were examined in the presence of amino acids. Typical dye structures that exhibit large binding constants to biomolecules were compared in order to optimize applications utilizing non-covalent interactions.

CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53

PubMed Central

Saldaña-Meyer, Ricardo; González-Buendía, Edgar; Guerrero, Georgina; Narendra, Varun; Bonasio, Roberto; Recillas-Targa, Félix; Reinberg, Danny

2014-01-01

The multifunctional CCCTC-binding factor (CTCF) protein exhibits a broad range of functions, including that of insulator and higher-order chromatin organizer. We found that CTCF comprises a previously unrecognized region that is necessary and sufficient to bind RNA (RNA-binding region [RBR]) and is distinct from its DNA-binding domain. Depletion of cellular CTCF led to a decrease in not only levels of p53 mRNA, as expected, but also those of Wrap53 RNA, an antisense transcript originated from the p53 locus. PAR-CLIP-seq (photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation [PAR-CLIP] combined with deep sequencing) analyses indicate that CTCF binds a multitude of transcripts genome-wide as well as to Wrap53 RNA. Apart from its established role at the p53 promoter, CTCF regulates p53 expression through its physical interaction with Wrap53 RNA. Cells harboring a CTCF mutant in its RBR exhibit a defective p53 response to DNA damage. Moreover, the RBR facilitates CTCF multimerization in an RNA-dependent manner, which may bear directly on its role in establishing higher-order chromatin structures in vivo. PMID:24696455

A spin transition mechanism for cooperative adsorption in metal-organic frameworks

NASA Astrophysics Data System (ADS)

Reed, Douglas A.; Keitz, Benjamin K.; Oktawiec, Julia; Mason, Jarad A.; Runčevski, Tomče; Xiao, Dianne J.; Darago, Lucy E.; Crocellà, Valentina; Bordiga, Silvia; Long, Jeffrey R.

2017-10-01

Cooperative binding, whereby an initial binding event facilitates the uptake of additional substrate molecules, is common in biological systems such as haemoglobin. It was recently shown that porous solids that exhibit cooperative binding have substantial energetic benefits over traditional adsorbents, but few guidelines currently exist for the design of such materials. In principle, metal-organic frameworks that contain coordinatively unsaturated metal centres could act as both selective and cooperative adsorbents if guest binding at one site were to trigger an electronic transformation that subsequently altered the binding properties at neighbouring metal sites. Here we illustrate this concept through the selective adsorption of carbon monoxide (CO) in a series of metal-organic frameworks featuring coordinatively unsaturated iron(II) sites. Functioning via a mechanism by which neighbouring iron(II) sites undergo a spin-state transition above a threshold CO pressure, these materials exhibit large CO separation capacities with only small changes in temperature. The very low regeneration energies that result may enable more efficient Fischer-Tropsch conversions and extraction of CO from industrial waste feeds, which currently underutilize this versatile carbon synthon. The electronic basis for the cooperative adsorption demonstrated here could provide a general strategy for designing efficient and selective adsorbents suitable for various separations.

Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

DOE PAGES

Ng, Simon; Lin, Edith; Kitov, Pavel I.; ...

2015-04-10

Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10 8 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3more » out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

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

Ng, Simon; Lin, Edith; Kitov, Pavel I.

Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10 8 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3more » out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

Curcumin as an amyloid-indicator dye in E. coli.

PubMed

McCrate, Oscar A; Zhou, Xiaoxue; Cegelski, Lynette

2013-05-14

We have demonstrated that curcumin is an amyloid-specific dye in E. coli. Curcumin binds to curliated whole cells and to isolated curli amyloid fibers. Similar to Congo red, curcumin exhibits a red-shift in absorbance and a significant increase in fluorescence upon binding to isolated curli.

Curcumin as an Amyloid-indicator Dye in E. coli †

PubMed Central

McCrate, Oscar A.; Zhou, Xiaoxue; Cegelski, Lynette

2013-01-01

We have demonstrated that curcumin is an amyloid-specific dye in E. coli. Curcumin binds to curliated whole cells and to isolated curli amyloid fibers. Similar to Congo red, curcumin exhibits a red-shift in absorbance and a significant increase in fluorescence upon binding to isolated curli. PMID:23287899

Alzheimer's Therapeutics Targeting Amyloid Beta 1–42 Oligomers I: Abeta 42 Oligomer Binding to Specific Neuronal Receptors Is Displaced by Drug Candidates That Improve Cognitive Deficits

PubMed Central

Izzo, Nicholas J.; Staniszewski, Agnes; To, Lillian; Fa, Mauro; Teich, Andrew F.; Saeed, Faisal; Wostein, Harrison; Walko, Thomas; Vaswani, Anisha; Wardius, Meghan; Syed, Zanobia; Ravenscroft, Jessica; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Finn, Patricia; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Miller, Miles; Johanson, Conrad; Stopa, Edward; Windisch, Manfred; Hutter-Paier, Birgit; Shamloo, Mehrdad; Arancio, Ottavio; LeVine, Harry; Catalano, Susan M.

2014-01-01

Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1–42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors - i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer's therapeutics. PMID:25390368

Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers I: Abeta 42 oligomer binding to specific neuronal receptors is displaced by drug candidates that improve cognitive deficits.

PubMed

Izzo, Nicholas J; Staniszewski, Agnes; To, Lillian; Fa, Mauro; Teich, Andrew F; Saeed, Faisal; Wostein, Harrison; Walko, Thomas; Vaswani, Anisha; Wardius, Meghan; Syed, Zanobia; Ravenscroft, Jessica; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Finn, Patricia; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Miller, Miles; Johanson, Conrad; Stopa, Edward; Windisch, Manfred; Hutter-Paier, Birgit; Shamloo, Mehrdad; Arancio, Ottavio; LeVine, Harry; Catalano, Susan M

2014-01-01

Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer's therapeutics.

Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP Binding: Requirement for Establishing Chronic Persistent Infection

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

Drumm, J.; Mi, K; Bilder, P

Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant failsmore » to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.« less

Modification of agonist binding moiety in hybrid derivative 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-1-ol/-2-amino versions: Impact on functional activity and selectivity for dopamine D2/D3 receptors

PubMed Central

Gopishetty, Bhaskar; Zhang, Suhong; Kharkar, Prashant S.; Antonio, Tamara; Reith, Maarten; Dutta, Aloke K.

2013-01-01

The goal of the present study was to explore, in our previously developed hybrid template, the effect of introduction of additional heterocyclic rings (mimicking catechol hydroxyl groups as bioisosteric replacement) on selectivity and affinity for the D3 versus D2 receptor. In addition, we wanted to explore the effect of derivatization of functional groups of the agonist binding moiety in compounds developed by us earlier from the hybrid template. Binding affinity (Ki) of the new compounds was measured with tritiated spiperone as the radioligand and HEK-293 cells expressing either D2 or D3 receptors. Functional activity of selected compounds was assessed in the GTPγS binding assay. In the imidazole series, compound 10a exhibited the highest D3 affinity whereas the indole derivative 13 exhibited similar high D3 affinity. Functionalization of the amino group in agonist (+)-9d with different sulfonamides derivatives improved the D3 affinity significantly with (+)-14f exhibiting the highest affinity. However, functionalization of the hydroxyl and amino groups of 15 and (+)-9d, known agonist and partial agonist, to sulfonate ester and amide in general modulated the affinity. In both cases loss of agonist potency resulted from such derivatization. PMID:23623679

Simultaneous binding to the tracking strand, displaced strand and the duplex of a DNA fork enhances unwinding by Dda helicase

PubMed Central

Aarattuthodiyil, Suja; Byrd, Alicia K.; Raney, Kevin D.

2014-01-01

Interactions between helicases and the tracking strand of a DNA substrate are well-characterized; however, the role of the displaced strand is a less understood characteristic of DNA unwinding. Dda helicase exhibited greater processivity when unwinding a DNA fork compared to a ss/ds DNA junction substrate. The lag phase in the unwinding progress curve was reduced for the forked DNA compared to the ss/ds junction. Fewer kinetic steps were required to unwind the fork compared to the ss/ds junction, suggesting that binding to the fork leads to disruption of the duplex. DNA footprinting confirmed that interaction of Dda with a fork leads to two base pairs being disrupted whereas no disruption of base pairing was observed with the ss/ds junction. Neutralization of the phosphodiester backbone resulted in a DNA-footprinting pattern similar to that observed with the ss/ds junction, consistent with disruption of the interaction between Dda and the displaced strand. Several basic residues in the 1A domain which were previously proposed to bind to the incoming duplex DNA were replaced with alanines, resulting in apparent loss of interaction with the duplex. Taken together, these results suggest that Dda interaction with the tracking strand, displaced strand and duplex coordinates DNA unwinding. PMID:25249618

Orbitofrontal Dopamine Depletion Upregulates Caudate Dopamine and Alters Behavior via Changes in Reinforcement Sensitivity

PubMed Central

Cardinal, R. N.; Rygula, R.; Hong, Y. T.; Fryer, T. D.; Sawiak, S. J.; Ferrari, V.; Cockcroft, G.; Aigbirhio, F. I.; Robbins, T. W.; Roberts, A. C.

2014-01-01

Schizophrenia is associated with upregulation of dopamine (DA) release in the caudate nucleus. The caudate has dense connections with the orbitofrontal cortex (OFC) via the frontostriatal loops, and both areas exhibit pathophysiological change in schizophrenia. Despite evidence that abnormalities in dopaminergic neurotransmission and prefrontal cortex function co-occur in schizophrenia, the influence of OFC DA on caudate DA and reinforcement processing is poorly understood. To test the hypothesis that OFC dopaminergic dysfunction disrupts caudate dopamine function, we selectively depleted dopamine from the OFC of marmoset monkeys and measured striatal extracellular dopamine levels (using microdialysis) and dopamine D2/D3 receptor binding (using positron emission tomography), while modeling reinforcement-related behavior in a discrimination learning paradigm. OFC dopamine depletion caused an increase in tonic dopamine levels in the caudate nucleus and a corresponding reduction in D2/D3 receptor binding. Computational modeling of behavior showed that the lesion increased response exploration, reducing the tendency to persist with a recently chosen response side. This effect is akin to increased response switching previously seen in schizophrenia and was correlated with striatal but not OFC D2/D3 receptor binding. These results demonstrate that OFC dopamine depletion is sufficient to induce striatal hyperdopaminergia and changes in reinforcement learning relevant to schizophrenia. PMID:24872570

Empirically Optimized Flow Cytometric Immunoassay Validates Ambient Analyte Theory

PubMed Central

Parpia, Zaheer A.; Kelso, David M.

2010-01-01

Ekins’ ambient analyte theory predicts, counter intuitively, that an immunoassay’s limit of detection can be improved by reducing the amount of capture antibody. In addition, it also anticipates that results should be insensitive to the volume of sample as well as the amount of capture antibody added. The objective of this study is to empirically validate all of the performance characteristics predicted by Ekins’ theory. Flow cytometric analysis was used to detect binding between a fluorescent ligand and capture microparticles since it can directly measure fractional occupancy, the primary response variable in ambient analyte theory. After experimentally determining ambient analyte conditions, comparisons were carried out between ambient and non-ambient assays in terms of their signal strengths, limits of detection, and their sensitivity to variations in reaction volume and number of particles. The critical number of binding sites required for an assay to be in the ambient analyte region was estimated to be 0.1VKd. As predicted, such assays exhibited superior signal/noise levels and limits of detection; and were not affected by variations in sample volume and number of binding sites. When the signal detected measures fractional occupancy, ambient analyte theory is an excellent guide to developing assays with superior performance characteristics. PMID:20152793

The ebola virus interferon antagonist VP24 directly binds STAT1 and has a novel, pyramidal fold.

PubMed

Zhang, Adrianna P P; Bornholdt, Zachary A; Liu, Tong; Abelson, Dafna M; Lee, David E; Li, Sheng; Woods, Virgil L; Saphire, Erica Ollmann

2012-02-01

Ebolaviruses cause hemorrhagic fever with up to 90% lethality and in fatal cases, are characterized by early suppression of the host innate immune system. One of the proteins likely responsible for this effect is VP24. VP24 is known to antagonize interferon signaling by binding host karyopherin α proteins, thereby preventing them from transporting the tyrosine-phosphorylated transcription factor STAT1 to the nucleus. Here, we report that VP24 binds STAT1 directly, suggesting that VP24 can suppress at least two distinct branches of the interferon pathway. Here, we also report the first crystal structures of VP24, derived from different species of ebolavirus that are pathogenic (Sudan) and nonpathogenic to humans (Reston). These structures reveal that VP24 has a novel, pyramidal fold. A site on a particular face of the pyramid exhibits reduced solvent exchange when in complex with STAT1. This site is above two highly conserved pockets in VP24 that contain key residues previously implicated in virulence. These crystal structures and accompanying biochemical analysis map differences between pathogenic and nonpathogenic viruses, offer templates for drug design, and provide the three-dimensional framework necessary for biological dissection of the many functions of VP24 in the virus life cycle.

In vitro studies on the relationship between the antioxidant activities of some berry extracts and their binding properties to serum albumin.

PubMed

Namiesnik, Jacek; Vearasilp, Kann; Nemirovski, Alina; Leontowicz, Hanna; Leontowicz, Maria; Pasko, Pawel; Martinez-Ayala, Alma Leticia; González-Aguilar, Gustavo A; Suhaj, Milan; Gorinstein, Shela

2014-03-01

The aim of this study was to investigate the possibility to use the bioactive components from cape gooseberry (Physalis peruviana), blueberry (Vaccinium corymbosum), and cranberry (Vaccinium macrocarpon) extracts as a novel source against oxidation in food supplementation. The quantitative analysis of bioactive compounds (polyphenols, flavonoids, flavanols, carotenoids, and chlorophyll) was based on radical scavenging spectrophometric assays and mass spectrometry. The total phenolic content was the highest (P < 0.05) in water extract of blueberries (46.6 ± 4.2 mg GAE/g DW). The highest antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay and Cupric reducing antioxidant capacity were in water extracts of blueberries, showing 108.1 ± 7.2 and 131.1 ± 9.6 μMTE/g DW with correlation coefficients of 0.9918 and 0.9925, and by β-carotene linoleate assay at 80.1 ± 6.6 % with correlation coefficient of 0.9909, respectively. The water extracts of berries exhibited high binding properties with human serum albumin in comparison with quercetin. In conclusion, the bioactive compounds from a relatively new source of gooseberries in comparison with blueberries and cranberries have the potential as food supplementation for human health. The antioxidant and binding activities of berries depend on their bioactive compounds.

Autoantibodies in Serum of Systemic Scleroderma Patients: Peptide-Based Epitope Mapping Indicates Increased Binding to Cytoplasmic Domains of CXCR3.

PubMed

Recke, Andreas; Regensburger, Ann-Katrin; Weigold, Florian; Müller, Antje; Heidecke, Harald; Marschner, Gabriele; Hammers, Christoph M; Ludwig, Ralf J; Riemekasten, Gabriela

2018-01-01

Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera ( N  = 32, with positive reactivity with CXCR3) to healthy controls ( N  = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients ( N  = 31) from normal healthy controls ( N  = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients.

Autoantibodies in Serum of Systemic Scleroderma Patients: Peptide-Based Epitope Mapping Indicates Increased Binding to Cytoplasmic Domains of CXCR3

PubMed Central

Recke, Andreas; Regensburger, Ann-Katrin; Weigold, Florian; Müller, Antje; Heidecke, Harald; Marschner, Gabriele; Hammers, Christoph M.; Ludwig, Ralf J.; Riemekasten, Gabriela

2018-01-01

Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera (N = 32, with positive reactivity with CXCR3) to healthy controls (N = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients (N = 31) from normal healthy controls (N = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients. PMID:29623076

Targeted delivery of hyaluronic acid to the ocular surface by a polymer-peptide conjugate system for dry eye disease.

PubMed

Lee, David; Lu, Qiaozhi; Sommerfeld, Sven D; Chan, Amanda; Menon, Nikhil G; Schmidt, Tannin A; Elisseeff, Jennifer H; Singh, Anirudha

2017-06-01

Hyaluronic acid (HA) solutions effectively lubricate the ocular surface and are used for the relief of dry eye related symptoms. However, HA undergoes rapid clearance due to limited adhesion, which necessitates frequent instillation. Conversely, highly viscous artificial tear formulations with HA blur vision and interfere with blinking. Here, we developed an HA-eye drop formulation that selectively binds and retains HA for extended periods of time on the ocular surface. We synthesized a heterobifunctional polymer-peptide system with one end binding HA while the other end binding either sialic acid-containing glycosylated transmembrane molecules on the ocular surface epithelium, or type I collagen molecule within the tissue matrix. HA solution was mixed with the polymer-peptide system and tested on both ex vivo and in vivo models to determine its ability to prolong HA retention. Furthermore, rabbit ocular surface tissues treated with binding peptides and HA solutions demonstrated superior lubrication with reduced kinetic friction coefficients compared to tissues treated with conventional HA solution. The results suggest that binding peptide-based solution can keep the ocular surface enriched with HA for prolonged times as well as keep it lubricated. Therefore, this system can be further developed into a more effective treatment for dry eye patients than a standard HA eye drop. Eye drop formulations containing HA are widely used to lubricate the ocular surface and relieve dry eye related symptoms, however its low residence time remains a challenge. We designed a polymer-peptide system for the targeted delivery of HA to the ocular surface using sialic acid or type I collagen as anchors for HA immobilization. The addition of the polymer-peptide system to HA eye drop exhibited a reduced friction coefficient, and it can keep the ocular surface enriched with HA for prolonged time. This system can be further developed into a more effective treatment for dry eye than a standard HA eye drop. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors.

PubMed

Alleti, Ramesh; Vagner, Josef; Dehigaspitiya, Dilani Chathurika; Moberg, Valerie E; Elshan, N G R D; Tafreshi, Narges K; Brabez, Nabila; Weber, Craig S; Lynch, Ronald M; Hruby, Victor J; Gillies, Robert J; Morse, David L; Mash, Eugene A

2013-09-01

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9nM and 4.2±0.48nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported. Copyright © 2013 Elsevier Ltd. All rights reserved.

Insights into the binding behavior of bovine serum albumin to black carbon nanoparticles and induced cytotoxicity.

PubMed

Wu, Hai; Chen, Miaomiao; Shang, Mengting; Li, Xiang; Mu, Kui; Fan, Suhua; Jiang, Shuanglin; Li, Wenyong

2018-07-05

Black carbon (BC) is a main component of particulate matter (PM 2.5 ). Due to their small size (<100nm), inhaled ultrafine BC nanoparticles may penetrate the lung alveoli, where they interact with surfactant proteins and lipids, causing more serious damage to human health. Here, BC was analyzed to investigate the binding mechanism of its interaction with protein and induction of cytotoxicity changes. The binding process and protein conformation between BC and a serum protein (bovine serum albumin, BSA) were monitored by using a fluorescence quenching technique and UV-vis absorption, Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies. The experimental results revealed that the fluorescence quenching of BSA induced by BC was a static quenching process and the hydrophobic force played the critical role in the interaction. The native conformation of BSA on the BC surface was slightly disturbed but obvious structural unfolding of the secondary structure did not occur. In the cytotoxicity study, BC nanoparticles with low concentrations exhibited strong toxicity towards BEAS-2B cells. However, the toxicity of BC nanoparticles could be mitigated by the presence of BSA. Therefore, proteins in biological fluids likely reduce the toxic effect of BC on human health. These findings delineated the binding mechanism and the toxicity between BC and the BSA-BC system, contributing to the understanding of the biological effects of BC exposure on human health in polluted atmospheres. Copyright © 2018 Elsevier B.V. All rights reserved.

RNA-binding properties and RNA chaperone activity of human peroxiredoxin 1

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

Kim, Ji-Hee; Lee, Jeong-Mi; Lee, Hae Na

2012-09-07

Highlights: Black-Right-Pointing-Pointer hPrx1 has RNA-binding properties. Black-Right-Pointing-Pointer hPrx1 exhibits helix-destabilizing activity. Black-Right-Pointing-Pointer Cold stress increases hPrx1 level in the nuclear fraction. Black-Right-Pointing-Pointer hPrx1 enhances the viability of cells exposed to cold stress. -- Abstract: Human peroxiredoxin 1 (hPrx1), a member of the peroxiredoxin family, detoxifies peroxide substrates and has been implicated in numerous biological processes, including cell growth, proliferation, differentiation, apoptosis, and redox signaling. To date, Prx1 has not been implicated in RNA metabolism. Here, we investigated the ability of hPrx1 to bind RNA and act as an RNA chaperone. In vitro, hPrx1 bound to RNA and DNA, and unwoundmore » nucleic acid duplexes. hPrx1 also acted as a transcription anti-terminator in an assay using an Escherichia coli strain containing a stem-loop structure upstream of the chloramphenicol resistance gene. The overall cellular level of hPrx1 expression was not increased at low temperatures, but the nuclear level of hPrx1 was increased. In addition, hPrx1 overexpression enhanced the survival of cells exposed to cold stress, whereas hPrx1 knockdown significantly reduced cell survival under the same conditions. These findings suggest that hPrx1 may perform biological functions as a RNA-binding protein, which are distinctive from known functions of hPrx1 as a reactive oxygen species scavenger.« less

MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding.

PubMed

Asamitsu, Kaori; Hirokawa, Takatsugu; Okamoto, Takashi

2017-01-01

In this study, we applied molecular dynamics (MD) simulation to analyze the dynamic behavior of the Tat/CycT1/CDK9 tri-molecular complex and revealed the structural changes of P-TEFb upon Tat binding. We found that Tat could deliberately change the local flexibility of CycT1. Although the structural coordinates of the H1 and H2 helices did not substantially change, H1', H2', and H3' exhibited significant changes en masse. Consequently, the CycT1 residues involved in Tat binding, namely Tat-recognition residues (TRRs), lost their flexibility with the addition of Tat to P-TEFb. In addition, we clarified the structural variation of CDK9 in complex with CycT1 in the presence or absence of Tat. Interestingly, Tat addition significantly reduced the structural variability of the T-loop, thus consolidating the structural integrity of P-TEFb. Finally, we deciphered the formation of the hidden catalytic cavity of CDK9 upon Tat binding. MD simulation revealed that the PITALRE signature sequence of CDK9 flips the inactive kinase cavity of CDK9 into the active form by connecting with Thr186, which is crucial for its activity, thus presumably recruiting the substrate peptide such as the C-terminal domain of RNA pol II. These findings provide vital information for the development of effective novel anti-HIV drugs with CDK9 catalytic activity as the target.

Isolation of an inhibitory insulin-like growth factor (IGF) binding protein from bone cell-conditioned medium: A potential local regulator of IGF action

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

Mohan, S.; Bautista, C.M.; Wergedal, J.

1989-11-01

Inhibitory insulin-like growth factor binding protein (In-IGF-BP) has been purified to homogeneity from medium conditioned by TE89 human osteosarcoma cells by two different methods using Sephadex G-100 gel filtration, FPLC Mono Q ion-exchange, HPLC C{sub 4} reverse-phase, HPLC CN reverse-phase and affinity chromatographies. In-IGF-BP thus purified appeared to be homogeneous and unique by the following criteria. (i) N-terminal sequence analysis yielded a unique sequence (Asp-Glu-Ala-Ile-His-Cys-Pro-Pro-Glu-Ser-Glu-Ala-Lys-Leu-Ala). (ii) Amino acid composition of In-IGF-BP revealed marked differences with the amino acid compositions of other known PBs. (iii) In-IGF-BP exhibited a single band with molecular mass of 25 kDa under reducing conditions on sodiummore » dodecyl sulfate/polyacrylamide gels. IGF-I and IGF-II but not insulin displaced the binding of {sup 125}I-labeled IGF-I or {sup 125}I-labeled IGF-II binding to In-IGF-BP. In-IGF-BP inhibited basal, IGF-stimulated bone cell proliferation and serum-stimulated bone cell proliferation. Forskolin increases synthesis of In-IGF-BP in TE85 human osteosarcoma cells in a dose-dependent manner. Based on these findings, the authors conclude that In-IGF-BP is a protein that has a unique sequence and significant biological actions on bone cells.« less

Contributions of a disulfide bond and a reduced cysteine side chain to the intrinsic activity of the high-density lipoprotein receptor SR-BI.

PubMed

Yu, Miao; Lau, Thomas Y; Carr, Steven A; Krieger, Monty

2012-12-18

The high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), binds HDL and mediates selective cholesteryl ester uptake. SR-BI's structure and mechanism are poorly understood. We used mass spectrometry to assign the two disulfide bonds in SR-BI that connect cysteines within the conserved Cys(321)-Pro(322)-Cys(323) (CPC) motif and connect Cys(280) to Cys(334). We used site-specific mutagenesis to evaluate the contributions of the CPC motif and the side chain of extracellular Cys(384) to HDL binding and lipid uptake. The effects of CPC mutations on activity were context-dependent. Full wild-type (WT) activity required Pro(322) and Cys(323) only when Cys(321) was present. Reduced intrinsic activities were observed for CXC and CPX, but not XXC, XPX, or XXX mutants (X ≠ WT residue). Apparently, a free thiol side chain at position 321 that cannot form an intra-CPC disulfide bond with Cys(323) is deleterious, perhaps because of aberrant disulfide bond formation. Pro(322) may stabilize an otherwise strained CPC disulfide bond, thus supporting WT activity, but this disulfide bond is not absolutely required for normal activity. C(384)X (X = S, T, L, Y, G, or A) mutants exhibited altered activities that varied with the side chain's size: larger side chains phenocopied WT SR-BI treated with its thiosemicarbazone inhibitor BLT-1 (enhanced binding, weakened uptake); smaller side chains produced almost inverse effects (increased uptake:binding ratio). C(384)X mutants were BLT-1-resistant, supporting the proposal that Cys(384)'s thiol interacts with BLT-1. We discuss the implications of our findings on the functions of the extracellular loop cysteines in SR-BI and compare our results to those presented by other laboratories.

A transcriptionally active estrogen receptor mutant is a novel type of dominant negative inhibitor of estrogen action.

PubMed

McInerney, E M; Ince, B A; Shapiro, D J; Katzenellenbogen, B S

1996-12-01

We have characterized a human estrogen receptor (ER) mutant, V364E, which has a single amino acid substitution in its hormone-binding domain. This ER mutant is fully active or even superactive at saturating levels of estradiol (10(-8) M E2) yet has the capacity to act as a strong dominant negative inhibitor of the wild type ER. In transient transfection assays using ER-negative Chinese hamster ovary (CHO) cells and two different estrogen response element (ERE)-containing promoter reporter genes, V364E treated with 10(-8) M E2 exhibited approximately 250% and 100% of the activity of the wild type ER with these two promoter contexts, respectively. Despite the high activity of V364E when present alone in cells, coexpression of both V364E and wild type ER causes a significant decrease in overall ER-mediated transcriptional activity. On the TATA promoter, where V364E was more inhibitory, estrogen-stimulated activity was reduced by approximately 50% at a 1:1 ratio of mutant to wild type ER expression vector, and at a 10:1 ratio, 75% of ER activity was inhibited. V364E was expressed at lower levels than wild type ER and has a approximately 40-fold lower affinity for E2 compared with wild type ER. In promoter interference assays, V364E exhibited a strict dependence upon E2 for binding to an ERE. Surprisingly, even when V364E was unable to bind to ERE DNA (i.e. either at low E2 concentration or by mutation of its DNA-binding domain), this mutant retained full dominant negative activity. This highly active ER mutant is, thus, able to repress ER-mediated transcription when the mutant and wild type ER are present together in cells, even without DNA binding. Since competition for ERE binding and the formation of inactive heterodimers cannot fully account for the dominant negative activity of V364E, it is probable that altered interactions with proteins important in ER-mediated transcription play a key role in the repression of transcription by V364E. The properties and probable mechanism of action of V364E distinguish it from other previously described dominant negative inhibitors, in which competition for cis-acting DNA elements by transcriptionally inactive receptors played a large role in the resultant dominant negative phenotype.

Antigenicity analysis of human parvovirus B19-VP1u protein in the induction of anti-phospholipid syndrome.

PubMed

Lin, Chun-Yu; Chiu, Chun-Ching; Cheng, Ju; Lin, Chia-Yun; Shi, Ya-Fang; Tsai, Chun-Chou; Tzang, Bor-Show; Hsu, Tsai-Ching

2018-01-01

Mounting evidence suggests a connection between human parvovirus B19 (B19) and autoimmune diseases, and especially an association between the B19-VP1 unique region (VP1u) and anti-phospholipid syndrome (APS). However, little is known about the antigenicity of B19-VP1u in the induction of APS-like syndrome. To elucidate the antigenicity of B19-VP1u in the induction of APS, N-terminal truncated B19-VP1u (tVP1u) proteins were prepared to immunize Balb/c mice to generate antibodies against B19-tVP1u proteins. The secreted phospholipase A2 (sPLA2) activities and binding specificity of mice anti-B19-tVP1u antibodies with cardiolipin (CL) and beta-2-glycoprotein I (β2GPI) were evaluated by performing immunoblot, ELISA and absorption experiments. A mice model of passively induced APS was adopted. Although sPLA2 activities were identified in all B19-tVP1u proteins, only amino acid residues 61-227 B19-tVP1u exhibited a higher sPLA2 activity. Autoantibodies against CL and β2GPI exhibited binding activities with all B19-tVP1u proteins. IgG that was purified from mice that had been immunized with amino acid residues 21-227 to 121-227 B19-tVP1u proteins exhibited significantly higher binding activity with CL. IgG that was purified from mice that had been immunized with amino acid residues 21-227, 31-227, 82-227 and 91-227 B19-tVP1u proteins exhibited significantly higher binding activity with β2GPI. Accordingly, significantly higher binding inhibition of CL was detected in the presence of amino acid residues 61-227 and 101-227 B19-tVP1u. Significantly higher binding inhibition of β2GPI was detected in the presence of amino acid residues 21-227, 31-227, 82-227 and 91-227 B19-tVP1u. The mice that received amino acid residues 31-227 or 61-227 anti-tB19-VP1u IgG revealed significant thrombocytopenia and those that received amino acid residues 21-227, 31-227, 61-227, 71-227, 82-227, 91-227, 101-227 or 114-227 anti-tB19-VP1u IgG exhibited significantly prolonged aPTT. These findings provide further information concerning the role of B19-VP1u antigenicity in APS-like autoimmunity.

Sugar-binding and crystallographic studies of an arabinose-binding protein mutant (Met108Leu) that exhibits enhanced affinity and altered specificity.

PubMed

Vermersch, P S; Lemon, D D; Tesmer, J J; Quiocho, F A

1991-07-16

In addition to hydrogen bonds, van der Waals forces contribute to the affinity of protein-carbohydrate interactions. Nonpolar van der Waals contacts in the complexes of the L-arabinose-binding protein (ABP) with monosaccharides have been studied by means of site-directed mutagenesis, equilibrium and rapid kinetic binding techniques, and X-ray crystallography. ABP, a periplasmic transport receptor of Escherichia coli, binds L-arabinose, D-galactose, and D-fucose with preferential affinity in the order of Ara greater than Gal much greater than Fuc. Well-refined, high-resolution structures of ABP complexed with the three sugars revealed that the structural differences in the ABP-sugar complexes are localized around C5 of the sugars, where the equatorial H of Ara has been substituted for CH3 (Fuc) or CH2OH (Gal). The side chain of Met108 undergoes a sterically dictated, ligand-specific, conformational change to optimize nonpolar interactions between its methyl group and the sugar. We found that the Met108Leu ABP binds Gal tighter than wild-type ABP binds Ara and exhibits a preference for ligand in the order of Gal much greater than Fuc greater than Ara. The differences in affinity can be attributed to differences in the dissociation rates of the ABP-sugar complexes. We have refined at better than 1.7-A resolution the crystal structures of the Met108Leu ABP complexed with each of the sugars and offer a molecular explanation for the altered binding properties.

Characterization of WY 14,643 and its Complex with Aldose Reductase

DOE PAGES

Sawaya, Michael R.; Verma, Malkhey; Balendiran, Vaishnavi; ...

2016-10-10

The peroxisome proliferator, WY 14,643 exhibits a pure non-competitive inhibition pattern in the aldehyde reduction and in alcohol oxidation activities of human Aldose reductase (hAR). Fluorescence emission measurements of the equilibrium dissociation constants, Kd, of oxidized (hAR•NADP+) and reduced (hAR•NADPH) holoenzyme complexes display a 2-fold difference between them. Kd values for the dissociation of WY 14,643 from the oxidized (hAR•NADP+•WY 14,643) and reduced (hAR•NADPH•WY 14,643) ternary complexes are comparable to each other. The ternary complex structure of hAR•NADP+•WY 14,643 reveals the first structural evidence of a fibrate class drug binding to hAR. These observations demonstrate how fibrate molecules such asmore » WY 14,643, besides being valued as agonists for PPAR, also inhibit hAR.« less

Characterization of WY 14,643 and its Complex with Aldose Reductase

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

Sawaya, Michael R.; Verma, Malkhey; Balendiran, Vaishnavi

The peroxisome proliferator, WY 14,643 exhibits a pure non-competitive inhibition pattern in the aldehyde reduction and in alcohol oxidation activities of human Aldose reductase (hAR). Fluorescence emission measurements of the equilibrium dissociation constants, Kd, of oxidized (hAR•NADP+) and reduced (hAR•NADPH) holoenzyme complexes display a 2-fold difference between them. Kd values for the dissociation of WY 14,643 from the oxidized (hAR•NADP+•WY 14,643) and reduced (hAR•NADPH•WY 14,643) ternary complexes are comparable to each other. The ternary complex structure of hAR•NADP+•WY 14,643 reveals the first structural evidence of a fibrate class drug binding to hAR. These observations demonstrate how fibrate molecules such asmore » WY 14,643, besides being valued as agonists for PPAR, also inhibit hAR.« less

Biomolecular interactions modulate macromolecular structure and dynamics in atomistic model of a bacterial cytoplasm.

PubMed

Yu, Isseki; Mori, Takaharu; Ando, Tadashi; Harada, Ryuhei; Jung, Jaewoon; Sugita, Yuji; Feig, Michael

2016-11-01

Biological macromolecules function in highly crowded cellular environments. The structure and dynamics of proteins and nucleic acids are well characterized in vitro, but in vivo crowding effects remain unclear. Using molecular dynamics simulations of a comprehensive atomistic model cytoplasm we found that protein-protein interactions may destabilize native protein structures, whereas metabolite interactions may induce more compact states due to electrostatic screening. Protein-protein interactions also resulted in significant variations in reduced macromolecular diffusion under crowded conditions, while metabolites exhibited significant two-dimensional surface diffusion and altered protein-ligand binding that may reduce the effective concentration of metabolites and ligands in vivo. Metabolic enzymes showed weak non-specific association in cellular environments attributed to solvation and entropic effects. These effects are expected to have broad implications for the in vivo functioning of biomolecules. This work is a first step towards physically realistic in silico whole-cell models that connect molecular with cellular biology.

Nickel-quinolones interaction. Part 4. Structure and biological evaluation of nickel(II)-enrofloxacin complexes compared to zinc(II) analogues.

PubMed

Skyrianou, Kalliopi C; Psycharis, Vassilis; Raptopoulou, Catherine P; Kessissoglou, Dimitris P; Psomas, George

2011-01-01

The nickel(II) complexes with the second-generation quinolone antibacterial agent enrofloxacin in the presence or absence of the nitrogen-donor heterocyclic ligands 1,10-phenanthroline, 2,2'-bipyridine or pyridine have been synthesized and characterized. Enrofloxacin acts as bidentate ligand coordinated to Ni(II) ion through the ketone oxygen and a carboxylato oxygen. The crystal structure of (1,10-phenanthroline)bis(enrofloxacinato)nickel(II) has been determined by X-ray crystallography. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that they bind to CT DNA and bis(pyridine)bis(enrofloxacinato)nickel(II) exhibits the highest binding constant to CT DNA. The cyclic voltammograms of the complexes have shown that in the presence of CT DNA the complexes can bind to CT DNA by the intercalative binding mode which has also been verified by DNA solution viscosity measurements. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. The complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values. The biological properties of the complexes have been evaluated in comparison to the corresponding Zn(II) enrofloxacinato complexes as well as Ni(II) complexes with the first-generation quinolone oxolinic acid. Copyright © 2010 Elsevier Inc. All rights reserved.

Electronic structure of Fe- vs. Ru-based dye molecules

NASA Astrophysics Data System (ADS)

Johnson, Phillip S.; Cook, Peter L.; Zegkinoglou, Ioannis; García-Lastra, J. M.; Rubio, Angel; Ruther, Rose E.; Hamers, Robert J.; Himpsel, F. J.

2013-01-01

In order to explore whether Ru can be replaced by inexpensive Fe in dye molecules for solar cells, the differences in the electronic structure of Fe- and Ru-based dyes are investigated by X-ray absorption spectroscopy and first-principles calculations. Molecules with the metal in a sixfold, octahedral N cage, such as tris(bipyridines) and tris(phenanthrolines), exhibit a systematic downward shift of the N 1s-to-π* transition when Ru is replaced by Fe. This shift is explained by an extra transfer of negative charge from the metal to the N ligands in the case of Fe, which reduces the binding energy of the N 1s core level. The C 1s-to-π* transitions show the opposite trend, with an increase in the transition energy when replacing Ru by Fe. Molecules with the metal in a fourfold, planar N cage (porphyrins) exhibit a more complex behavior due to a subtle competition between the crystal field, axial ligands, and the 2+ vs. 3+ oxidation states.

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

Iwasaki, Yuko; Iwasaki, Hitoshi; Yatoh, Shigeru

Transgenic mice expressing nuclear sterol regulatory element-binding protein-1a under the control of the insulin promoter were generated to determine the role of SREBP-1a in pancreatic {beta}-cells. Only low expressors could be established, which exhibited mild hyperglycemia, impaired glucose tolerance, and reduced plasma insulin levels compared to C57BL/6 controls. The islets isolated from the transgenic mice were fewer and smaller, and had decreased insulin content and unaltered glucagon staining. Both glucose- and potassium-stimulated insulin secretions were decreased. The transgenic islets consistently expressed genes for fatty acids and cholesterol synthesis, resulting in accumulation of triglycerides but not cholesterol. PDX-1, {beta}{epsilon}{tau}{alpha}2, MafA, andmore » IRS-2 were suppressed, partially explaining the loss and dysfunction of {beta}-cell mass. The transgenic mice on a high fat/high sucrose diet still exhibited impaired insulin secretion and continuous {beta}-cell growth defect. Therefore, nuclear SREBP-1a, even at a low level, strongly disrupts {beta}-cell mass and function.« less

Stuttering Min oscillations within E. coli bacteria: a stochastic polymerization model

NASA Astrophysics Data System (ADS)

Sengupta, Supratim; Derr, Julien; Sain, Anirban; Rutenberg, Andrew D.

2012-10-01

We have developed a 3D off-lattice stochastic polymerization model to study the subcellular oscillation of Min proteins in the bacteria Escherichia coli, and used it to investigate the experimental phenomenon of Min oscillation stuttering. Stuttering was affected by the rate of immediate rebinding of MinE released from depolymerizing filament tips (processivity), protection of depolymerizing filament tips from MinD binding and fragmentation of MinD filaments due to MinE. Processivity, protection and fragmentation each reduce stuttering, speed oscillations and MinD filament lengths. Neither processivity nor tip protection were, on their own, sufficient to produce fast stutter-free oscillations. While filament fragmentation could, on its own, lead to fast oscillations with infrequent stuttering; high levels of fragmentation degraded oscillations. The infrequent stuttering observed in standard Min oscillations is consistent with short filaments of MinD, while we expect that mutants that exhibit higher stuttering frequencies will exhibit longer MinD filaments. Increased stuttering rate may be a useful diagnostic to find observable MinD polymerization under experimental conditions.

The capsule plays an important role in Escherichia coli K1 interactions with Acanthamoeba.

PubMed

Jung, Suk-Yul; Matin, Abdul; Kim, Kwang Sik; Khan, Naveed Ahmed

2007-03-01

Escherichia coli K1 is shown to bind to, associate with, invade and survive inside Acanthamoeba, but the precise mechanisms associated with these events are unclear. We have previously shown that outer membrane protein A and lipopolysaccharide are critical bacterial determinants involved in E. coli K1 interactions with Acanthamoeba. Using an isogenic K1 capsule-deletion mutant (lacking the neuDB genes cluster that is necessary for the production of cytoplasmic precursors to the exopolysaccharide capsule), we observed that the capsule modulates and enhances E. coli K1 association and survival inside Acanthamoeba. The capsule-deletion mutant exhibited significantly reduced association compared with the wild type strain, E44. Similarly, the K1 capsule-deletion mutant exhibited limited ability for invasion/uptake by and survival inside Acanthamoeba. Next, we determined whether E. coli K1 survive inside Acanthamoeba during the encystment process and that viable bacteria can be isolated from the mature cysts. Using encystment assays, our findings revealed that E. coli K1, but not its capsule-deletion mutant, exhibit survival inside Acanthamoeba cysts. We believe this is the first demonstration that the K1 capsule plays an important role in E. coli K1 interactions with Acanthamoeba.

Linear scaffolds for multivalent targeting of melanocortin receptors.

PubMed

Dehigaspitiya, Dilani Chathurika; Anglin, Bobbi L; Smith, Kara R; Weber, Craig S; Lynch, Ronald M; Mash, Eugene A

2015-12-21

Molecules bearing one, two, three, or four copies of the tetrapeptide His-dPhe-Arg-Trp were attached to scaffolds based on ethylene glycol, glycerol, and d-mannitol by means of the copper-assisted azide-alkyne cyclization. The abilities of these compounds to block binding of a probe at the melanocortin 4 receptor were evaluated using a competitive binding assay. All of the multivalent molecules studied exhibited 30- to 40-fold higher apparent affinites when compared to a monovalent control. These results are consistent with divalent binding to receptor dimers. No evidence for tri- or tetravalent binding was obtained. Differences in the interligand spacing required for divalent binding, as opposed to tri- or tetravalent binding, may be responsible for these results.

Novel Autophosphorylation Sites of Src Family Kinases Regulate Kinase Activity and SH2 Domain Binding Capacity

PubMed Central

Weir, Marion E.; Mann, Jacqueline E.; Corwin, Thomas; Fulton, Zachary W.; Hao, Jennifer M.; Maniscalco, Jeanine F.; Kenney, Marie C.; Roque, Kristal M. Roman; Chapdelaine, Elizabeth F.; Stelzl, Ulrich; Deming, Paula B.; Ballif, Bryan A.; Hinkle, Karen L.

2016-01-01

Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly-regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly-phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the site C-terminal to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. PMID:27001024

Effects of asparagine mutagenesis of conserved aspartic acids in helix two (D2.50) and three (D3.32) of M1 – M4 muscarinic receptors on the irreversible binding of nitrogen mustard analogs of acetylcholine and McN-A-343

PubMed Central

Suga, Hinako; Ehlert, Frederick J.

2013-01-01

We investigated how asparagine mutagenesis of conserved aspartic acids in helix two (D2.50) and three (D3.32) of M1 – M4 muscarinic receptors alters the irreversible binding of acetylcholine mustard and BR384 (4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate), a nitrogen mustard derivative of McN-A-343 ([4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl] trimethylammonium chloride). The D2.50N mutation moderately increased the affinity of the aziridinium ions of acetylcholine mustard and BR384 for M2 – M4 receptors and had little effect on the rate constant for receptor alkylation. The D3.32N mutation greatly reduced the rate constant for receptor alkylation by acetylcholine mustard, but not by BR384, although the affinity of BR384 was reduced. The combination of both mutations (D2.50N/D3.32N) substantially reduced the rate constant for receptor alkylation by BR384 relative to wild type and mutant D2.50N and D3.32N receptors. The change in binding affinity caused by the mutations suggests that the D2.50N mutation alters the interaction of acetylcholine mustard with D3.32 of M1 and M3 receptors, but not that of the M4 receptor. BR384 exhibited the converse relationship. The simplest explanation is that acetylcholine mustard and BR384 alkylate at least two residues on M1 – M4 receptors and that the D2.50N mutation alters the rate of alkylation of D3.32 relative to another residue, perhaps D2.50 itself. PMID:23826889

Effect of Methamphetamine on Spectral Binding, Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4

PubMed Central

Ande, Anusha; Wang, Lei; Vaidya, Naveen K.; Li, Weihua; Kumar, Santosh; Kumar, Anil

2016-01-01

Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δAmax and KD of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δAmax (0.004±0.0003 vs. 0.0068±0.0001) and KD (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δAmax (0.0038±0.0003 vs. 0.0055±0.0003) and KD (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced KD in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir-boosted antiretroviral therapy, in HIV-1-infected individuals who abuse methamphetamine. PMID:26741368

Insights into resistance mechanism of the macrolide biosensor protein MphR(A) binding to macrolide antibiotic erythromycin by molecular dynamics simulation

NASA Astrophysics Data System (ADS)

Feng, Tingting; Zhang, Yanjun; Ding, Jing-Na; Fan, Song; Han, Ju-Guang

2015-12-01

Macrolide biosensor protein MphR(A) has been known as a key regulatory protein in metabolite sensing and genetic expression regulating. MphR(A) protein binds to macrolide antibiotic erythromycin (Ery) and releases the gene operon, thus activates expression of the mphA gene and initiates Ery resistance. The two mutant amino acid residues (V66L and V126L) might potentially disrupt Ery binding to MphR(A). In these studies, the binding of macrolide antibiotic Ery to wild type (Wt) MphR(A) and double mutant (V66L/V126L) MphR(A) are explored by molecular dynamics simulations. Compared to the Apo-MphR(A) protein and Wt-MphR(A)-Ery complex, many interesting effects owing to the double mutant (V66L/V126L) are discovered. In the case of Ery, Helix I which plays an important role in transcription shows itself a right-hand α helix in Wt-MphR(A)-Ery, whereas the activated helix is broken down in double mutant-V66L/V126L-MphR(A)-Ery. The calculated results exhibit that the double mutant V66L/V126L reduces the binding affinity of the V66L/V126L-MphR(A) to Ery, resulting in the block of Ery resistance. The binding free energy decomposition analysis reveals that the decrease of the binding affinity for the variant V66L/V126L-MphR(A)-Ery is mainly attributed to the gas phase electrostatic energies. The residue Leu66, Thr154, and Arg122 enhance the binding affinity of V66L/V126L-MphR(A) to Ery. The residues Tyr103 and His147 contributes mainly to binding energies in the Wt-MphR(A)-Ery complex, whereas the two residues have no contribution to the binding free energy inV66L/V126L-MphR(A)-Ery complex. Our study gives useful insights into the nature of amino acids mutation effect, the mechanism of blocking drug resistance at the atomic level and the characteristics in binding affinity for Ery to double mutant (V66L/V126L) MphR(A), which will contribute to the design of more effective macrolide antibiotics.

Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM family.

PubMed

Nallapareddy, Sreedhar R; Weinstock, George M; Murray, Barbara E

2003-03-01

A collagen-binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen-binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin-resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI-binding vancomycin-resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non-CI-binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration-dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm-specific IgGs purified from anti-recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen-binding clinical isolates of E. faecium tested, but in none of the strains with a non-functional pseudo acm gene. Introduction of a functional acm gene into two non-CI-binding natural acm mutant strains conferred a CI-binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection-derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the ability of E. faecium to bind collagen.

Rearranged diterpenoids from the biotransformation of ent-trachyloban-18-oic acid by Rhizopus arrhizus.

PubMed

Leverrier, Aurélie; Martin, Marie-Thérèse; Servy, Claudine; Ouazzani, Jamal; Retailleau, Pascal; Awang, Khalijah; Mukhtar, Mat Ropi; Guéritte, Françoise; Litaudon, Marc

2010-06-25

In our search for inhibitors of the antiapoptotic protein Bcl-xL, investigation of Xylopia caudata afforded a new diterpenoid, ent-trachyloban-4beta-ol (2), and five known ent-trachylobane or ent-atisane compounds. Only ent-trachyloban-18-oic acid (1) exhibited weak binding activity to Bcl-xL. These compounds exhibited cytotoxicity against KB and HCT-116 cell lines with IC(50) values between 10 and 30 microM. Bioconversion of compound 1 by Rhizopus arrhizus afforded new hydroxylated metabolites (3-7) of the ent-trachylobane and ent-kaurene type and compound 8, with a rearranged pentacyclic carbon framework that was named rhizopene. Compounds 3-8 were noncytotoxic to the two cancer cell lines, and compounds 3 and 5 exhibited only weak binding affinity for Bcl-xL.

Crystal structure correlations with the intrinsic thermodynamics of human carbonic anhydrase inhibitor binding

PubMed Central

Smirnov, Alexey; Zubrienė, Asta; Manakova, Elena; Gražulis, Saulius

2018-01-01

The structure-thermodynamics correlation analysis was performed for a series of fluorine- and chlorine-substituted benzenesulfonamide inhibitors binding to several human carbonic anhydrase (CA) isoforms. The total of 24 crystal structures of 16 inhibitors bound to isoforms CA I, CA II, CA XII, and CA XIII provided the structural information of selective recognition between a compound and CA isoform. The binding thermodynamics of all structures was determined by the analysis of binding-linked protonation events, yielding the intrinsic parameters, i.e., the enthalpy, entropy, and Gibbs energy of binding. Inhibitor binding was compared within structurally similar pairs that differ by para- or meta-substituents enabling to obtain the contributing energies of ligand fragments. The pairs were divided into two groups. First, similar binders—the pairs that keep the same orientation of the benzene ring exhibited classical hydrophobic effect, a less exothermic enthalpy and a more favorable entropy upon addition of the hydrophobic fragments. Second, dissimilar binders—the pairs of binders that demonstrated altered positions of the benzene rings exhibited the non-classical hydrophobic effect, a more favorable enthalpy and variable entropy contribution. A deeper understanding of the energies contributing to the protein-ligand recognition should lead toward the eventual goal of rational drug design where chemical structures of ligands could be designed based on the target protein structure. PMID:29503769

Supramolecular approach to enantioselective DNA recognition using enantiomerically resolved cationic 4-amino-1,8-naphthalimide-based Tröger's bases.

PubMed

Banerjee, Swagata; Bright, Sandra A; Smith, Jayden A; Burgeat, Jeremy; Martinez-Calvo, Miguel; Williams, D Clive; Kelly, John M; Gunnlaugsson, Thorfinnur

2014-10-03

The synthesis and photophysical studies of two cationic Tröger's base (TB)-derived bis-naphthalimides 1 and 2 and the TB derivative 6, characterized by X-ray crystallography, are presented. The enantiomers of 1 and 2 are separated by cation-exchange chromatography on Sephadex C25 using sodium (-)-dibenzoyl-l-tartarate as the chiral mobile phase. The binding of enantiomers with salmon testes (st)-DNA and synthetic polynucleotides are studied by a variety of spectroscopic methods including UV/vis absorbance, circular dichroism, linear dichroism, and ethidium bromide displacement assays, which demonstrated binding of these compounds to the DNA grooves with very high affinity (K ∼ 10(6) M(-1)) and preferential binding of (-)-enantiomer. In all cases, binding to DNA resulted in a significant stabilization of the double-helical structure of DNA against thermal denaturation. Compound (±)-2 and its enantiomers possessed significantly higher binding affinity for double-stranded DNA compared to 1, possibly due to the presence of the methyl group, which allows favorable hydrophobic and van der Waals interactions with DNA. The TB derivatives exhibited marked preference for AT rich sequences, where the binding affinities follow the order (-)-enantiomer > (±) > (+)-enantiomer. The compounds exhibited significant photocleavage of plasmid DNA upon visible light irradiation and are rapidly internalized into malignant cell lines.

Haptoglobin preferentially binds β but not α subunits cross-linked hemoglobin tetramers with minimal effects on ligand and redox reactions.

PubMed

Jia, Yiping; Wood, Francine; Buehler, Paul W; Alayash, Abdu I

2013-01-01

Human hemoglobin (Hb) and haptoglobin (Hp) exhibit an extremely high affinity for each other, and the dissociation of Hb tetramers into dimers is generally believed to be a prerequisite for complex formation. We have investigated Hp interactions with native Hb, αα, and ββ cross-linked Hb (ααXLHb and ββXLHb, respectively), and rapid kinetics of Hb ligand binding as well as the redox reactivity in the presence of and absence of Hp. The quaternary conformation of ββ subunit cross-linking results in a higher binding affinity than that of αα subunit cross-linked Hb. However, ββ cross-linked Hb exhibits a four fold slower association rate constant than the reaction rate of unmodified Hb with Hp. The Hp contact regions in the Hb dimer interfaces appear to be more readily exposed in ββXLHb than ααXLHb. In addition, apart from the functional changes caused by chemical modifications, Hp binding does not induce appreciable effects on the ligand binding and redox reactions of ββXLHb. Our findings may therefore be relevant to the design of safer Hb-based oxygen therapeutics by utilizing this preferential binding of ββXLHb to Hp. This may ultimately provide a safe oxidative inactivation and clearance pathway for chemically modified Hbs in circulation.

WalK(S221P), a naturally occurring mutation, confers vancomycin resistance in VISA strain XN108.

PubMed

Peng, Huagang; Hu, Qiwen; Shang, Weilong; Yuan, Jizhen; Zhang, Xiaopeng; Liu, Hui; Zheng, Ying; Hu, Zhen; Yang, Yi; Tan, Li; Li, Shu; Hu, Xiaomei; Li, Ming; Rao, Xiancai

2017-04-01

Vancomycin-intermediate Staphylococcus aureus (VISA) strains have spread globally. We previously isolated an ST239 VISA (XN108) with a vancomycin MIC of 12 mg/L. The mechanism for XN108 resistance to vancomycin was investigated in this study. Genome comparison was performed to characterize mutations that might contribute to the XN108 resistance phenotype. The novel mutation WalK(S221P) was identified and investigated using allelic replacement experiments. Vancomycin susceptibilities, autolytic activities and morphologies of the strains were examined. Autophosphorylation activities of WalK and the WalK(S221P) mutant were determined in vitro with [λ- 32 P]ATP, and binding activity of WalK(S221P)-activated WalR to the promoter region of its target gene lytM was determined by electrophoretic mobility shift assay. Genome comparison revealed three mutations, GraS(T136I), RpoB(H481N) and WalK(S221P), which might be responsible for vancomycin resistance in XN108. The introduction of WalK(S221P) to the vancomycin-susceptible strain N315 increased its vancomycin MIC from 1.5 to 8 mg/L, whereas the allelic replacement of WalK(S221P) with the native N315 WalK allele in XN108 decreased its vancomycin MIC from 12 to 4 mg/L. The VISA strains have thickened cell walls and decreased autolysis, consistent with observed changes in the expression of genes involved in cell wall metabolism and virulence regulation. WalK(S221P) exhibited reduced autophosphorylation, which may lead to reduced phosphorylation of WalR. WalK(S221P)-phosphorylated WalR also exhibited a reduced capacity to bind to the lytM promoter. The naturally occurring WalK(S221P) mutation plays a key role in vancomycin resistance in XN108. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The Role of Aging in Intra-Item and Item-Context Binding Processes in Visual Working Memory

ERIC Educational Resources Information Center

Peterson, Dwight J.; Naveh-Benjamin, Moshe

2016-01-01

Aging is accompanied by declines in both working memory and long-term episodic memory processes. Specifically, important age-related memory deficits are characterized by performance impairments exhibited by older relative to younger adults when binding distinct components into a single integrated representation, despite relatively intact memory…

An in vitro study on the anti-adherence effect of Brucea javanica and Piper betle extracts towards oral Candida.

PubMed

Nordin, Mohd-Al-Faisal; Wan Harun, Wan Himratul Aznita; Abdul Razak, Fathilah

2013-10-01

The adherence of Candida to mucosal surfaces is the initial step for successful invasive process of the oral cavity. The study aimed to investigate the effect of two plant extracts on the non-specific and specific bindings of oral candida. In the former, adsorption to hexadecane was used to measure the hydrophobic interaction of the candida cells. In the later, glass beads coated with saliva represented the experimental pellicles in specific adhesion of oral candida to hard tissue surface. Candida krusei, Candida dubliniensis and Candida tropicalis showed the highest adsorption to hexadecane at 30.23%, 26.19% and 19.70%, respectively, while the others within the range of 7-10%. All candidal species were significantly affected by the extracts (P<0.05) with Brucea javanica exhibited more than 60% reduction of CSH than Piper betle. Candida parapsilosis showed the highest affinity in specific-bindings to pellicle with 18.72±0.71×10(5)CFU/ml. Exposing to P. betle-treated pellicle has drastically reduced the adherence of C. tropicalis, Candida albicans and C. krusei by 86.01%, 61.41% and 56.34%, respectively. B. javanica exhibited similar effect on C. tropicalis (89.86%), Candida lusitaniae (88.95%), C. albicans (79.74%), Candida glabrata (76.85%) and C. krusei (67.61%). The extracts demonstrated anti-adherence activities by modifying the CSH and the characteristics of the experimental pellicle. Copyright © 2013 Elsevier Ltd. All rights reserved.

Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl, oxygen-rebound mechanism

PubMed Central

Kim, Seonah; Ståhlberg, Jerry; Sandgren, Mats; Paton, Robert S.; Beckham, Gregg T.

2014-01-01

Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η1-superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η1) to copper, and that a copper-oxyl–mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds. PMID:24344312

Alteration of blood clotting and lung damage by protamine are avoided using the heparin and polyphosphate inhibitor UHRA

PubMed Central

Abraham, Libin; Kapopara, Piyushkumar R.; Lai, Benjamin F. L.; Shenoi, Rajesh A.; Rosell, Federico I.; Conway, Edward M.; Pryzdial, Edward L. G.; Haynes, Charles A.

2017-01-01

Anticoagulant therapy–associated bleeding and pathological thrombosis pose serious risks to hospitalized patients. Both complications could be mitigated by developing new therapeutics that safely neutralize anticoagulant activity and inhibit activators of the intrinsic blood clotting pathway, such as polyphosphate (polyP) and extracellular nucleic acids. The latter strategy could reduce the use of anticoagulants, potentially decreasing bleeding events. However, previously described cationic inhibitors of polyP and extracellular nucleic acids exhibit both nonspecific binding and adverse effects on blood clotting that limit their use. Indeed, the polycation used to counteract heparin-associated bleeding in surgical settings, protamine, exhibits adverse effects. To address these clinical shortcomings, we developed a synthetic polycation, Universal Heparin Reversal Agent (UHRA), which is nontoxic and can neutralize the anticoagulant activity of heparins and the prothrombotic activity of polyP. Sharply contrasting protamine, we show that UHRA does not interact with fibrinogen, affect fibrin polymerization during clot formation, or abrogate plasma clotting. Using scanning electron microscopy, confocal microscopy, and clot lysis assays, we confirm that UHRA does not incorporate into clots, and that clots are stable with normal fibrin morphology. Conversely, protamine binds to the fibrin clot, which could explain how protamine instigates clot lysis and increases bleeding after surgery. Finally, studies in mice reveal that UHRA reverses heparin anticoagulant activity without the lung injury seen with protamine. The data presented here illustrate that UHRA could be safely used as an antidote during adverse therapeutic modulation of hemostasis. PMID:28034889

Functional characterization of mongoose nicotinic acetylcholine receptor alpha-subunit: resistance to alpha-bungarotoxin and high sensitivity to acetylcholine.

PubMed

Asher, O; Lupu-Meiri, M; Jensen, B S; Paperna, T; Fuchs, S; Oron, Y

1998-07-24

The mongoose is resistant to snake neurotoxins. The mongoose muscle nicotinic acetylcholine receptor (AChR) alpha-subunit contains a number of mutations in the ligand-binding domain and exhibits poor binding of alpha-bungarotoxin (alpha-BTX). We characterized the functional properties of a hybrid (alpha-mongoose/beta gamma delta-rat) AChR. Hybrid AChRs, expressed in Xenopus oocytes, respond to acetylcholine with depolarizing current, the mean maximal amplitude of which was greater than that mediated by the rat AChR. The IC50 of alpha-BTX to the hybrid AChR was 200-fold greater than that of the rat, suggesting much lower affinity for the toxin. Hybrid AChRs exhibited an apparent higher rate of desensitization and higher affinity for ACh (EC50 1.3 vs. 23.3 microM for the rat AChR). Hence, changes in the ligand-binding domain of AChR not only affect the binding properties of the receptor, but also result in marked changes in the characteristics of the current.

C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger.

PubMed

Xu, Wenxuan; Liu, Yajuan; Ye, Yanxin; Liu, Meng; Han, Laichuang; Song, Andong; Liu, Liangwei

2016-10-01

The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module. A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn. C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.

Region-specific associations between sex, social status, and oxytocin receptor density in the brains of eusocial rodents.

PubMed

Mooney, S J; Coen, C W; Holmes, M M; Beery, A K

2015-09-10

Naturally occurring variations in neuropeptide receptor distributions in the brain contribute to numerous mammalian social behaviors. In naked mole-rats, which live in large social groups and exhibit remarkable reproductive skew, colony-related social behaviors vary with reproductive status. Here we examined whether variation in social status is associated with variations in the location and/or density of oxytocin binding in this species. Autoradiography was performed to assess forebrain oxytocin receptor (OTR) densities in breeding and non-breeding naked mole-rats of both sexes. Overall, males exhibited higher OTR binding in the medial amygdala in comparison to females. While there were no main effects of reproductive status in any region, a sex difference in OTR binding in the nucleus accumbens was mediated by status. Specifically, breeding males tended to have more OTR binding than breeding females in the nucleus accumbens, while no sex difference was observed in subordinates. These effects suggest that oxytocin may act in a sex- and region-specific way that corresponds to reproductive status and associated social behaviors. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Toxoplasma gondii peptide ligands open the gate of the HLA class I binding groove

PubMed Central

McMurtrey, Curtis; Trolle, Thomas; Sansom, Tiffany; Remesh, Soumya G; Kaever, Thomas; Bardet, Wilfried; Jackson, Kenneth; McLeod, Rima; Sette, Alessandro; Nielsen, Morten; Zajonc, Dirk M; Blader, Ira J; Peters, Bjoern; Hildebrand, William

2016-01-01

HLA class I presentation of pathogen-derived peptide ligands is essential for CD8+ T-cell recognition of Toxoplasma gondii infected cells. Currently, little data exist pertaining to peptides that are presented after T. gondii infection. Herein we purify HLA-A*02:01 complexes from T. gondii infected cells and characterize the peptide ligands using LCMS. We identify 195 T. gondii encoded ligands originating from both secreted and cytoplasmic proteins. Surprisingly, T. gondii ligands are significantly longer than uninfected host ligands, and these longer pathogen-derived peptides maintain a canonical N-terminal binding core yet exhibit a C-terminal extension of 1–30 amino acids. Structural analysis demonstrates that binding of extended peptides opens the HLA class I F’ pocket, allowing the C-terminal extension to protrude through one end of the binding groove. In summary, we demonstrate that unrealized structural flexibility makes MHC class I receptive to parasite-derived ligands that exhibit unique C-terminal peptide extensions. DOI: http://dx.doi.org/10.7554/eLife.12556.001 PMID:26824387

Mechanism and Substrate Recognition of Human Holo ACP Synthase

PubMed Central

Bunkoczi, Gabor; Pasta, Saloni; Joshi, Anil; Wu, Xiaoqiu; Kavanagh, Kathryn L.; Smith, Stuart; Oppermann, Udo

2007-01-01

Summary Mammals utilize a single phosphopantetheinyl transferase for the posttranslational modification of at least three different apoproteins: the carrier protein components of cytosolic and mitochondrial fatty acid synthases and the aminoadipate semialdehyde reductase involved in lysine degradation. We determined the crystal structure of the human phosphopantetheinyl transferase, a eukaryotic phosphopantetheinyl transferase characterized, complexed with CoA and Mg2+, and in ternary complex with CoA and ACP. The involvement of key residues in ligand binding and catalysis was confirmed by mutagenesis and kinetic analysis. Human phosphopantetheinyl transferase exhibits an α/β fold and 2-fold pseudosymmetry similar to the Sfp phosphopantetheinyl transferase from Bacillus subtilis. Although the bound ACP exhibits a typical four-helix structure, its binding is unusual in that it is facilitated predominantly by hydrophobic interactions. A detailed mechanism is proposed describing the substrate binding and catalytic process. PMID:18022563

Photocontrol of the expression of genes encoding chlorophyll a/b binding proteins and small subunit of ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (L. ) and Nicotiana tabacum (L. )

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

Wehmeyer, B.; Cashmore, A.R.; Schaefer, E.

Phytochrome and the blue ultraviolet-A photoreceptor control light-induced expression of genes encoding the chlorophyll a/b binding protein of photosystem II and photosystem I and the genes for the small subunit of the ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (tomato) and Nicotiana tabacum (tobacco). A high irradiance response also controls the induction of these genes. Genes encoding photosystem II- and I-associated chlorophyll a/b binding proteins both exhibit a transient rapid increase in expression in response to light pulse or to continuous irradiation. In contrast, genes encoding the small subunit exhibit a continuous increase in expression in response to light.more » These distinct expression characteristics are shown to reflect differences at the level of transcription.« less

Relationship between L-DOPA-induced reduction in motor and exploratory activity and degree of DAT binding in the rat

PubMed Central

Nikolaus, Susanne; Beu, Markus; De Souza Silva, Angelica Maria; Huston, Joseph P.; Hautzel, Hubertus; Chao, Owen Y.; Antke, Christina; Müller, Hans-Wilhelm

2014-01-01

Purpose: The present study assessed the influence of L-DOPA administration on neostriatal dopamine (DA) transporter (DAT) binding in relation to motor and exploratory behaviors in the rat. Methods: Rats received injections of 5 mg/kg L-DOPA, 10 mg/kg L-DOPA or vehicle. Motor and exploratory behaviors were assessed for 30 min in an open field prior to administration of [123I]FP-CIT. Dopamine transporter binding was measured with small animal single-photon emission computed tomography (SPECT) 2 h after radioligand administration for 60 min. Results: Both L-DOPA doses significantly reduced DAT binding and led to significantly less head-shoulder motility and more sitting relative to vehicle. Moreover, 10 mg/kg L-DOPA induced less distance traveled and ambulation than 5 mg/kg L-DOPA. Analysis of time-behavior (t-b) curves showed that L-DOPA-treated animals relative to vehicle exhibited (1) a faster rate of increase in duration of sitting; (2) a slower rate of increase in duration of head-shoulder motility; and (3) a slower rate of decrease in frequency of head-shoulder motility. Conclusions: The reductions of striatal DAT binding after L-DOPA challenges reflected elevated concentrations of synaptic DA. L-DOPA-treated animals showed less head-shoulder motility and more sitting than vehicle-treated animals, indicating an association between less behavioral activity and increased availability of striatal DA. The faster increase of sitting duration to a higher final level and the slower increase of head-shoulder motility to a lower final level relative to controls may be interpreted in terms on behavioral habituation to a novel environment. PMID:25566000

Neutralizing Epitopes in the Membrane-Proximal External Region of HIV-1 gp41 Are Influenced by the Transmembrane Domain and the Plasma Membrane

PubMed Central

Montero, Marinieve; Klaric, Kristina-Ana; Donald, Jason E.; Lepik, Christa; Wu, Sampson; Tsai, Sue; Julien, Jean-Philippe; Hessell, Ann J.; Wang, Shixia; Lu, Shan; Burton, Dennis R.; Pai, Emil F.; DeGrado, William F.

2012-01-01

Failure to elicit broadly neutralizing (bNt) antibodies (Abs) against the membrane-proximal external region of HIV-1 gp41 (MPER) reflects the difficulty of mimicking its neutralization-competent structure (NCS). Here, we analyzed MPER antigenicity in the context of the plasma membrane and identified a role for the gp41 transmembrane domain (TM) in exposing the epitopes of three bNt monoclonal Abs (MAbs) (2F5, 4E10, and Z13e1). We transiently expressed DNA constructs encoding gp41 ectodomain fragments fused to either the TM of the platelet-derived growth factor receptor (PDGFR) or the gp41 TM and cytoplasmic tail domain (CT). Constructs encoding the MPER tethered to the gp41 TM followed by a 27-residue CT fragment (MPER-TM1) produced optimal MAb binding. Critical binding residues for the three Nt MAbs were identified using a panel of 24 MPER-TM1 mutants bearing single amino acid substitutions in the MPER; many were previously shown to affect MAb-mediated viral neutralization. Moreover, non-Nt mutants of MAbs 2F5 and 4E10 exhibited a reduction in binding to MPER-TM1 and yet maintained binding to synthetic MPER peptides, indicating that MPER-TM1 better approximates the MPER NCS than peptides. Replacement of the gp41 TM and CT of MPER-TM1 with the PDGFR TM reduced binding by MAb 4E10, but not 2F5, indicating that the gp41 TM plays a pivotal role in orienting the 4E10 epitope, and more globally, in affecting MPER exposure. PMID:22238313

Analysis of the Borrelia burgdorferi Cyclic-di-GMP-Binding Protein PlzA Reveals a Role in Motility and Virulence ▿

PubMed Central

Pitzer, Joshua E.; Sultan, Syed Z.; Hayakawa, Yoshihiro; Hobbs, Gerry; Miller, Michael R.; Motaleb, Md A.

2011-01-01

The cyclic-dimeric-GMP (c-di-GMP)-binding protein PilZ has been implicated in bacterial motility and pathogenesis. Although BB0733 (PlzA), the only PilZ domain-containing protein in Borrelia burgdorferi, was reported to bind c-di-GMP, neither its role in motility or virulence nor it's affinity for c-di-GMP has been reported. We determined that PlzA specifically binds c-di-GMP with high affinity (dissociation constant [Kd], 1.25 μM), consistent with Kd values reported for c-di-GMP-binding proteins from other bacteria. Inactivation of the monocistronically transcribed plzA resulted in an opaque/solid colony morphology, whereas the wild-type colonies were translucent. While the swimming pattern of mutant cells appeared normal, on swarm plates, mutant cells exhibited a significantly reduced swarm diameter, demonstrating a role of plzA in motility. Furthermore, the plzA mutant cells were significantly less infectious in experimental mice (as determined by 50% infectious dose [ID50]) relative to wild-type spirochetes. The mutant also had survival rates in fed ticks lower than those of the wild type. Consequently, plzA mutant cells failed to complete the mouse-tick-mouse infection cycle, indicating plzA is essential for the enzootic life cycle of B. burgdorferi. All of these defects were corrected when the mutant was complemented in cis. We propose that failure of plzA mutant cells to infect mice was due to altered motility; however, the possibility that an unidentified factor(s) contributed to interruption of the B. burgdorferi enzootic life cycle cannot yet be excluded. PMID:21357718

Structural insights into selective agonist actions of tamoxifen on human estrogen receptor alpha.

PubMed

Chakraborty, Sandipan; Biswas, Pradip Kumar

2014-08-01

Tamoxifen-an anti-estrogenic ligand in breast tissues used as a first-line treatment in estrogen receptor (ER)-positive breast cancers-is associated with the development of resistance followed by resumption of tumor growth in about 30 % of cases. Whether tamoxifen assists in proliferation in such cases or whether any ligand-independent pathway to transcription exists is not fully understood; also, no ERα mutants have been detected so far that could lead to tamoxifen resistance. Using in silico conformational analysis of the ERα ligand binding domain (LBD), in the absence and presence of selective agonist (diethylstilbestrol; DES), antagonist (Faslodex; ICI), and selective estrogen receptor modulator (SERM; 4-hydroxy tamoxifen; 4-OHT) ligands, we have elucidated ligand-responsive structural modulations of the ERα-LBD dimer in its agonist and antagonist complexes to address the issue of "tamoxifen resistance". DES and ICI were found to stabilize the dimer in their agonist and antagonist conformations, respectively. The ERα-LBD dimer without the presence of any bound ligand also led to a stable structure in agonist conformation. However, binding of 4-OHT to the antagonist structure led to a flexible conformation allowing the protein to visit conformations populated by agonists as was evident from principal component analysis and radius of gyration plots. Further, the relaxed conformations of the 4-OHT bound protein exhibited a diminished size of the co-repressor binding pocket in the LBD, thus signaling a partial blockage of the co-repressor binding motif. Thus, the ability of 4-OHT-bound ERα-LBD to assume flexible conformations visited by agonists and reduced co-repressor binding surface at the LBD provide crucial structural insights into tamoxifen-resistance that complement our existing understanding.

VKORC1 and VKORC1L1 have distinctly different oral anticoagulant dose-response characteristics and binding sites

PubMed Central

Czogalla, Katrin J.; Liphardt, Kerstin; Höning, Klara; Hornung, Veit; Biswas, Arijit; Watzka, Matthias

2018-01-01

Vitamin K reduction is catalyzed by 2 enzymes in vitro: the vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) and its isozyme VKORC1-like1 (VKORC1L1). In vivo, VKORC1 reduces vitamin K to sustain γ-carboxylation of vitamin K-dependent proteins, including coagulation factors. Inhibition of VKORC1 by oral anticoagulants (OACs) is clinically used in therapy and in prevention of thrombosis. However, OACs also inhibit VKORC1L1, which was previously shown to play a role in intracellular redox homeostasis in vitro. Here, we report data for the first time on specific inhibition of both VKOR enzymes for various OACs and rodenticides examined in a cell-based assay. Effects on endogenous VKORC1 and VKORC1L1 were independently investigated in genetically engineered HEK 293T cells that were knocked out for the respective genes by CRISPR/Cas9 technology. In general, dose-responses for 4-hydroxycoumarins and 1,3-indandiones were enzyme-dependent, with lower susceptibility for VKORC1L1 compared with VKORC1. In contrast, rodenticides exhibited nearly identical dose-responses for both enzymes. To explain the distinct inhibition pattern, we performed in silico modeling suggesting different warfarin binding sites for VKORC1 and VKORC1L1. We identified arginine residues at positions 38, 42, and 68 in the endoplasmatic reticulum luminal loop of VKORC1L1 responsible for charge-stabilized warfarin binding, resulting in a binding pocket that is diametrically opposite to that of VKORC1. In conclusion, our findings provide insight into structural and molecular drug binding on VKORC1, and especially on VKORC1L1. PMID:29581108

Altered G Protein Coupling in Olfactory Neuroepithelial Cells From Patients With Schizophrenia

PubMed Central

Borgmann-Winter, Karin E.; Wang, Hoau-Yan; Ray, Rabindranath; Willis, Brooke R.; Moberg, Paul J.; Rawson, Nancy E.; Gur, Raquel E.; Turetsky, Bruce I.; Hahn, Chang-Gyu

2016-01-01

Increasing evidence suggests that olfactory dysfunction is an endophenotype of schizophrenia, and thus the olfactory system can be studied both in relation to this sensory dysfunction and also as a means of examining pathophysiologic mechanisms of schizophrenia. In this study, we examined human olfactory neuroepithelial (ON) biopsy tissues and their in vitro culture cells for ligand-induced guanine nucleotide-binding protein (G protein) activation and downstream signaling. We assessed the binding of a nonhydrolyzable GTP analogue [35S]GTPγS binding to specific G protein subtypes in response to odorants, dopamine, or serotonin in ON cell membranes from matched schizophrenia-control subjects. In response to odorant mixtures, we found decreased [35S]GTPγS binding to Gαs/olf in schizophrenia patients. These changes were not mediated by mRNA expression of key molecules of G protein coupling, including adenylate cyclase III (ACIII), protein kinase A (PKA), protein kinase Cγ (PKCγ), or Gαs or Gαolf in ON cells or ON biopsy tissues. In contrast, dopamine (DA)- and serotonin (5HT)-induced S35-GTPγS binding to Gαs/olf and Gαq/11 were significantly increased in schizophrenia cases, while these parameters were strikingly reduced by in vitro treatment with antipsychotics. Patients with schizophrenia exhibit increases in electrolfactogram (EOG) recordings, suggesting enhanced odorant-induced activation. Our results of decreased odorant-induced G protein activation may point further downstream for underlying mechanisms for increased EOG measures. Increased G protein activation in response to DA and 5HT may suggest increased postreceptor DA or 5HT signaling as an additional mechanism of dopaminergic or serotonergic dysregulation in schizophrenia. PMID:26373539

Function and CO binding properties of the NiFe complex in carbon monoxide dehydrogenase from Clostridium thermoaceticum.

PubMed

Shin, W; Lindahl, P A

1992-12-29

Adding 1,10-phenanthroline to carbon monoxide dehydrogenase from Clostridium thermoaceticum results in the complete loss of the NiFeC EPR signal and the CO/acetyl-CoA exchange activity. Other EPR signals characteristic of the enzyme (the gav = 1.94 and gav = 1.86 signals) and the CO oxidation activity are completely unaffected by the 1,10-phenanthroline treatment. This indicates that there are two catalytic sites on the enzyme; the NiFe complex is required for catalyzing the exchange and acetyl-CoA synthase reactions, while some other site is responsible for CO oxidation. The strength of CO binding to the NiFe complex was examined by titrating dithionite-reduced enzyme with CO. During the titration, the NiFeC EPR signal developed to a final spin intensity of 0.23 spin/alpha beta. The resulting CO titration curve (NiFeC spins/alpha beta vs CO pha beta) was fitted using two reactions: binding of CO to the oxidized NiFe complex, and reduction of the CO-bound species to a form that exhibits the NiFeC signal. Best fits yielded apparent binding constants between 6000 and 14,000 M-1 (Kd = 70-165 microM). This sizable range is due to uncertainty whether CO binds to all or only a small fraction (approximately 23%) of the NiFe complexes. Reduction of the CO-bound NiFe complex is apparently required to activate it for catalysis. The electron used for this reduction originates from the CO oxidation site, suggesting that delivery of a low-potential electron to the CO-bound NiFe complex is the physiological function of the CO oxidation reaction catalyzed by this enzyme.

A Water‐Soluble Tetraazaperopyrene Dye as Strong G‐Quadruplex DNA Binder

PubMed Central

Hahn, Lena

2016-01-01

Abstract The interactions of the water‐soluble tetraazaperopyrene dye 1 with ct‐DNA, duplex‐[(dAdT)12 ⋅(dAdT)12], duplex‐[(dGdC)12 ⋅(dGdC)12] as well as with two G‐quadruplex‐forming sequences, namely the human telomeric 22AG and the promotor sequence c‐myc, were investigated by means of UV/visible and fluorescence spectroscopy, isothermal titration calorimetry (ITC) and molecular docking studies. Dye 1 exhibits a high affinity for G‐quadruplex structures over duplex DNA structures. Furthermore, the ligand shows promising G‐quadruplex discrimination, with an affinity towards c‐myc of 2×107  m −1 (i.e., K d=50 nm), which is higher than for 22AG (4×106  m −1). The ITC data reveal that compound 1 interacts with c‐myc in a stoichiometric ratio of 1:1 but also indicate the presence of two identical lower affinity secondary binding sites per quadruplex. In 22AG, there are two high affinity binding sites per quadruplex, that is, one on each side, with a further four weaker binding sites. For both quadruplex structures, the high affinity interactions between compound 1 and the quadruplex‐forming nucleic acid structures are weakly endothermic. Molecular docking studies suggest an end‐stacking binding mode for compound 1 interacting with quadruplex structures, and a higher affinity for the parallel conformation of c‐myc than for the mixed‐hybrid conformation of 22AG. In addition, docking studies also suggest that the reduced affinity for duplex DNA structures is due to the non‐viability of an intercalative binding mode. PMID:26997208

Characterization of ligand binding to melanocortin 4 receptors using fluorescent peptides with improved kinetic properties.

PubMed

Link, Reet; Veiksina, Santa; Rinken, Ago; Kopanchuk, Sergei

2017-03-15

Melanocortin 4 (MC 4 ) receptors are important drug targets as they regulate energy homeostasis, eating behaviour and sexual functions. The ligand binding process to these G protein-coupled receptors is subject to considerable complexity. Different steps in the complex dynamic regulation can be characterized by ligand binding kinetics. Optimization of these kinetic parameters in terms of on-rate and residence time can increase the rapid onset of drug action and reduce off-target effects. Fluorescence anisotropy (FA) is one of the homogeneous fluorescence-based assays that enable continuous online monitoring of ligand binding kinetics. FA has been implemented for the kinetic study of melanocortin MC 4 receptors expressed on budded baculoviruses. However, the slow dissociation of the fluorescently labelled peptide NDP-α-MSH does not enable reaching equilibrium nor enable more in-depth study of the binding mechanisms. To overcome this problem, two novel red-shifted fluorescent ligands were designed. These cyclized heptapeptide derivatives (UTBC101 and UTBC102) exhibited nanomolar affinity toward melanocortin MC 4 receptors but had relatively different kinetic properties. The dissociation half-lives of UTBC101 (τ 1/2 =160min) and UTBC102 (τ 1/2 =7min) were shorter compared to that what was previously reported for Cy3B-NDP-α-MSH (τ 1/2 =224min). The significantly shorter dissociation half-life of UTBC102 enables equilibrium in screening assays, whereas the higher affinity of UTBC101 helps to resolve a wider range of competitor potencies. These two ligands are suitable for further kinetic screening of novel melanocortin MC 4 receptor specific ligands and could complement each other in these studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots.

PubMed

Baker, Christopher L; Petkova, Pavlina; Walker, Michael; Flachs, Petr; Mihola, Ondrej; Trachtulec, Zdenek; Petkov, Petko M; Paigen, Kenneth

2015-09-01

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9+/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape.

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots

PubMed Central

Baker, Christopher L.; Petkova, Pavlina; Walker, Michael; Flachs, Petr; Mihola, Ondrej; Trachtulec, Zdenek; Petkov, Petko M.; Paigen, Kenneth

2015-01-01

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9 +/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape. PMID:26368021

Tuning the specificity of a Two-in-One Fab against three angiogenic antigens by fully utilizing the information of deep mutational scanning.

PubMed

Koenig, Patrick; Sanowar, Sarah; Lee, Chingwei V; Fuh, Germaine

Monoclonal antibodies developed for therapeutic or diagnostic purposes need to demonstrate highly defined binding specificity profiles. Engineering of an antibody to enhance or reduce binding to related antigens is often needed to achieve the desired biologic activity without safety concern. Here, we describe a deep sequencing-aided engineering strategy to fine-tune the specificity of an angiopoietin-2 (Ang2)/vascular endothelial growth factor (VEGF) dual action Fab, 5A12.1 for the treatment of age-related macular degeneration. This antibody utilizes overlapping complementarity-determining region (CDR) sites for dual Ang2/VEGF interaction with K D in the sub-nanomolar range. However, it also exhibits significant (K D of 4 nM) binding to angiopoietin-1, which has high sequence identity with Ang2. We generated a large phage-displayed library of 5A12.1 Fab variants with all possible single mutations in the 6 CDRs. By tracking the change of prevalence of each mutation during various selection conditions, we identified 35 mutations predicted to decrease the affinity for Ang1 while maintaining the affinity for Ang2 and VEGF. We confirmed the specificity profiles for 25 of these single mutations as Fab protein. Structural analysis showed that some of the Fab mutations cluster near a potential Ang1/2 epitope residue that differs in the 2 proteins, while others are up to 15 Å away from the antigen-binding site and likely influence the binding interaction remotely. The approach presented here provides a robust and efficient method for specificity engineering that does not require prior knowledge of the antigen antibody interaction and can be broadly applied to antibody specificity engineering projects.

Modification of Titanium Substrates with Chimeric Peptides Comprising Antimicrobial and Titanium-Binding Motifs Connected by Linkers To Inhibit Biofilm Formation.

PubMed

Liu, Zihao; Ma, Shiqing; Duan, Shun; Xuliang, Deng; Sun, Yingchun; Zhang, Xi; Xu, Xinhua; Guan, Binbin; Wang, Chao; Hu, Meilin; Qi, Xingying; Zhang, Xu; Gao, Ping

2016-03-02

Bacterial adhesion and biofilm formation are the primary causes of implant-associated infection, which is difficult to eliminate and may induce failure in dental implants. Chimeric peptides with both binding and antimicrobial motifs may provide a promising alternative to inhibit biofilm formation on titanium surfaces. In this study, chimeric peptides were designed by connecting an antimicrobial motif (JH8194: KRLFRRWQWRMKKY) with a binding motif (minTBP-1: RKLPDA) directly or via flexible/rigid linkers to modify Ti surfaces. We evaluated the binding behavior of peptides using quartz crystal microbalance (QCM) and atomic force microscopy (AFM) techniques and investigated the effect of the modification of titanium surfaces with these peptides on the bioactivity of Streptococcus gordonii (S. gordonii) and Streptococcus sanguis (S. sanguis). Compared with the flexible linker (GGGGS), the rigid linker (PAPAP) significantly increased the adsorption of the chimeric peptide on titanium surfaces (p < 0.05). Concentration-dependent adsorption is consistent with a single Langmuir model, whereas time-dependent adsorption is in line with a two-domain Langmuir model. Additionally, the chimeric peptide with the rigid linker exhibited more effective antimicrobial ability than the peptide with the flexible linker. This finding was ascribed to the ability of the rigid linker to separate functional domains and reduce their interference to the maximum extent. Consequently, the performance of chimeric peptides with specific titanium-binding motifs and antimicrobial motifs against bacteria can be optimized by the proper selection of linkers. This rational design of chimeric peptides provides a promising alternative to inhibit the formation of biofilms on titanium surfaces with the potential to prevent peri-implantitis and peri-implant mucositis.

Bean Metal-Responsive Element-Binding Transcription Factor Confers Cadmium Resistance in Tobacco1

PubMed Central

Sun, Na; Liu, Meng; Zhang, Wentao; Yang, Wanning; Bei, Xiujuan; Ma, Hui; Qiao, Fan; Qi, Xiaoting

2015-01-01

Cadmium (Cd) is highly toxic to plants. Modulation of Cd-responsive transcription is an important way for Cd detoxification in plants. Metal-responsive element (MRE) is originally described in animal metallothionein genes. Although functional MREs also exist in Cd-regulated plant genes, specific transcription factors that bind MRE to regulate Cd tolerance have not been identified. Previously, we showed that Cd-inducible bean (Phaseolus vulgaris) stress-related gene2 (PvSR2) produces a short (S) PvSR2 transcript (S-PvSR2) driven by an intronic promoter. Here, we demonstrate that S-PvSR2 encodes a bean MRE-binding transcription factor1 (PvMTF-1) that confers Cd tolerance in tobacco (Nicotiana tabacum). PvMTF-1 expression was up-regulated by Cd at the levels of RNA and protein. Importantly, expression of PvMTF-1 in tobacco enhanced Cd tolerance, indicating its role in regulating Cd resistance in planta. This was achieved through direct regulation of a feedback-insensitive Anthranilate Synthase α-2 chain gene (ASA2), which catalyzes the first step for tryptophan biosynthesis. In vitro and in vivo DNA-protein interaction studies further revealed that PvMTF-1 directly binds to the MRE in the ASA2 promoter, and this binding depends on the zinc finger-like motif of PvMTF-1. Through modulating ASA2 up-regulation by Cd, PvMTF-1 increased free tryptophan level and subsequently reduced Cd accumulation, thereby enhancing Cd tolerance of transgenic tobacco plants. Consistent with this observation, tobacco transiently overexpressing ASA2 also exhibited increased tolerance to Cd. We conclude that PvMTF-1 is a zinc finger-like transcription factor that links MRE to Cd resistance in transgenic tobacco through activation of tryptophan biosynthesis. PMID:25624396

Pulsed ultraviolet light reduces immunoglobulin E binding to Atlantic white shrimp (Litopenaeus setiferus) extract.

PubMed

Shriver, Sandra; Yang, Wade; Chung, Si-Yin; Percival, Susan

2011-07-01

Pulsed ultraviolet light (PUV), a novel food processing and preservation technology, has been shown to reduce allergen levels in peanut and soybean samples. In this study, the efficacy of using PUV to reduce the reactivity of the major shrimp allergen, tropomyosin (36-kDa), and to attenuate immunoglobulin E (IgE) binding to shrimp extract was examined. Atlantic white shrimp (Litopenaeus setiferus) extract was treated with PUV (3 pulses/s, 10 cm from light source) for 4 min. Tropomyosin was compared in the untreated, boiled, PUV-treated and [boiled+PUV]-treated samples, and changes in the tropomyosin levels were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). IgE binding of the treated extract was analyzed via immunoblot and enzyme-linked immunosorbent assay (ELISA) using pooled human plasma containing IgE antibodies against shrimp allergens. Results showed that levels of tropomyosin and IgE binding were reduced following PUV treatment. However, boiling increased IgE binding, while PUV treatment could offset the increased allergen reactivity caused by boiling. In conclusion, PUV treatment reduced the reactivity of the major shrimp allergen, tropomyosin, and decreased the IgE binding capacity of the shrimp extract.

Autoradiographic analysis of tritiated imipramine binding in the human brain post mortem: effects of suicide

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

Gross-Isseroff, R.; Israeli, M.; Biegon, A.

In vitro quantitative autoradiography of high-affinity tritiated imipramine binding sites was performed on brains of 12 suicide victims and 12 matched controls. Region-specific differences in imipramine binding were found between the two groups. Thus, the pyramidal and molecular layers of the cornu ammoni hippocampal fields and the hilus of the dentate gyrus exhibited 80%, 60%, and 90% increases in binding in the suicide group, respectively. The postcentral cortical gyrus, insular cortex, and claustrum had 45%, 28%, and 75% decreases in binding in the suicide group, respectively. No difference in imipramine binding was observed in prefrontal cortical regions, in the basalmore » ganglia, and in mesencephalic nuclei. No sex and postmortem delay effects on imipramine binding were found. Imipramine binding was positively correlated with age, the effect of age being most pronounced in portions of the basal ganglia and temporal cortex.« less

Compositions and methods related to serotonin 5-HT1A receptors

DOEpatents

Mukherjee, Jogeshwar; Saigal, Neil

2010-06-08

Contemplated substituted arylpiperazinyl compounds, and most preferably ¹⁸F-Mefway, exhibit desirable in vitro and in vivo binding characteristics to the 5-HT1A receptor. Among other advantageous parameters, contemplated compounds retain high binding affinity, display optimal lipophilicity, and are radiolabeled efficiently with ¹⁸F-fluorine in a single step. Still further, contemplated compounds exhibit high target to non-target ratios in receptor-rich regions both in vitro and in vivo, and selected compounds can be effectively and sensitively displaced by serotonin, thus providing a quantitative tool for measuring 5-HT1A receptors and serotonin concentration changes in the living brain.

Compositions and methods related to serotonin 5-HT1A receptors

DOEpatents

Mukherjee, Jogeshwar [Irvine, CA; Saigal, Neil [Fresno, CA; Saigal, legal representative, Harsh

2012-09-25

Contemplated substituted arylpiperazinyl compounds, and most preferably .sup.18F-Mefway, exhibit desirable in vitro and in vivo binding characteristics to the 5-HT1A receptor. Among other advantageous parameters, contemplated compounds retain high binding affinity, display optimal lipophilicity, and are radiolabeled efficiently with .sup.18F-fluorine in a single step. Still further, contemplated compounds exhibit high target to non-target ratios in receptor-rich regions both in vitro and in vivo, and selected compounds can be effectively and sensitively displaced by serotonin, thus providing a quantitative tool for measuring 5-HT1A receptors and serotonin concentration changes in the living brain.

Compositions and methods related to serotonin 5-HT1A receptors

DOEpatents

Mukherjee, Jogeshwar; Saigal, Neil; Saigal, legal representative, Harsh

2012-09-25

Contemplated substituted arylpiperazinyl compounds, and most preferably ¹⁸F-Mefway, exhibit desirable in vitro and in vivo binding characteristics to the 5-HT1A receptor. Among other advantageous parameters, contemplated compounds retain high binding affinity, display optimal lipophilicity, and are radiolabeled efficiently with ¹⁸F-fluorine in a single step. Still further, contemplated compounds exhibit high target to non-target ratios in receptor-rich regions both in vitro and in vivo, and selected compounds can be effectively and sensitively displaced by serotonin, thus providing a quantitative tool for measuring 5-HT1A receptors and serotonin concentration changes in the living brain.

High-Affinity Rb Binding, p53 Inhibition, Subcellular Localization, and Transformation by Wild-Type or Tumor-Derived Shortened Merkel Cell Polyomavirus Large T Antigens

PubMed Central

Borchert, Sophie; Czech-Sioli, Manja; Neumann, Friederike; Schmidt, Claudia; Wimmer, Peter; Dobner, Thomas

2014-01-01

ABSTRACT Interference with tumor suppressor pathways by polyomavirus-encoded tumor antigens (T-Ags) can result in transformation. Consequently, it is thought that T-Ags encoded by Merkel cell polyomavirus (MCPyV), a virus integrated in ∼90% of all Merkel cell carcinoma (MCC) cases, are major contributors to tumorigenesis. The MCPyV large T-Ag (LT-Ag) has preserved the key functional domains present in all family members but has also acquired unique regions that flank the LxCxE motif. As these regions may mediate unique functions, or may modulate those shared with T-Ags of other polyomaviruses, functional studies of MCPyV T-Ags are required. Here, we have performed a comparative study of full-length or MCC-derived truncated LT-Ags with regard to their biochemical characteristics, their ability to bind to retinoblastoma (Rb) and p53 proteins, and their transforming potential. We provide evidence that full-length MCPyV LT-Ag may not directly bind to p53 but nevertheless can significantly reduce p53-dependent transcription in reporter assays. Although early region expression constructs harboring either full-length or MCC-derived truncated LT-Ag genes can transform primary baby rat kidney cells, truncated LT-Ags do not bind to p53 or reduce p53-dependent transcription. Interestingly, shortened LT-Ags exhibit a very high binding affinity for Rb, as shown by coimmunoprecipitation and in vitro binding studies. Additionally, we show that truncated MCPyV LT-Ag proteins are expressed at higher levels than those for the wild-type protein and are able to partially relocalize Rb to the cytoplasm, indicating that truncated LT proteins may have gained additional features that distinguish them from the full-length protein. IMPORTANCE MCPyV is one of the 12 known polyomaviruses that naturally infect humans. Among these, it is of particular interest since it is the only human polyomavirus known to be involved in tumorigenesis. MCPyV is thought to be causally linked to MCC, a rare skin tumor. In these tumors, viral DNA is monoclonally integrated into the genome of the tumor cells in up to 90% of all MCC cases, and the integrated MCV genomes, furthermore, harbor signature mutations in the so-called early region that selectively abrogate viral replication while preserving cell cycle deregulating functions of the virus. This study describes comparative studies of early region T-Ag protein characteristics, their ability to bind to Rb and p53, and their transforming potential. PMID:24371076

Differential Receptor Binding and Regulatory Mechanisms for the Lymphangiogenic Growth Factors Vascular Endothelial Growth Factor (VEGF)-C and -D*

PubMed Central

Davydova, Natalia; Harris, Nicole C.; Roufail, Sally; Paquet-Fifield, Sophie; Ishaq, Musarat; Streltsov, Victor A.; Williams, Steven P.; Karnezis, Tara; Stacker, Steven A.; Achen, Marc G.

2016-01-01

VEGF-C and VEGF-D are secreted glycoproteins that induce angiogenesis and lymphangiogenesis in cancer, thereby promoting tumor growth and spread. They exhibit structural homology and activate VEGFR-2 and VEGFR-3, receptors on endothelial cells that signal for growth of blood vessels and lymphatics. VEGF-C and VEGF-D were thought to exhibit similar bioactivities, yet recent studies indicated distinct signaling mechanisms (e.g. tumor-derived VEGF-C promoted expression of the prostaglandin biosynthetic enzyme COX-2 in lymphatics, a response thought to facilitate metastasis via the lymphatic vasculature, whereas VEGF-D did not). Here we explore the basis of the distinct bioactivities of VEGF-D using a neutralizing antibody, peptide mapping, and mutagenesis to demonstrate that the N-terminal α-helix of mature VEGF-D (Phe93–Arg108) is critical for binding VEGFR-2 and VEGFR-3. Importantly, the N-terminal part of this α-helix, from Phe93 to Thr98, is required for binding VEGFR-3 but not VEGFR-2. Surprisingly, the corresponding part of the α-helix in mature VEGF-C did not influence binding to either VEGFR-2 or VEGFR-3, indicating distinct determinants of receptor binding by these growth factors. A variant of mature VEGF-D harboring a mutation in the N-terminal α-helix, D103A, exhibited enhanced potency for activating VEGFR-3, was able to promote increased COX-2 mRNA levels in lymphatic endothelial cells, and had enhanced capacity to induce lymphatic sprouting in vivo. This mutant may be useful for developing protein-based therapeutics to drive lymphangiogenesis in clinical settings, such as lymphedema. Our studies shed light on the VEGF-D structure/function relationship and provide a basis for understanding functional differences compared with VEGF-C. PMID:27852824

Structure-Function Analysis of Friedreich's Ataxia Mutants Reveals Determinants of Frataxin Binding and Activation of the Fe-S Assembly Complex

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

Bridwell-Rabb, Jennifer; Winn, Andrew M; Barondeau, David P

2012-08-01

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-Smore » cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a k cat/K M higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest k cat/K M of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.« less

Immune depression in Rhodnius prolixus by seco-steroids, physalins.

PubMed

Castro, D P; Figueiredo, M B; Ribeiro, I M; Tomassini, T C B; Azambuja, P; Garcia, E S

2008-03-01

A comparative study of the effects of physalins, seco-steroidal substances of Physalis angulata (Solanaceae), on the immune reactions of R. prolixus was carried out. Ecdysis and mortality were not affected by treatment with physalins B, D, F or G (1-10 microg/ml of blood meal). R. prolixus larvae fed with blood containing physalins and inoculated with 1 microl of Enterobacter cloacae beta12 (5 x 10(3)/insect) exhibited mortality rates three times higher than controls. The insects treated with physalin B, and F (1 microg/ml) and inoculated with E. cloacae beta12 showed significant differences on lysozyme activity in the hemolymph compared to untreated insects. Furthermore, physalin D (1 microg/ml) significantly reduced the antibacterial activity. Concerning cellular immune reactions, all insects treated with physalins (1 microg/ml), exhibited drastic reductions in the quantity of yeast cell-hemocyte binding and subsequent internalization. Insects inoculated with bacteria and treated with physalins B, F and G showed reductions of microaggregate formation but physalin D did not. Physalins B and F also reduced total hemocyte count in the hemolymph. These results suggest that, in different ways, probably due to their different chemical structures, physalin B, D, F and G are immunomodulatory substances for the bloodsucking insect, R. prolixus.

Ferroelectric triggering of carbon monoxide adsorption on lead zirco-titanate (001) surfaces

PubMed Central

Tănase, Liviu Cristian; Apostol, Nicoleta Georgiana; Abramiuc, Laura Elena; Tache, Cristian Alexandru; Hrib, Luminița; Trupină, Lucian; Pintilie, Lucian; Teodorescu, Cristian Mihail

2016-01-01

Atomically clean lead zirco-titanate PbZr0.2Ti0.8O3 (001) layers exhibit a polarization oriented inwards P(−), visible by a band bending of all core levels towards lower binding energies, whereas as introduced layers exhibit P(+) polarization under air or in ultrahigh vacuum. The magnitude of the inwards polarization decreases when the temperature is increased at 700 K. CO adsorption on P(−) polarized surfaces saturates at about one quarter of a monolayer of carbon, and occurs in both molecular (oxidized) and dissociated (reduced) states of carbon, with a large majority of reduced state. The sticking of CO on the surface in ultrahigh vacuum is found to be directly related to the P(−) polarization state of the surface. A simple electrostatic mechanism is proposed to explain these dissociation processes and the sticking of carbon on P(−) polarized areas. Carbon desorbs also when the surface is irradiated with soft X-rays. Carbon desorption when the polarization is lost proceeds most probably in form of CO2. Upon carbon desorption cycles, the ferroelectric surface is depleted in oxygen and at some point reverses its polarization, owing to electrons provided by oxygen vacancies which are able to screen the depolarization field produced by positive fixed charges at the surface. PMID:27739461

Genetically obese (ob/ob) mice are resistant to the lethal effects of thioacetamide hepatotoxicity

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

Won, Young-Suk; Song, Ji-Won; Lim, Jong-Hwan

Obesity increases the risk of chronic liver diseases, including viral hepatitis, alcohol-induced liver disease, and non-alcoholic steatohepatitis. In this study, we investigated the effects of obesity in acute hepatic failure using a murine model of thioacetamide (TA)-induced liver injury. Genetically obese ob/ob mice, together with non-obese ob/+ littermates, were subjected to a single intraperitoneal injection of TA, and examined for signs of hepatic injury. ob/ob mice showed a significantly higher survival rate, lower levels of serum alanine aminotransferase and aspartate aminotransferase, and less hepatic necrosis and apoptosis, compared with ob/+ mice. In addition, ob/ob mice exhibited significantly lower levels ofmore » malondialdehyde and significantly higher levels of glutathione and antioxidant enzyme activities compared with their ob/+ counterparts. Bioactivation analyses revealed reduced plasma clearance of TA and covalent binding of [{sup 14}C]TA to liver macromolecules in ob/ob mice. Together, these data demonstrate that genetically obese mice are resistant to TA-induced acute liver injury through diminished bioactivation of TA and antioxidant effects. - Highlights: • ob/ob mice are resistant to lethal doses of thioacetamide, compared to ob/+ mice. • ob/ob mice show reduced oxidative stress and enhanced antioxidant enzyme activity. • ob/ob mice exhibit diminished bioactivation of thioacetamide.« less

Sublingual immunization with the phosphate-binding-protein (PstS) reduces oral colonization by Streptococcus mutans.

PubMed

Ferreira, E L; Batista, M T; Cavalcante, R C M; Pegos, V R; Passos, H M; Silva, D A; Balan, A; Ferreira, L C S; Ferreira, R C C

2016-10-01

Bacterial ATP-binding cassette (ABC) transporters play a crucial role in the physiology and pathogenicity of different bacterial species. Components of ABC transporters have also been tested as target antigens for the development of vaccines against different bacterial species, such as those belonging to the Streptococcus genus. Streptococcus mutans is the etiological agent of dental caries, and previous studies have demonstrated that deletion of the gene encoding PstS, the substrate-binding component of the phosphate uptake system (Pst), reduced the adherence of the bacteria to abiotic surfaces. In the current study, we generated a recombinant form of the S. mutans PstS protein (rPstS) with preserved structural features, and we evaluated the induction of antibody responses in mice after sublingual mucosal immunization with a formulation containing the recombinant protein and an adjuvant derived from the heat-labile toxin from enterotoxigenic Escherichia coli strains. Mice immunized with rPstS exhibited systemic and secreted antibody responses, measured by the number of immunoglobulin A-secreting cells in draining lymph nodes. Serum antibodies raised in mice immunized with rPstS interfered with the adhesion of bacteria to the oral cavity of naive mice challenged with S. mutans. Similarly, mice actively immunized with rPstS were partially protected from oral colonization after challenge with the S. mutans NG8 strain. Therefore, our results indicate that S. mutans PstS is a potential target antigen capable of inducing specific and protective antibody responses after sublingual administration. Overall, these observations raise interesting perspectives for the development of vaccines to prevent dental caries. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Combination of two insulin-like growth factor-I receptor inhibitory antibodies targeting distinct epitopes leads to an enhanced antitumor response.

PubMed

Dong, Jianying; Demarest, Stephen J; Sereno, Arlene; Tamraz, Susan; Langley, Emma; Doern, Adam; Snipas, Tracey; Perron, Keli; Joseph, Ingrid; Glaser, Scott M; Ho, Steffan N; Reff, Mitchell E; Hariharan, Kandasamy

2010-09-01

The insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor tyrosine kinase that mediates cell survival signaling and supports tumor progression in multiple tumor types. We identified a spectrum of inhibitory IGF-IR antibodies with diverse binding epitopes and ligand-blocking properties. By binding distinct inhibitory epitopes, two of these antibodies, BIIB4 and BIIB5, block both IGF-I and IGF-II binding to IGF-IR using competitive and allosteric mechanisms, respectively. Here, we explored the inhibitory effects of combining BIIB4 and BIIB5. In biochemical assays, the combination of BIIB4 and BIIB5 improved both the potency and extent of IGF-I and IGF-II blockade compared with either antibody alone. In tumor cells, the combination of BIIB4 and BIIB5 accelerated IGF-IR downregulation and more efficiently inhibited IGF-IR activation as well as downstream signaling, particularly AKT phosphorylation. In several carcinoma cell lines, the antibody combination more effectively inhibited ligand-driven cell growth than either BIIB4 or BIIB5 alone. Notably, the enhanced tumor growth-inhibitory activity of the BIIB4 and BIIB5 combination was much more pronounced at high ligand concentrations, where the individual antibodies exhibited substantially reduced activity. Compared with single antibodies, the BIIB4 and BIIB5 combination also significantly further enhanced the antitumor activity of the epidermal growth factor receptor inhibitor erlotinib and the mTOR inhibitor rapamycin. Moreover, in osteosarcoma and hepatocellular carcinoma xenograft models, the BIIB4 and BIIB5 combination significantly reduced tumor growth to a greater degree than each single antibody. Taken together, our results suggest that targeting multiple distinct inhibitory epitopes on IGF-IR may be a more effective strategy of affecting the IGF-IR pathway in cancer.

Pyridine Nucleotide Complexes with Bacillus anthracis Coenzyme A-Disulfide Reductase: A Structural Analysis of Dual NAD(P)H Specificity

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

Wallen,J.; Paige, C.; Mallett, T.

2008-01-01

We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis, and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 Angstroms resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides.more » The structures of the NADH and NADPH complexes at ca. 2.3 Angstroms resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2'-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367'-OH and Tyr425'-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367, 425F BACoADR mutants indicate that Tyr425' is the primary proton donor in catalysis, with Tyr367' functioning as a cryptic alternate donor in the absence of Tyr425'.« less

RNA-binding proteins regulate cell respiration and coenzyme Q biosynthesis by post-transcriptional regulation of COQ7.

PubMed

Cascajo, María V; Abdelmohsen, Kotb; Noh, Ji Heon; Fernández-Ayala, Daniel J M; Willers, Imke M; Brea, Gloria; López-Lluch, Guillermo; Valenzuela-Villatoro, Marina; Cuezva, José M; Gorospe, Myriam; Siendones, Emilio; Navas, Plácido

2016-07-02

Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain carrying electrons from complexes I and II to complex III and it is an intrinsic component of the respirasome. CoQ concentration is highly regulated in cells in order to adapt the metabolism of the cell to challenges of nutrient availability and stress stimuli. At least 10 proteins have been shown to be required for CoQ biosynthesis in a multi-peptide complex and COQ7 is a central regulatory factor of this pathway. We found that the first 765 bp of the 3'-untranslated region (UTR) of COQ7 mRNA contains cis-acting elements of interaction with RNA-binding proteins (RBPs) HuR and hnRNP C1/C2. Binding of hnRNP C1/C2 to COQ7 mRNA was found to require the presence of HuR, and hnRNP C1/C2 silencing appeared to stabilize COQ7 mRNA modestly. By contrast, lowering HuR levels by silencing or depriving cells of serum destabilized and reduced the half-life of COQ7 mRNA, thereby reducing COQ7 protein and CoQ biosynthesis rate. Accordingly, HuR knockdown decreased oxygen consumption rate and mitochondrial production of ATP, and increased lactate levels. Taken together, our results indicate that a reduction in COQ7 mRNA levels by HuR depletion causes mitochondrial dysfunction and a switch toward an enhanced aerobic glycolysis, the characteristic phenotype exhibited by primary deficiency of CoQ10. Thus HuR contributes to efficient oxidative phosphorylation by regulating of CoQ10 biosynthesis.

Small-Molecule Inhibitor of the Shigella flexneri Master Virulence Regulator VirF

PubMed Central

Koppolu, Veerendra; Osaka, Ichie; Skredenske, Jeff M.; Kettle, Bria; Hefty, P. Scott; Li, Jiaqin

2013-01-01

VirF is an AraC family transcriptional activator that is required for the expression of virulence genes associated with invasion and cell-to-cell spread by Shigella flexneri, including multiple components of the type three secretion system (T3SS) machinery and effectors. We tested a small-molecule compound, SE-1 (formerly designated OSSL_051168), which we had identified as an effective inhibitor of the AraC family proteins RhaS and RhaR, for its ability to inhibit VirF. Cell-based reporter gene assays with Escherichia coli and Shigella, as well as in vitro DNA binding assays with purified VirF, demonstrated that SE-1 inhibited DNA binding and transcription activation (likely by blocking DNA binding) by VirF. Analysis of mRNA levels using real-time quantitative reverse transcription-PCR (qRT-PCR) further demonstrated that SE-1 reduced the expression of the VirF-dependent virulence genes icsA, virB, icsB, and ipaB in Shigella. We also performed eukaryotic cell invasion assays and found that SE-1 reduced invasion by Shigella. The effect of SE-1 on invasion required preincubation of Shigella with SE-1, in agreement with the hypothesis that SE-1 inhibited the expression of VirF-activated genes required for the formation of the T3SS apparatus and invasion. We found that the same concentrations of SE-1 had no detectable effects on the growth or metabolism of the bacterial cells or the eukaryotic host cells, respectively, indicating that the inhibition of invasion was not due to general toxicity. Overall, SE-1 appears to inhibit transcription activation by VirF, exhibits selectivity toward AraC family proteins, and has the potential to be developed into a novel antibacterial agent. PMID:24002059

Evaluation of the Schistosoma mansoni Y-box-binding protein (SMYB1) potential as a vaccine candidate against schistosomiasis.

PubMed

Dias, Sílvia R C; Boroni, Mariana; Rocha, Elizângela A; Dias, Thomaz L; de Laet Souza, Daniela; Oliveira, Fabrício M S; Bitar, Mainá; Macedo, Andrea M; Machado, Carlos R; Caliari, Marcelo V; Franco, Glória R

2014-01-01

Schistosomiasis is a neglected tropical disease, and after malaria, is the second most important tropical disease in public health. A vaccine that reduces parasitemia is desirable to achieve mass treatment with a low cost. Although potential antigens have been identified and tested in clinical trials, no effective vaccine against schistosomiasis is available. Y-box-binding proteins (YBPs) regulate gene expression and participate in a variety of cellular processes, including transcriptional and translational regulation, DNA repair, cellular proliferation, drug resistance, and stress responses. The Schistosoma mansoni ortholog of the human YB-1, SMYB1, is expressed in all stages of the parasite life cycle. Although SMYB1 binds to DNA or RNA oligonucleotides, immunohistochemistry assays demonstrated that it is primarily localized in the cytoplasm of parasite cells. In addition, SMYB1 interacts with a protein involved in mRNA processing, suggesting that SMYB1 functions in the turnover, transport, and/or stabilization of RNA molecules during post-transcriptional gene regulation. Here we report the potential of SMYB1 as a vaccine candidate. We demonstrate that recombinant SMYB1 stimulates the production of high levels of specific IgG1 antibodies in a mouse model. The observed levels of specific IgG1 and IgG2a antibodies indicate an actual protection against cercariae challenge. Animals immunized with rSMYB1 exhibited a 26% reduction in adult worm burden and a 28% reduction in eggs retained in the liver. Although proteins from the worm tegument are considered optimal targets for vaccine development, this study demonstrates that unexposed cytoplasmic proteins can reduce the load of intestinal worms and the number of eggs retained in the liver.

Structure–function analyses of a pertussis-like toxin from pathogenic Escherichia coli reveal a distinct mechanism of inhibition of trimeric G-proteins

PubMed Central

Littler, Dene R.; Ang, Sheng Y.; Moriel, Danilo G.; Kocan, Martina; Kleifeld, Oded; Johnson, Matthew D.; Tran, Mai T.; Paton, Adrienne W.; Paton, James C.; Summers, Roger J.; Schembri, Mark A.; Rossjohn, Jamie; Beddoe, Travis

2017-01-01

Pertussis-like toxins are secreted by several bacterial pathogens during infection. They belong to the AB5 virulence factors, which bind to glycans on host cell membranes for internalization. Host cell recognition and internalization are mediated by toxin B subunits sharing a unique pentameric ring-like assembly. Although the role of pertussis toxin in whooping cough is well-established, pertussis-like toxins produced by other bacteria are less studied, and their mechanisms of action are unclear. Here, we report that some extra-intestinal Escherichia coli pathogens (i.e. those that reside in the gut but can spread to other bodily locations) encode a pertussis-like toxin that inhibits mammalian cell growth in vitro. We found that this protein, EcPlt, is related to toxins produced by both nontyphoidal and typhoidal Salmonella serovars. Pertussis-like toxins are secreted as disulfide-bonded heterohexamers in which the catalytic ADP-ribosyltransferase subunit is activated when exposed to the reducing environment in mammalian cells. We found here that the reduced EcPlt exhibits large structural rearrangements associated with its activation. We noted that inhibitory residues tethered within the NAD+-binding site by an intramolecular disulfide in the oxidized state dissociate upon the reduction and enable loop restructuring to form the nucleotide-binding site. Surprisingly, although pertussis toxin targets a cysteine residue within the α subunit of inhibitory trimeric G-proteins, we observed that activated EcPlt toxin modifies a proximal lysine/asparagine residue instead. In conclusion, our results reveal the molecular mechanism underpinning activation of pertussis-like toxins, and we also identified differences in host target specificity. PMID:28663369

Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition.

PubMed

Neth, O; Jack, D L; Dodds, A W; Holzel, H; Klein, N J; Turner, M W

2000-02-01

Mannose-binding lectin (MBL) is a collagenous serum lectin believed to be of importance in innate immunity. Genetically determined low levels of the protein are known to predispose to infections. In this study the binding of purified MBL to pathogens isolated from immunocompromised children was investigated by flow cytometry. Diverse Candida species, Aspergillus fumigatus, Staphylococcus aureus, and beta-hemolytic group A streptococci exhibited strong binding of MBL, whereas Escherichia coli, Klebsiella species, and Haemophilus influenzae type b were characterized by heterogeneous binding patterns. In contrast, beta-hemolytic group B streptococci, Streptococcus pneumoniae, and Staphylococcus epidermidis showed low levels of binding. Bound MBL was able to promote C4 deposition in a concentration-dependent manner. We conclude that MBL may be of importance in first-line immune defense against several important pathogens.

Dioxin mediates downregulation of the reduced folate carrier transport activity via the arylhydrocarbon receptor signalling pathway

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

Halwachs, Sandra, E-mail: halwachs@vetmed.uni-leipzig.d; Lakoma, Cathleen; Gebhardt, Rolf

2010-07-15

Dioxins such as 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) are common environmental contaminants known to regulate several genes via activation of the transcription factor aryl hydrocarbon receptor (AhR) associated with the development of numerous adverse biological effects. However, comparatively little is known about the molecular mechanisms by which dioxins display their toxic effects in vertebrates. The 5' untranslated region of the hepatocellular Reduced folate carrier (Rfc1; Slc19a1) exhibits AhR binding sites termed dioxin responsive elements (DRE) that have as yet only been found in the promoter region of prototypical TCDD target genes. Rfc1 mediated transport of reduced folates and antifolate drugs such as methotrexatemore » (MTX) plays an essential role in physiological folate homeostasis and MTX cancer chemotherapy. In order to determine whether this carrier represents a target gene of dioxins we have investigated the influence of TCDD on functional Rfc1 activity in rat liver. Pre-treatment of rats with TCDD significantly diminished hepatocellular Rfc1 uptake activity in a time- and dose-dependent manner. In further mechanistic studies we demonstrated that this reduction was due to TCDD-dependent activation of the AhR signalling pathway. We additionally showed that binding of the activated receptor to DRE motifs in the Rfc1 promoter resulted in downregulation of Rfc1 gene expression and reduced carrier protein levels. As downregulation of pivotal Rfc1 activity results in functional folate deficiency associated with an elevated risk of cardiovascular diseases or carcinogenesis, our results indicate that deregulation of this essential transport pathway represents a novel regulatory mechanism how dioxins display their toxic effects through the Ah receptor.« less

Hydrogen Sulfide Alleviates Aluminum Toxicity via Decreasing Apoplast and Symplast Al Contents in Rice

PubMed Central

Zhu, Chun Q.; Zhang, Jun H.; Sun, Li M.; Zhu, Lian F.; Abliz, Buhailiqem; Hu, Wen J.; Zhong, Chu; Bai, Zhi G.; Sajid, Hussain; Cao, Xiao C.; Jin, Qian Y.

2018-01-01

Hydrogen sulfide (H2S) plays a vital role in Al3+ stress resistance in plants, but the underlying mechanism is unclear. In the present study, pretreatment with 2 μM of the H2S donor NaHS significantly alleviated the inhibition of root elongation caused by Al toxicity in rice roots, which was accompanied by a decrease in Al contents in root tips under 50 μM Al3+ treatment. NaHS pretreatment decreased the negative charge in cell walls by reducing the activity of pectin methylesterase and decreasing the pectin and hemicellulose contents in rice roots. This treatment also masked Al-binding sites in the cell wall by upregulating the expression of OsSATR1 and OsSTAR2 in roots and reduced Al binding in the cell wall by stimulating the expression of the citrate acid exudation gene OsFRDL4 and increasing the secretion of citrate acid. In addition, NaHS pretreatment decreased the symplasmic Al content by downregulating the expression of OsNRAT1, and increasing the translocation of cytoplasmic Al to the vacuole via upregulating the expression of OsALS1. The increment of antioxidant enzyme [superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD)] activity with NaHS pretreatment significantly decreased the MDA and H2O2 content in rice roots, thereby reducing the damage of Al3+ toxicity on membrane integrity in rice. H2S exhibits crosstalk with nitric oxide (NO) in response to Al toxicity, and through reducing NO content in root tips to alleviate Al toxicity. Together, this study establishes that H2S alleviates Al toxicity by decreasing the Al content in the apoplast and symplast of rice roots. PMID:29559992

A topical treatment containing heat-treated Lactobacillus johnsonii NCC 533 reduces Staphylococcus aureus adhesion and induces antimicrobial peptide expression in an in vitro reconstructed human epidermis model.

PubMed

Rosignoli, Carine; Thibaut de Ménonville, Séverine; Orfila, Danielle; Béal, Méline; Bertino, Béatrice; Aubert, Jérôme; Mercenier, Annick; Piwnica, David

2018-04-01

Staphylococcus aureus colonization is thought to contribute to the pathophysiology of atopic dermatitis (AD). AD patients exhibit reduced levels of cutaneous antimicrobial peptides (AMPs), which may explain their increased susceptibility to infections. Using an in vitro reconstructed human epidermis (RHE) model, we sought to determine whether topical application of a non-replicating probiotic, heat-treated Lactobacillus johnsonii NCC 533 (HT La1), could inhibit S. aureus adhesion to skin and boost cutaneous innate immunity. We found that application of HT La1 suspension to RHE samples reduced the binding of radiolabelled S. aureus by up to 74%. To investigate a potential effect of HT La1 on innate immunity, we analysed the expression of nine AMP genes, including those encoding beta defensins and S100 proteins, following topical application of HT La1 in suspension or in a daily moisturizer lotion. Analysed genes were induced by up to fourfold in a dose-dependent manner by HT La1 in suspension and by up to 2.4-fold by HT La1 in the moisturizer lotion. Finally, using ELISA and immunohistochemical detection, we evaluated the expression and secretion of the AMPs hBD-2 and psoriasin and determined that both proteins were induced by topical HT La1, particularly in the stratum corneum of the RHE. These findings demonstrate that a topically applied, non-replicating probiotic can modulate endogenous AMP expression and inhibit binding of S. aureus to an RHE model in vitro. Moreover, they suggest that a topical formulation containing HT La1 could benefit atopic skin by enhancing cutaneous innate immunity and reducing S. aureus colonization. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effect of pH on Cl sup minus transport in TAL of Henle's loop

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

Kondo, Yoshiaki; Yoshitomi, Koji; Imai, Masashi

1987-12-01

To further characterize the mechanism of Cl{sup {minus}} transport across the hamster thin ascending limb (TAL) of Henle's loop, the authors examined effects of pH on Cl{sup {minus}} permeability as determined by either the choline chloride diffusion voltage or the lumen-to-bath {sup 36}Cl flux in the isolated segments perfused in vitro. When pH of the bathing fluid or the perfusate was reduced from 7.4 to 5.8, the Cl{sup {minus}}-Na{sup +} permeability ratio was reduced from 2.77 {plus minus} 0.21 to 0.48 {plus minus} 0.02 or from 2.55 {plus minus} 0.15 to 0.81 {plus minus} 0.11, respectively. At 37{degree}C, when themore » pH of the bathing fluid was reduced from 7.4 to 6.2, the lumen-to-bath flux coefficient for {sup 36}Cl was reduced from 84.8 {plus minus} 7.5 to 20.4 {plus minus} 3.2, whereas the value for {sup 22}Na was unchanged. From the pH titration curves for P{sub Cl}/P{sub Na}, pK{sub a} values for proton binding were 6.31 and 5.78, and Hill's coefficients were 2.1 and 2.3 on the basolateral side and on the luminal side, respectively. Intracellular acidification with o-nitrophenylacetate also decreased the Cl{sup {minus}} permeability. They conclude that (1) Cl{sup {minus}} transport across the TAL is mediated by a process that is sensitive to ambient pH, (2) the basolateral side is more sensitive to pH, (3) the proton binding exhibits positive cooperativity in the modulation of Cl{sup {minus}} transport, and (4) intracellular pH might also modulate Cl{sup {minus}} transport.« less

Interleukin (IL)-18 Binding Protein Deficiency Disrupts Natural Killer Cell Maturation and Diminishes Circulating IL-18

PubMed Central

Harms, Robert Z.; Creer, Austin J.; Lorenzo-Arteaga, Kristina M.; Ostlund, Katie R.; Sarvetnick, Nora E.

2017-01-01

The cytokine interleukin (IL)-18 is a crucial amplifier of natural killer (NK) cell function. IL-18 signaling is regulated by the inhibitory effects of IL-18 binding protein (IL-18BP). Using mice deficient in IL-18BP (IL-18BPKO), we investigated the impact of mismanaged IL-18 signaling on NK cells. We found an overall reduced abundance of splenic NK cells in the absence of IL-18BP. Closer examination of NK cell subsets in spleen and bone marrow using CD27 and CD11b expression revealed that immature NK cells were increased in abundance, while the mature population of NK cells was reduced. Also, NK cells were polarized to greater production of TNF-α, while dedicated IFN-γ producers were reduced. A novel subset of IL-18 receptor α− NK cells contributed to the expansion of immature NK cells in IL-18BPKO mice. Splenocytes cultured with IL-18 resulted in alterations similar to those observed in IL-18BP deficiency. NK cell changes were associated with significantly reduced levels of circulating plasma IL-18. However, IL-18BPKO mice exhibited normal weight gain and responded to LPS challenge with a >10-fold increase in IFN-γ compared to wild type. Finally, we identified that the source of splenic IL-18BP was among dendritic cells/macrophage localized to the T cell-rich regions of the spleen. Our results demonstrate that IL-18BP is required for normal NK cell abundance and function and also contributes to maintaining steady-state levels of circulating IL-18. Thus, IL-18BP appears to have functions suggestive of a carrier protein, not just an inhibitor. PMID:28900426

Hydrogen Sulfide Alleviates Aluminum Toxicity via Decreasing Apoplast and Symplast Al Contents in Rice.

PubMed

Zhu, Chun Q; Zhang, Jun H; Sun, Li M; Zhu, Lian F; Abliz, Buhailiqem; Hu, Wen J; Zhong, Chu; Bai, Zhi G; Sajid, Hussain; Cao, Xiao C; Jin, Qian Y

2018-01-01

Hydrogen sulfide (H 2 S) plays a vital role in Al 3+ stress resistance in plants, but the underlying mechanism is unclear. In the present study, pretreatment with 2 μM of the H 2 S donor NaHS significantly alleviated the inhibition of root elongation caused by Al toxicity in rice roots, which was accompanied by a decrease in Al contents in root tips under 50 μM Al 3+ treatment. NaHS pretreatment decreased the negative charge in cell walls by reducing the activity of pectin methylesterase and decreasing the pectin and hemicellulose contents in rice roots. This treatment also masked Al-binding sites in the cell wall by upregulating the expression of OsSATR1 and OsSTAR2 in roots and reduced Al binding in the cell wall by stimulating the expression of the citrate acid exudation gene OsFRDL4 and increasing the secretion of citrate acid. In addition, NaHS pretreatment decreased the symplasmic Al content by downregulating the expression of OsNRAT1 , and increasing the translocation of cytoplasmic Al to the vacuole via upregulating the expression of OsALS1 . The increment of antioxidant enzyme [superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD)] activity with NaHS pretreatment significantly decreased the MDA and H 2 O 2 content in rice roots, thereby reducing the damage of Al 3+ toxicity on membrane integrity in rice. H 2 S exhibits crosstalk with nitric oxide (NO) in response to Al toxicity, and through reducing NO content in root tips to alleviate Al toxicity. Together, this study establishes that H 2 S alleviates Al toxicity by decreasing the Al content in the apoplast and symplast of rice roots.

Variola Virus IL-18 Binding Protein Interacts with Three Human IL-18 Residues That Are Part of a Binding Site for Human IL-18 Receptor Alpha Subunit

PubMed Central

Meng, Xiangzhi; Leman, Michael; Xiang, Yan

2007-01-01

Interleukin-18 (IL-18) plays an important role in host defense against microbial pathogens. Many poxviruses encode homologous IL-18 binding proteins (IL-18BP) that neutralize IL-18 activity. Here, we examined whether IL-18BP neutralizes IL-18 activity by binding to the same region of IL-18 where IL-18 receptor (IL-18R) binds. We introduced alanine substitutions to known receptor binding sites of human IL18, and found that only the substitution of Leu5 reduced the binding affinity of IL-18 with IL-18BP of variola virus (varvIL-18BP) by more than 4-fold. The substitutions of Lys53 and Ser55, which were not previously known to be part of the receptor binding site but that are spatially adjacent to Leu5, reduced the binding affinity to varvIL-18BP by approximately 100- and 7-fold, respectively. These two substitutions also reduced the binding affinity with human IL-18R alpha subunit (hIL-18Rα) by 4- and 2-fold, respectively. Altogether, our data shows that varvIL-18BP prevents IL-18 from binding to IL-18R by interacting with three residues that are part of the binding site for hIL-18Rα. PMID:16979683

Screening of biologically important Zn2 + by a chemosensor with fluorescent turn on-off mechanism

NASA Astrophysics Data System (ADS)

Khan, Tanveer A.; Sheoran, Monika; Nikhil Raj M., Venkata; Jain, Surbhi; Gupta, Diksha; Naik, Sunil G.

2018-01-01

Reported herein the synthesis, characterization and biologically important zinc ion binding propensity of a weakly fluorescent chemosensor, 4-methyl-2,6-bis((E)-(2-(4-phenylthiazol-2-yl)hydrazono)methyl)phenol (1). 1H NMR spectroscopic titration experiment reveals the binding knack of 1 to the essential Zn2 +. The photo-physical studies of 1 exhibit an enhancement in the fluorescence by several folds upon binding with the zinc ions attributed to PET-off process, with a binding constant value of 5.22 × 103 M- 1. 1 exhibits an excellent detection range for Zn2 + with lower detection limit value of 2.31 × 10- 8 M. The selectivity of 1 was studied with various mono and divalent metal cations and it was observed that most cations either quenches the fluorescence or remains unchanged except for Cd2 +, which shows a slight enhancement in fluorescence intensity of 1. The ratiometric displacement of Cd2 + ions by Zn2 + ions shows an excellent selectivity towards in-situ detection of Zn2 + ions. Photo-physical studies also support the reversible binding of 1 to Zn2 + ions having on and off mechanism in presence of EDTA. Such recognition of the biologically important zinc ions finds potential application in live cell imaging.

The evolutionarily conserved interaction between LC3 and p62 selectively mediates autophagy-dependent degradation of mutant huntingtin.

PubMed

Tung, Ying-Tsen; Hsu, Wen-Ming; Lee, Hsinyu; Huang, Wei-Pang; Liao, Yung-Feng

2010-07-01

Mammalian p62/sequestosome-1 protein binds to both LC3, the mammalian homologue of yeast Atg8, and polyubiquitinated cargo proteins destined to undergo autophagy-mediated degradation. We previously identified a cargo receptor-binding domain in Atg8 that is essential for its interaction with the cargo receptor Atg19 in selective autophagic processes in yeast. We, thus, sought to determine whether this interaction is evolutionally conserved from yeast to mammals. Using an amino acid replacement approach, we demonstrate that cells expressing mutant LC3 (LC3-K30D, LC3-K51A, or LC3-L53A) all exhibit defective lipidation of LC3, a disrupted LC3-p62 interaction, and impaired autophagic degradation of p62, suggesting that the p62-binding site of LC3 is localized within an evolutionarily conserved domain. Importantly, whereas cells expressing these LC3 mutants exhibited similar overall autophagic activity comparable to that of cells expressing wild-type LC3, autophagy-mediated clearance of the aggregation-prone mutant Huntingtin was defective in the mutant-expressing cells. Together, these results suggest that p62 directly binds to the evolutionarily conserved cargo receptor-binding domain of Atg8/LC3 and selectively mediates the clearance of mutant Huntingtin.

Functional somatostatin receptors on a rat pancreatic acinar cell line

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

Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.

1988-07-01

Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of {sup 125}I-(Tyr{sup 11})Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 {plus minus} 20 fmol/10{sup 6} cells. Somatostatin receptor structure was analyzed by covalently cross-linking {sup 125}I-(Tyr{sup 11})somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibitionmore » of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N{sub i} to inhibit adenylate cyclase.« less

Analysis of Paracoccidioides secreted proteins reveals fructose 1,6-bisphosphate aldolase as a plasminogen-binding protein.

PubMed

Chaves, Edilânia Gomes Araújo; Weber, Simone Schneider; Báo, Sonia Nair; Pereira, Luiz Augusto; Bailão, Alexandre Melo; Borges, Clayton Luiz; Soares, Célia Maria de Almeida

2015-02-27

Despite being important thermal dimorphic fungi causing Paracoccidioidomycosis, the pathogenic mechanisms that underlie the genus Paracoccidioides remain largely unknown. Microbial pathogens express molecules that can interact with human plasminogen, a protein from blood plasma, which presents fibrinolytic activity when activated into plasmin. Additionally, plasmin exhibits the ability of degrading extracellular matrix components, favoring the pathogen spread to deeper tissues. Previous work from our group demonstrated that Paracoccidioides presents enolase, as a protein able to bind and activate plasminogen, increasing the fibrinolytic activity of the pathogen, and the potential for adhesion and invasion of the fungus to host cells. By using proteomic analysis, we aimed to identify other proteins of Paracoccidioides with the ability of binding to plasminogen. In the present study, we employed proteomic analysis of the secretome, in order to identify plasminogen-binding proteins of Paracoccidioides, Pb01. Fifteen proteins were present in the fungal secretome, presenting the ability to bind to plasminogen. Those proteins are probable targets of the fungus interaction with the host; thus, they could contribute to the invasiveness of the fungus. For validation tests, we selected the protein fructose 1,6-bisphosphate aldolase (FBA), described in other pathogens as a plasminogen-binding protein. The protein FBA at the fungus surface and the recombinant FBA (rFBA) bound human plasminogen and promoted its conversion to plasmin, potentially increasing the fibrinolytic capacity of the fungus, as demonstrated in fibrin degradation assays. The addition of rFBA or anti-rFBA antibodies was capable of reducing the interaction between macrophages and Paracoccidioides, possibly by blocking the binding sites for FBA. These data reveal the possible participation of the FBA in the processes of cell adhesion and tissue invasion/dissemination of Paracoccidioides. These data indicate that Paracoccidioides is a pathogen that has several plasminogen-binding proteins that likely play important roles in pathogen-host interaction. In this context, FBA is a protein that might be involved somehow in the processes of invasion and spread of the fungus during infection.

Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: a [35S]GTPgammaS binding study.

PubMed

Newman-Tancredi, A; Gavaudan, S; Conte, C; Chaput, C; Touzard, M; Verrièle, L; Audinot, V; Millan, M J

1998-08-21

Recombinant human (h) 5-HT1A receptor-mediated G-protein activation was characterised in membranes of transfected Chinese hamster ovary (CHO) cells by use of guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS binding). The potency and efficacy of 21 5-HT receptor agonists and antagonists was determined. The agonists, 5-CT (carboxamidotryptamine) and flesinoxan displayed high affinity (subnanomolar Ki values) and high efficacy (Emax > 90%, relative to 5-HT = 100%). In contrast, ipsapirone, zalospirone and buspirone displayed partial agonist activity. EC50s for agonist stimulation of [35S]GTPgammaS binding correlated well with Ki values from competition binding (r = +0.99). Among the compounds tested for antagonist activity, methiothepin and (+)butaclamol exhibited 'inverse agonist' behaviour, inhibiting basal [35S]GTPgammaS binding. The actions of 17 antipsychotic agents were investigated. Clozapine and several putatively 'atypical' antipsychotic agents, including ziprasidone, quetiapine and tiospirone, exhibited partial agonist activity and marked affinity at h5-HT1A receptors, similar to their affinity at hD2 dopamine receptors. In contrast, risperidone and sertindole displayed low affinity at h5-HT1A receptors and behaved as 'neutral' antagonists, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Likewise the 'typical' neuroleptics, haloperidol, pimozide, raclopride and chlorpromazine exhibited relatively low affinity and 'neutral' antagonist activity at h5-HT1A receptors with Ki values which correlated with their respective Kb values. The present data show that (i) [35S]GTPgammaS binding is an effective method to evaluate the efficacy and potency of agonists and antagonists at recombinant human 5-HT1A receptors. (ii) Like clozapine, several putatively 'atypical' antipsychotic drugs display balanced serotonin h5-HT1A/dopamine hD2 receptor affinity and partial agonist activity at h5-HT1A receptors. (iii) Several 'typical' and some putatively 'atypical' antipsychotic agents displayed antagonist properties at h5-HT1A sites with generally much lower affinity than at hD2 dopamine receptors. It is suggested that agonist activity at 5-HT1A receptors may be of utility for certain antipsychotic agents.

Modelling dynamics in protein crystal structures by ensemble refinement

PubMed Central

Burnley, B Tom; Afonine, Pavel V; Adams, Paul D; Gros, Piet

2012-01-01

Single-structure models derived from X-ray data do not adequately account for the inherent, functionally important dynamics of protein molecules. We generated ensembles of structures by time-averaged refinement, where local molecular vibrations were sampled by molecular-dynamics (MD) simulation whilst global disorder was partitioned into an underlying overall translation–libration–screw (TLS) model. Modeling of 20 protein datasets at 1.1–3.1 Å resolution reduced cross-validated Rfree values by 0.3–4.9%, indicating that ensemble models fit the X-ray data better than single structures. The ensembles revealed that, while most proteins display a well-ordered core, some proteins exhibit a ‘molten core’ likely supporting functionally important dynamics in ligand binding, enzyme activity and protomer assembly. Order–disorder changes in HIV protease indicate a mechanism of entropy compensation for ordering the catalytic residues upon ligand binding by disordering specific core residues. Thus, ensemble refinement extracts dynamical details from the X-ray data that allow a more comprehensive understanding of structure–dynamics–function relationships. DOI: http://dx.doi.org/10.7554/eLife.00311.001 PMID:23251785

Mutagenesis of threonine to serine in the active site of Mycobacterium tuberculosis fructose-1,6-bisphosphatase (Class II) retains partial enzyme activity.

PubMed

Bondoc, Jasper Marc G; Wolf, Nina M; Ndichuck, Michael; Abad-Zapatero, Celerino; Movahedzadeh, Farahnaz

2017-09-01

The glpX gene encodes for the Class II fructose-1,6-bisphosphatase enzyme in Mycobacterium tuberculosis ( Mt ), an essential enzyme for pathogenesis. We have performed site directed mutagenesis to introduce two mutations at residue Thr84, T84A and T84S, to explore the binding affinity of the substrate and the catalytic mechanism. The T84A mutant fully abolishes enzyme activity while retaining substrate binding affinity. In contrast, the T84S mutant retains some activity having a 10 times reduction in V max and exhibited similar sensitivity to lithium when compared to the wildtype. Homology modeling using the Escherichia coli enzyme structure suggests that the replacement of the critical nucleophile OH - in the Thr84 residue of the wildtype of Mt FBPase by Ser84 results in subtle alterations of the position and orientation that reduce the catalytic efficiency. This mutant could be used to trap reaction intermediates, through crystallographic methods, facilitating the design of potent inhibitors via structure-based drug design.

The lectin from Musa paradisiaca binds with the capsid protein of tobacco mosaic virus and prevents viral infection.

PubMed

Liu, Xiao-Yu; Li, Huan; Zhang, Wei

2014-05-04

It has been demonstrated that the lectin from Musa paradisiaca (BanLec-1) could inhibit the cellular entry of human immunodeficiency virus (HIV). In order to evaluate its effects on tobacco mosaic virus (TMV), the banlec-1 gene was cloned and transformed into Escherichia coli and tobacco, respectively. Recombinant BanLec-1 showed metal ions dependence, and higher thermal and pH stability. Overexpression of banlec-1 in tobacco resulted in decreased leaf size, and higher resistance to TMV infection, which includes reduced TMV cellular entry, more stable chlorophyll contents, and enhanced antioxidant enzymes. BanLec-1 was found to bind directly to the TMV capsid protein in vitro , and to inhibit TMV infection in a dose-dependent manner. In contrast to limited prevention in vivo , purified rBanLec-1 exhibited more significant effects on TMV infection in vitro . Taken together, our study indicated that BanLec-1 could prevent TMV infection in tobacco, probably through the interaction between BanLec-1 and TMV capsid protein.

The lectin from Musa paradisiaca binds with the capsid protein of tobacco mosaic virus and prevents viral infection

PubMed Central

Liu, Xiao-Yu; Li, Huan; Zhang, Wei

2014-01-01

It has been demonstrated that the lectin from Musa paradisiaca (BanLec-1) could inhibit the cellular entry of human immunodeficiency virus (HIV). In order to evaluate its effects on tobacco mosaic virus (TMV), the banlec-1 gene was cloned and transformed into Escherichia coli and tobacco, respectively. Recombinant BanLec-1 showed metal ions dependence, and higher thermal and pH stability. Overexpression of banlec-1 in tobacco resulted in decreased leaf size, and higher resistance to TMV infection, which includes reduced TMV cellular entry, more stable chlorophyll contents, and enhanced antioxidant enzymes. BanLec-1 was found to bind directly to the TMV capsid protein in vitro, and to inhibit TMV infection in a dose-dependent manner. In contrast to limited prevention in vivo, purified rBanLec-1 exhibited more significant effects on TMV infection in vitro. Taken together, our study indicated that BanLec-1 could prevent TMV infection in tobacco, probably through the interaction between BanLec-1 and TMV capsid protein. PMID:26019527

The glycine-rich motif of Pyrococcus abyssi DNA polymerase D is critical for protein stability.

PubMed

Castrec, Benoît; Laurent, Sébastien; Henneke, Ghislaine; Flament, Didier; Raffin, Jean-Paul

2010-03-05

A glycine-rich motif described as being involved in human polymerase delta proliferating cell nuclear antigen (PCNA) binding has also been identified in all euryarchaeal DNA polymerase D (Pol D) family members. We redefined the motif as the (G)-PYF box. In the present study, Pol D (G)-PYF box motif mutants from Pyrococcus abyssi were generated to investigate its role in functional interactions with the cognate PCNA. We demonstrated that this motif is not essential for interactions between PabPol D (P. abyssi Pol D) and PCNA, using surface plasmon resonance and primer extension studies. Interestingly, the (G)-PYF box is located in a hydrophobic region close to the active site. The (G)-PYF box mutants exhibited altered DNA binding properties. In addition, the thermal stability of all mutants was reduced compared to that of wild type, and this effect could be attributed to increased exposure of the hydrophobic region. These studies suggest that the (G)-PYF box motif mediates intersubunit interactions and that it may be crucial for the thermostability of PabPol D. (c) 2010 Elsevier Ltd. All rights reserved.

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

Stier, Andreas V.; McCreary, Kathleen M.; Jonker, Berend T.

In bulk and quantum-confined semiconductors, magneto-optical studies have historically played an essential role in determining the fundamental parameters of excitons (size, binding energy, spin, dimensionality and so on). Here we report low-temperature polarized reflection spectroscopy of atomically thin WS 2 and MoS 2 in high magnetic fields to 65 T. Both the A and B excitons exhibit similar Zeeman splittings of approximately –230 μeV T–1 (g-factor ≃–4), thereby quantifying the valley Zeeman effect in monolayer transition-metal disulphides. Crucially, these large fields also allow observation of the small quadratic diamagnetic shifts of both A and B excitons in monolayer WS 2,more » from which radii of ~1.53 and ~1.16 nm are calculated. Further, when analysed within a model of non-local dielectric screening, these diamagnetic shifts also constrain estimates of the A and B exciton binding energies (410 and 470 meV, respectively, using a reduced A exciton mass of 0.16 times the free electron mass). Lastly, these results highlight the utility of high magnetic fields for understanding new two-dimensional materials.« less

Exciton diamagnetic shifts and valley Zeeman effects in monolayer WS2 and MoS2 to 65 Tesla

NASA Astrophysics Data System (ADS)

Stier, Andreas V.; McCreary, Kathleen M.; Jonker, Berend T.; Kono, Junichiro; Crooker, Scott A.

2016-02-01

In bulk and quantum-confined semiconductors, magneto-optical studies have historically played an essential role in determining the fundamental parameters of excitons (size, binding energy, spin, dimensionality and so on). Here we report low-temperature polarized reflection spectroscopy of atomically thin WS2 and MoS2 in high magnetic fields to 65 T. Both the A and B excitons exhibit similar Zeeman splittings of approximately -230 μeV T-1 (g-factor ~=-4), thereby quantifying the valley Zeeman effect in monolayer transition-metal disulphides. Crucially, these large fields also allow observation of the small quadratic diamagnetic shifts of both A and B excitons in monolayer WS2, from which radii of ~1.53 and ~1.16 nm are calculated. Further, when analysed within a model of non-local dielectric screening, these diamagnetic shifts also constrain estimates of the A and B exciton binding energies (410 and 470 meV, respectively, using a reduced A exciton mass of 0.16 times the free electron mass). These results highlight the utility of high magnetic fields for understanding new two-dimensional materials.

Tumor-homing peptides as tools for targeted delivery of payloads to the placenta

PubMed Central

King, Anna; Ndifon, Cornelia; Lui, Sylvia; Widdows, Kate; Kotamraju, Venkata R.; Agemy, Lilach; Teesalu, Tambet; Glazier, Jocelyn D.; Cellesi, Francesco; Tirelli, Nicola; Aplin, John D.; Ruoslahti, Erkki; Harris, Lynda K.

2016-01-01

The availability of therapeutics to treat pregnancy complications is severely lacking mainly because of the risk of causing harm to the fetus. As enhancement of placental growth and function can alleviate maternal symptoms and improve fetal growth in animal models, we have developed a method for targeted delivery of payloads to the placenta. We show that the tumor-homing peptide sequences CGKRK and iRGD bind selectively to the placental surface of humans and mice and do not interfere with normal development. Peptide-coated nanoparticles intravenously injected into pregnant mice accumulated within the mouse placenta, whereas control nanoparticles exhibited reduced binding and/or fetal transfer. We used targeted liposomes to efficiently deliver cargoes of carboxyfluorescein and insulin-like growth factor 2 to the mouse placenta; the latter significantly increased mean placental weight when administered to healthy animals and significantly improved fetal weight distribution in a well-characterized model of fetal growth restriction. These data provide proof of principle for targeted delivery of drugs to the placenta and provide a novel platform for the development of placenta-specific therapeutics. PMID:27386551

Induction of expression and co-localization of heat shock polypeptides with the polyalanine expansion mutant of poly(A)-binding protein N1 after chemical stress

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

Wang Qishan; Bag, Jnanankur

Formation of nuclear inclusions consisting of aggregates of a polyalanine expansion mutant of nuclear poly(A)-binding protein (PABPN1) is the hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant disease. Patients with this disorder exhibit progressive swallowing difficulty and drooping of their eye lids, which starts around the age of 50. Previously we have shown that treatment of cells expressing the mutant PABPN1 with a number of chemicals such as ibuprofen, indomethacin, ZnSO{sub 4}, and 8-hydroxy-quinoline induces HSP70 expression and reduces PABPN1 aggregation. In these studies we have shown that expression of additional HSPs including HSP27, HSP40,more » and HSP105 were induced in mutant PABPN1 expressing cells following exposure to the chemicals mentioned above. Furthermore, all three additional HSPs were translocated to the nucleus and probably helped to properly fold the mutant PABPN1 by co-localizing with this protein.« less

Fungal melanins and their interactions with metals.

PubMed

Fogarty, R V; Tobin, J M

1996-09-01

Fungal melanins are dark brown or black pigments located in cell walls. They also exist as extracellular polymers. Melanized fungi possess increased virulence and resistance to microbial attack as well as enhanced survival while under environmental stress. Melanins contain various functional groups which provide an array of multiple nonequivalent binding sites for metal ions. Pigmented Cladosporium cladosporoides was shown to biosorb 2.5- to four-fold more Ni, Cu, Zn, Cd, and Pb than albino Penicillium digitatum and at four- to six-fold higher rates. Metal desorption was significantly lower for extracellular melanin than from pigmented or albino biomass which indicated the strength of the melanin-metal bond. At equilibrium, tributyltin chloride (TBTC) concentrations of 2.5 mM, pigmented and albino Aureobasidium pullulans absorbed approximately 0.9 and 0.7 mumol TBTC mg -1 dry wt, respectively, whereas purified extracellular melanin exhibited uptake levels of approximately 22 mumol TBTC mg-1 dry wt at an equilibrium concentration of only 0.4 mM. Addition of melanin to the growth medium reduced the toxic effect of CuSO4 and TBTC due to melanin metal binding and sequestration.

In Silico Identification of Potent Pancreatic Triacylglycerol Lipase Inhibitors from Traditional Chinese Medicine

PubMed Central

Chen, Kuan-Yu; Chang, Su-Sen; Chen, Calvin Yu-Chian

2012-01-01

Pancreatic triacylglycerol lipase (PNLIP) are primary lipases that are critical for triacylglyceride digestion in human. Since reduced metabolism of triacylglyceride might be a plausible concept for weight loss, we screened for potential PNLIP inhibitors from traditional Chinese medicine (TCM) with the aim to identify weight loss candidate compounds. TCM candidates Aurantiamide, Cnidiadin, and 2-hexadecenoic acid exhibited higher Dock Scores than the commercial drug Orlistat, and were also predicted to have inhibitory characteristics against PNLIP using constructed MLR (R2 = 0.8664) and SVM (R2 = 0.9030) models. Molecular dynamics indicated that the TCM-PNLIP complexes formed were stable. We identified that the PNLIP binding site has several residues that can serve as anchors, and a hydrophobic corridor that provides additional stability to the complex. Aurantiamide, Cnidiadin, and 2-hexadecenoic acid all have features that correspond to these binding site features, indicating their potential as candidates for PNLIP inhibitors. The information presented in this study may provide helpful insights to designing novel weight-control drugs. PMID:22970152

Alpha-amylase inhibitor, CS-1036 binds to serum amylase in a concentration-dependent and saturable manner.

PubMed

Honda, Tomohiro; Kaneno-Urasaki, Yoko; Ito, Takashi; Kimura, Takako; Matsushima, Nobuko; Okabe, Hiromi; Yamasaki, Atsushi; Izumi, Takashi

2014-03-01

(2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036), which is an α-amylase inhibitor, exhibited biphasic and sustained elimination with a long t1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t1/2, the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (KD) of 8.95 and 27.2 nM, and maximal binding capacity (Bmax) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (KD 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (≤10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro KD and Bmax, the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats.

Dynamics of Fos-Jun-NFAT1 complexes

PubMed Central

Ramirez-Carrozzi, Vladimir R.; Kerppola, Tom K.

2001-01-01

Transcription initiation in eukaryotes is controlled by nucleoprotein complexes formed through cooperative interactions among multiple transcription regulatory proteins. These complexes may be assembled via stochastic collisions or defined pathways. We investigated the dynamics of Fos-Jun-NFAT1 complexes by using a multicolor fluorescence resonance energy transfer assay. Fos-Jun heterodimers can bind to AP-1 sites in two opposite orientations, only one of which is populated in mature Fos-Jun-NFAT1 complexes. We studied the reversal of Fos-Jun binding orientation in response to NFAT1 by measuring the efficiencies of energy transfer from donor fluorophores linked to opposite ends of an oligonucleotide to an acceptor fluorophore linked to one subunit of the heterodimer. The reorientation of Fos-Jun by NFAT1 was not inhibited by competitor oligonucleotides or heterodimers. The rate of Fos-Jun reorientation was faster than the rate of heterodimer dissociation at some binding sites. The facilitated reorientation of Fos-Jun heterodimers therefore can enhance the efficiency of Fos-Jun-NFAT1 complex formation. We also examined the influence of the preferred orientation of Fos-Jun binding on the stability and transcriptional activity of Fos-Jun-NFAT1 complexes. Complexes formed at sites where Fos-Jun favored the same binding orientation in the presence and absence of NFAT1 exhibited an 8-fold slower dissociation rate than complexes formed at sites where Fos-Jun favored the opposite binding orientation. Fos-Jun-NFAT1 complexes also exhibited greater transcription activation at promoter elements that favored the same orientation of Fos-Jun binding in the presence and absence of NFAT1. Thus, the orientation of heterodimer binding can influence both the dynamics and promoter selectivity of multiprotein transcription regulatory complexes. PMID:11320240

Dynamics of Fos-Jun-NFAT1 complexes.

PubMed

Ramirez-Carrozzi, V R; Kerppola, T K

2001-04-24

Transcription initiation in eukaryotes is controlled by nucleoprotein complexes formed through cooperative interactions among multiple transcription regulatory proteins. These complexes may be assembled via stochastic collisions or defined pathways. We investigated the dynamics of Fos-Jun-NFAT1 complexes by using a multicolor fluorescence resonance energy transfer assay. Fos-Jun heterodimers can bind to AP-1 sites in two opposite orientations, only one of which is populated in mature Fos-Jun-NFAT1 complexes. We studied the reversal of Fos-Jun binding orientation in response to NFAT1 by measuring the efficiencies of energy transfer from donor fluorophores linked to opposite ends of an oligonucleotide to an acceptor fluorophore linked to one subunit of the heterodimer. The reorientation of Fos-Jun by NFAT1 was not inhibited by competitor oligonucleotides or heterodimers. The rate of Fos-Jun reorientation was faster than the rate of heterodimer dissociation at some binding sites. The facilitated reorientation of Fos-Jun heterodimers therefore can enhance the efficiency of Fos-Jun-NFAT1 complex formation. We also examined the influence of the preferred orientation of Fos-Jun binding on the stability and transcriptional activity of Fos-Jun-NFAT1 complexes. Complexes formed at sites where Fos-Jun favored the same binding orientation in the presence and absence of NFAT1 exhibited an 8-fold slower dissociation rate than complexes formed at sites where Fos-Jun favored the opposite binding orientation. Fos-Jun-NFAT1 complexes also exhibited greater transcription activation at promoter elements that favored the same orientation of Fos-Jun binding in the presence and absence of NFAT1. Thus, the orientation of heterodimer binding can influence both the dynamics and promoter selectivity of multiprotein transcription regulatory complexes.

Gallium-67-labeled lactam bridge-cyclized alpha-MSH peptides with enhanced melanoma uptake and reduced renal uptake.

PubMed

Guo, Haixun; Gallazzi, Fabio; Miao, Yubin

2012-06-20

The purpose of this study was to examine the melanoma targeting and pharmacokinetic properties of (67)Ga-DOTA-GGNle-CycMSHhex {(67)Ga-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and (67)Ga-NOTA-GGNle-CycMSHhex {(67)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and compare with (67)Ga-DOTA-GlyGlu-CycMSH {(67)Ga-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]} we previously reported. DOTA-GGNle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSHhex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSHhex. The melanoma targeting and pharmacokinetic properties of (67)Ga-NOTA-GGNle-CycMSHhex and (67)Ga-DOTA-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex displayed comparable MC1 receptor binding affinities (1.6 vs 2.1 nM) in B16/F1 melanoma cells. Both (67)Ga-NOTA-GGNle-CycMSHhex and (67)Ga-DOTA-GGNle-CycMSHhex exhibited dramatically enhanced melanoma uptake and reduced renal uptake than (67)Ga-DOTA-GlyGlu-CycMSH in B16/F1 melanoma-bearing C57 mice. Furthermore, (67)Ga-NOTA-GGNle-CycMSHhex exhibited more favorable radiolabeling conditions (>85% radiolabeling yields started at 37 °C), as well as higher tumor/kidney uptake ratios than (67)Ga-DOTA-GGNle-CycMSHhex at 0.5, 2, and 24 h postinjection. High melanoma uptake coupled with low renal uptake highlighted the potential of (67)Ga-NOTA-GGNle-CycMSHhex for melanoma imaging and therapy.

Apigenin inhibits prostate cancer progression in TRAMP mice via targeting PI3K/Akt/FoxO pathway

PubMed Central

Gupta, Sanjay

2014-01-01

Forkhead box O (FoxO) transcription factors play an important role as tumor suppressor in several human malignancies. Disruption of FoxO activity due to loss of phosphatase and tensin homolog and activation of phosphatidylinositol-3 kinase (PI3K)/Akt are frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits antiproliferative and anticarcinogenic activities through mechanisms, which are not fully defined. In the present study, we show that apigenin suppressed prostate tumorigenesis in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice through the PI3K/Akt/FoxO-signaling pathway. Apigenin-treated TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate as well as completely abolished distant organ metastasis. Apigenin treatment resulted in significant decrease in the weight of genitourinary apparatus (P < 0.0001), dorsolateral (P < 0.0001) and ventral prostate (P < 0.028), compared with the control group. Apigenin-treated mice showed reduced phosphorylation of Akt (Ser473) and FoxO3a (Ser253), which correlated with its increased nuclear retention and decreased binding of FoxO3a with 14-3-3. These events lead to reduced proliferation as assessed by Ki-67 and cyclin D1, along with upregulation of FoxO-responsive proteins BIM and p27/Kip1. Complementing in vivo results, similar observations were noted in human prostate cancer LNCaP and PC-3 cells after apigenin treatment. Furthermore, binding of FoxO3a with p27/Kip1 was markedly increased after 10 and 20 μM apigenin treatment resulting in G0/G1-phase cell cycle arrest, which was consistent with the effects elicited by PI3K/Akt inhibitor, LY294002. These results provide convincing evidence that apigenin effectively suppressed prostate cancer progression, at least in part, by targeting the PI3K/Akt/FoxO-signaling pathway. PMID:24067903

Multimechanistic Monoclonal Antibodies (MAbs) Targeting Staphylococcus aureus Alpha-Toxin and Clumping Factor A: Activity and Efficacy Comparisons of a MAb Combination and an Engineered Bispecific Antibody Approach.

PubMed

Tkaczyk, C; Kasturirangan, S; Minola, A; Jones-Nelson, O; Gunter, V; Shi, Y Y; Rosenthal, K; Aleti, V; Semenova, E; Warrener, P; Tabor, D; Stover, C K; Corti, D; Rainey, G; Sellman, B R

2017-08-01

Secreted alpha-toxin and surface-localized clumping factor A (ClfA) are key virulence determinants in Staphylococcus aureus bloodstream infections. We previously demonstrated that prophylaxis with a multimechanistic monoclonal antibody (MAb) combination against alpha-toxin (MEDI4893*) and ClfA (11H10) provided greater strain coverage and improved efficacy in an S. aureus lethal bacteremia model. Subsequently, 11H10 was found to exhibit reduced affinity and impaired inhibition of fibrinogen binding to ClfA002 expressed by members of a predominant hospital-associated methicillin-resistant S. aureus (MRSA) clone, ST5. Consequently, we identified another anti-ClfA MAb (SAR114) from human tonsillar B cells with >100-fold increased affinity for three prominent ClfA variants, including ClfA002, and potent inhibition of bacterial agglutination by 112 diverse clinical isolates. We next constructed bispecific Abs (BiSAbs) comprised of 11H10 or SAR114 as IgG scaffolds and grafted anti-alpha-toxin (MEDI4893*) single-chain variable fragment to the amino or carboxy terminus of the anti-ClfA heavy chains. Although the BiSAbs exhibited in vitro potencies similar to those of the parental MAbs, only 11H10-BiSAb, but not SAR114-BiSAb, showed protective activity in murine infection models comparable to the respective MAb combination. In vivo activity with SAR114-BiSAb was observed in infection models with S. aureus lacking ClfA. Our data suggest that high-affinity binding to ClfA sequesters the SAR114-BiSAb to the bacterial surface, thereby reducing both alpha-toxin neutralization and protection in vivo These results indicate that a MAb combination targeting ClfA and alpha-toxin is more promising for future development than the corresponding BiSAb. Copyright © 2017 Tkaczyk et al.

Solubilization and characterization of haloperidol-sensitive (+)-( sup 3 H)SKF-10,047 binding sites (sigma sites) from rat liver membranes

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

McCann, D.J.; Su, T.P.

1991-05-01

The zwitterionic detergent 3-((3-cholamidopropyl)dimethylamino)-1-propanesulfonate (CHAPS) produced optimal solubilization of (+)-({sup 3}H)SKF-10,047 binding sites from rat liver membranes at a concentration of 0.2%, well below the critical micellular concentration of the detergent. The pharmacological selectivity of the liver (+)-({sup 3}H)SKF-10,047 binding sites corresponds to that of sigma sites from rat and guinea pig brain. When the affinities of 18 different drugs at (+)-({sup 3}H)SKF-10,047 binding sites in membranes and solubilized preparations were compared, a correlation coefficient of 0.99 and a slope of 1.03 were obtained, indicating that the pharmacological selectivity of rat liver sigma sites is retained after solubilization. In addition,more » the binding of 20 nM ({sup 3}H)progesterone to solubilized rat liver preparations was found to exhibit a pharmacological selectivity appropriate for sigma sites. A stimulatory effect of phenytoin on (+)-({sup 3}H)SKF-10,047 binding to sigma sites persisted after solubilization. When the solubilized preparation was subjected to molecular sizing chromatography, a single peak exhibiting specific (+)-({sup 3}H)SKF-10,047 binding was obtained. The binding activity of this peak was stimulated symmetrically when assays were performed in the presence of 300 microM phenytoin. The molecular weight of the CHAPS-solubilized sigma site complex was estimated to be 450,000 daltons. After solubilization with CHAPS, rat liver sigma sites were enriched to 12 pmol/mg of protein. The present results demonstrate a successful solubilization of sigma sites from rat liver membranes and provide direct evidence that the gonadal steroid progesterone binds to sigma sites. The results also suggest that the anticonvulsant phenytoin binds to an associated allosteric site on the sigma site complex.« less

High-Content Microfluidic Screening Platform Used To Identify σ2R/Tmem97 Binding Ligands that Reduce Age-Dependent Neurodegeneration in C. elegans SC_APP Model.

PubMed

Mondal, Sudip; Hegarty, Evan; Sahn, James J; Scott, Luisa L; Gökçe, Sertan Kutal; Martin, Chris; Ghorashian, Navid; Satarasinghe, Praveen Navoda; Iyer, Sangeetha; Sae-Lee, Wisath; Hodges, Timothy R; Pierce, Jonathan T; Martin, Stephen F; Ben-Yakar, Adela

2018-05-16

The nematode Caenorhabditis elegans, with tractable genetics and a well-defined nervous system, provides a unique whole-animal model system to identify novel drug targets and therapies for neurodegenerative diseases. Large-scale drug or target screens in models that recapitulate the subtle age- and cell-specific aspects of neurodegenerative diseases are limited by a technological requirement for high-throughput analysis of neuronal morphology. Recently, we developed a single-copy model of amyloid precursor protein (SC_APP) induced neurodegeneration that exhibits progressive degeneration of select cholinergic neurons. Our previous work with this model suggests that small molecule ligands of the sigma 2 receptor (σ2R), which was recently cloned and identified as transmembrane protein 97 (TMEM97), are neuroprotective. To determine structure-activity relationships for unexplored chemical space in our σ2R/Tmem97 ligand collection, we developed an in vivo high-content screening (HCS) assay to identify potential drug leads. The HCS assay uses our recently developed large-scale microfluidic immobilization chip and automated imaging platform. We discovered norbenzomorphans that reduced neurodegeneration in our C. elegans model, including two compounds that demonstrated significant neuroprotective activity at multiple doses. These findings provide further evidence that σ2R/Tmem97-binding norbenzomorphans may represent a new drug class for treating neurodegenerative diseases.

Functional adaptation between yeast actin and its cognate myosin motors.

PubMed

Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S; Rubenstein, Peter A; Lord, Matthew

2011-09-02

We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins.

Anomalous versus Slowed-Down Brownian Diffusion in the Ligand-Binding Equilibrium

PubMed Central

Soula, Hédi; Caré, Bertrand; Beslon, Guillaume; Berry, Hugues

2013-01-01

Measurements of protein motion in living cells and membranes consistently report transient anomalous diffusion (subdiffusion) that converges back to a Brownian motion with reduced diffusion coefficient at long times after the anomalous diffusion regime. Therefore, slowed-down Brownian motion could be considered the macroscopic limit of transient anomalous diffusion. On the other hand, membranes are also heterogeneous media in which Brownian motion may be locally slowed down due to variations in lipid composition. Here, we investigate whether both situations lead to a similar behavior for the reversible ligand-binding reaction in two dimensions. We compare the (long-time) equilibrium properties obtained with transient anomalous diffusion due to obstacle hindrance or power-law-distributed residence times (continuous-time random walks) to those obtained with space-dependent slowed-down Brownian motion. Using theoretical arguments and Monte Carlo simulations, we show that these three scenarios have distinctive effects on the apparent affinity of the reaction. Whereas continuous-time random walks decrease the apparent affinity of the reaction, locally slowed-down Brownian motion and local hindrance by obstacles both improve it. However, only in the case of slowed-down Brownian motion is the affinity maximal when the slowdown is restricted to a subregion of the available space. Hence, even at long times (equilibrium), these processes are different and exhibit irreconcilable behaviors when the area fraction of reduced mobility changes. PMID:24209851

PARP-1 dependent recruitment of the amyotrophic lateral sclerosis-associated protein FUS/TLS to sites of oxidative DNA damage

PubMed Central

Rulten, Stuart L.; Rotheray, Amy; Green, Ryan L.; Grundy, Gabrielle J.; Moore, Duncan A. Q.; Gómez-Herreros, Fernando; Hafezparast, Majid; Caldecott, Keith W

2014-01-01

Amyotrophic lateral sclerosis (ALS) is associated with progressive degeneration of motor neurons. Several of the genes associated with this disease encode proteins involved in RNA processing, including fused-in-sarcoma/translocated-in-sarcoma (FUS/TLS). FUS is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins that bind thousands of pre-mRNAs and can regulate their splicing. Here, we have examined the possibility that FUS is also a component of the cellular response to DNA damage. We show that both GFP-tagged and endogenous FUS re-localize to sites of oxidative DNA damage induced by UVA laser, and that FUS recruitment is greatly reduced or ablated by an inhibitor of poly (ADP-ribose) polymerase activity. Consistent with this, we show that recombinant FUS binds directly to poly (ADP-ribose) in vitro, and that both GFP-tagged and endogenous FUS fail to accumulate at sites of UVA laser induced damage in cells lacking poly (ADP-ribose) polymerase-1. Finally, we show that GFP-FUSR521G, harbouring a mutation that is associated with ALS, exhibits reduced ability to accumulate at sites of UVA laser-induced DNA damage. Together, these data suggest that FUS is a component of the cellular response to DNA damage, and that defects in this response may contribute to ALS. PMID:24049082

RNA Aptamer-Based Functional Ligands of the Neurotrophin Receptor, TrkB

PubMed Central

Huang, Yang Zhong; Hernandez, Frank J.; Gu, Bin; Stockdale, Katie R.; Nanapaneni, Kishore; Scheetz, Todd E.; Behlke, Mark A.; Peek, Andrew S.; Bair, Thomas; Giangrande, Paloma H.

2012-01-01

Many cell surface signaling receptors, such as the neurotrophin receptor, TrkB, have emerged as potential therapeutic targets for diverse diseases. Reduced activation of TrkB in particular is thought to contribute to neurodegenerative diseases. Unfortunately, development of therapeutic reagents that selectively activate particular cell surface receptors such as TrkB has proven challenging. Like many cell surface signaling receptors, TrkB is internalized upon activation; in this proof-of-concept study, we exploited this fact to isolate a pool of nuclease-stabilized RNA aptamers enriched for TrkB agonists. One of the selected aptamers, C4-3, was characterized with recombinant protein-binding assays, cell-based signaling and functional assays, and, in vivo in a seizure model in mice. C4-3 binds the extracellular domain of TrkB with high affinity (KD ∼2 nM) and exhibits potent TrkB partial agonistic activity and neuroprotective effects in cultured cortical neurons. In mice, C4-3 activates TrkB upon infusion into the hippocampus; systemic administration of C4-3 potentiates kainic acid-induced seizure development. We conclude that C4-3 is a potentially useful therapeutic agent for neurodegenerative diseases in which reduced TrkB activation has been implicated. We anticipate that the cell-based aptamer selection approach used here will be broadly applicable to the identification of aptamer-based agonists for a variety of cell-surface signaling receptors. PMID:22752556

Murine glomerulotropic monoclonal antibodies are highly oligoclonal and exhibit distinctive molecular features.

PubMed

Lefkowith, J B; Di Valerio, R; Norris, J; Glick, G D; Alexander, A L; Jackson, L; Gilkeson, G S

1996-08-01

We recently produced a panel of seven glomerular-binding mAbs from a nephritic MRL-lpr mouse that bind to histones/nucleosomes (group I) or DNA (group II) adherent to glomerular basement membrane. To elucidate the molecular basis of their binding and ontogeny, we sequenced their variable (V) regions, analyzed the apparent somatic mutations, and predicted their three-dimensional structures. There were two clonally related sets (3 of 4 in group I, 3 of 3 in group II) both of the VHJ1558 family, and one mAb of the VH 7183 family. V region somatic mutations within clonally related sets had little effect on glomerular binding and did not appear to be selected for based on glomerular binding. The VH regions were most homologous with those from autoantibodies to histones, DNA, or IgG (i.e., rheumatoid factors), the Vkappa regions, with those from autoantibodies to small nuclear ribonucleoproteins (snRNP). The VH regions also exhibited an unusual VD junction (in the group I clonally related set) and an overall high content of charged amino acids (arginine, aspartic acid) in complementarity-determining regions (CDRs), particularly in CDR3. Molecular modeling studies suggested that the Fv regions of these mAbs converge to form a flat, open surface with a net positive charge. The CDR arginines in group I mAbs; appear to be located in Ag contact regions of the binding cleft. In sum, these data suggest that glomerulotropic mAbs are a highly restricted set of Abs with distinctive molecular features that may mediate their binding to glomeruli.

2.2 A resolution structure analysis of two refined N-acetylneuraminyl-lactose--wheat germ agglutinin isolectin complexes.

PubMed

Wright, C S

1990-10-20

The crystal structures of complexes of isolectins 1 and 2 of wheat germ agglutinin (WGA1 and WGA2) with N-acetylneuraminyl-lactose (NeuNAc-alpha(2-3)-Gal-beta(1-4)-Glc) have been refined on the basis of data in the 8 to 2.2 A resolution range to final crystallographic R-factors of 17.2% and 15.3% (Fo greater than 1 sigma), respectively. Specific binding interactions and water association, as well as changes in conformation and mobility of the structure upon ligand binding, were compared in the two complexes. The temperature factors (B = 16.3 A2 and 18.4 A2) were found to be much lower compared with those of their respective native structures (19 to 22 A2). Residues involved in sugar binding, dimerization and in lattice contacts exhibit the largest decreases in B-value, suggesting that sugar binding reduces the overall mobility of the protein molecules in the crystal lattice. The binding mode of this sialyl-trisaccharide, an important cell receptor analogue, has been compared in the two isolectins. Only one of the two unique binding sites (4 per dimer), located in the subunit/subunit interface, is occupied in the crystals. This site, termed the "primary" binding site, contains one of the five amino acid substitutions that differentiate WGA1 and WGA2. Superposition of the refined models in each of the independent crystallographic environments indicates a close match only of the terminal non-reducing NeuNAc residue (root-mean-square delta r of 0.5 to 0.6 A). The Gal-Glc portion was found to superimpose poorly, lack electron density, and possess high atomic thermal factors. In both complexes NeuNAc is stabilized through contact with six amino acid side-chains (Ser114 and Glu115 of subunit 1 and Ser62, Tyr64, Tyr(His)66 and Tyr73 of subunit 2), involving all NeuNAc ring substituents. Refinement has allowed accurate assessment of the contact distances for four hydrogen bonds, a strong buried non-polar contact with the acetamido CH3 group and a large number of van der Waals' interactions with the three aromatic side-chains. The higher affinity of N-acetylneuraminyl-lactose observed by nuclear magnetic resonance studies for WGA1 can be explained by the more favorable binding interactions that occur when residue 66 is a Tyr. The tyrosyl side-chain provides a larger surface for van der Waals' stacking against the NeuNAc pyranose ring than His66 and a hydrogen bond contact with Gal (C2-OH), not possible in WGA2.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of Squalene Epoxidase of Saccharomyces cerevisiae by Applying Terbinafine-Sensitive Variants▿

PubMed Central

Ruckenstuhl, Christoph; Lang, Silvia; Poschenel, Andrea; Eidenberger, Armin; Baral, Pravas Kumar; Kohút, Peter; Hapala, Ivan; Gruber, Karl; Turnowsky, Friederike

2007-01-01

Squalene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies to p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of the ERG1 gene, which codes for the Saccharomyces cerevisiae SE, we isolated erg1 alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed in erg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G25S, D335X (W, F, P), and G210A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G30S and L37P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase in erg1 mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R269G variant exhibited moderately reduced SE activity and a 5- to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines. PMID:17043127

Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes.

PubMed

Subasinghe, Supundi; Unabia, Sharon; Barrow, Colin J; Mok, Su San; Aguilar, Marie-Isabel; Small, David H

2003-02-01

Accumulation of beta amyloid (Abeta) in the brain is central to the pathogenesis of Alzheimer's disease. Abeta can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Abeta binding to membranes. Abeta peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Abeta peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Abeta peptides and their membrane binding. 'Ageing' the Abeta peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Abeta analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Abeta to purified plasma membrane preparations but also reduced Abeta toxicity. The results support the view that Abeta toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Abeta-membrane binding.

Structural analysis of determinants of histo-blood group antigen binding specificity in genogroup I noroviruses.

PubMed

Shanker, Sreejesh; Czako, Rita; Sankaran, Banumathi; Atmar, Robert L; Estes, Mary K; Prasad, B V Venkataram

2014-06-01

Human noroviruses (NoVs) cause acute epidemic gastroenteritis. Susceptibility to the majority of NoV infections is determined by genetically controlled secretor-dependent expression of histo-blood group antigens (HBGAs), which are also critical for NoV attachment to host cells. Human NoVs are classified into two major genogroups (genogroup I [GI] and GII), with each genogroup further divided into several genotypes. GII NoVs are more prevalent and exhibit periodic emergence of new variants, suggested to be driven by altered HBGA binding specificities and antigenic drift. Recent epidemiological studies show increased activity among GI NoVs, with some members showing the ability to bind nonsecretor HBGAs. NoVs bind HBGAs through the protruding (P) domain of the major capsid protein VP1. GI NoVs, similar to GII, exhibit significant sequence variations in the P domain; it is unclear how these variations affect HBGA binding specificities. To understand the determinants of possible strain-specific HBGA binding among GI NoVs, we determined the structure of the P domain of a GI.7 clinical isolate and compared it to the previously determined P domain structures of GI.1 and GI.2 strains. Our crystallographic studies revealed significant structural differences, particularly in the loop regions of the GI.7 P domain, altering its surface topography and electrostatic landscape and potentially indicating antigenic variation. The GI.7 strain bound to H- and A-type, Lewis secretor, and Lewis nonsecretor families of HBGAs, allowing us to further elucidate the structural determinants of nonsecretor HBGA binding among GI NoVs and to infer several contrasting and generalizable features of HBGA binding in the GI NoVs. Human noroviruses (NoVs) cause acute epidemic gastroenteritis. Recent epidemiological studies have shown increased prevalence of genogroup I (GI) NoVs. Although secretor-positive status is strongly correlated with NoV infection, cases of NoV infection associated with secretor-negative individuals are reported. Biochemical studies have shown that GI NoVs exhibit genotype-dependent binding to nonsecretor histo-blood group antigens (HBGAs). From our crystallographic studies of a GI.7 NoV, in comparison with previous studies on GI.1 and GI.2 NoVs, we show that genotypic differences translate to extensive structural changes in the loop regions that significantly alter the surface topography and electrostatic landscape of the P domain; these features may be indicative of antigenic variations contributing to serotypic differentiation in GI NoVs and also differential modulation of the HBGA binding characteristics. A significant finding is that the threshold length and the structure of one of the loops are critical determinants in the binding of GI NoVs to nonsecretor HBGAs.

Development of a strategy and computational application to select candidate protein analogues with reduced HLA binding and immunogenicity.

PubMed

Dhanda, Sandeep Kumar; Grifoni, Alba; Pham, John; Vaughan, Kerrie; Sidney, John; Peters, Bjoern; Sette, Alessandro

2018-01-01

Unwanted immune responses against protein therapeutics can reduce efficacy or lead to adverse reactions. T-cell responses are key in the development of such responses, and are directed against immunodominant regions within the protein sequence, often associated with binding to several allelic variants of HLA class II molecules (promiscuous binders). Herein, we report a novel computational strategy to predict 'de-immunized' peptides, based on previous studies of erythropoietin protein immunogenicity. This algorithm (or method) first predicts promiscuous binding regions within the target protein sequence and then identifies residue substitutions predicted to reduce HLA binding. Further, this method anticipates the effect of any given substitution on flanking peptides, thereby circumventing the creation of nascent HLA-binding regions. As a proof-of-principle, the algorithm was applied to Vatreptacog α, an engineered Factor VII molecule associated with unintended immunogenicity. The algorithm correctly predicted the two immunogenic peptides containing the engineered residues. As a further validation, we selected and evaluated the immunogenicity of seven substitutions predicted to simultaneously reduce HLA binding for both peptides, five control substitutions with no predicted reduction in HLA-binding capacity, and additional flanking region controls. In vitro immunogenicity was detected in 21·4% of the cultures of peptides predicted to have reduced HLA binding and 11·4% of the flanking regions, compared with 46% for the cultures of the peptides predicted to be immunogenic. This method has been implemented as an interactive application, freely available online at http://tools.iedb.org/deimmunization/. © 2017 John Wiley & Sons Ltd.

Geraniin extracted from the rind of Nephelium lappaceum binds to dengue virus type-2 envelope protein and inhibits early stage of virus replication.

PubMed

Abdul Ahmad, Siti Aisyah; Palanisamy, Uma D; Tejo, Bimo A; Chew, Miaw Fang; Tham, Hong Wai; Syed Hassan, Sharifah

2017-11-21

The rapid rise and spread in dengue cases, together with the unavailability of safe vaccines and effective antiviral drugs, warrant the need to discover and develop novel anti-dengue treatments. In this study the antiviral activity of geraniin, extracted from the rind of Nephelium lappaceum, against dengue virus type-2 (DENV-2) was investigated. Geraniin was prepared from Nephelium lappaceum rind by reverse phase C-18 column chromatography. Cytotoxicity of geraniin towards Vero cells was evaluated using MTT assay while IC 50 value was determined by plaque reduction assay. The mode-of-action of geraniin was characterized using the virucidal, attachment, penetration and the time-of-addition assays'. Docking experiments with geraniin molecule and the DENV envelope (E) protein was also performed. Finally, recombinant E Domain III (rE-DIII) protein was produced to physiologically test the binding of geraniin to DENV-2 E-DIII protein, through ELISA competitive binding assay. Cytotoxicity assay confirmed that geraniin was not toxic to Vero cells, even at the highest concentration tested. The compound exhibited DENV-2 plaque formation inhibition, with an IC 50 of 1.75 μM. We further revealed that geraniin reduced viral infectivity and inhibited DENV-2 from attaching to the cells but had little effect on its penetration. Geraniin was observed to be most effective when added at the early stage of DENV-2 infection. Docking experiments showed that geraniin binds to DENV E protein, specifically at the DIII region, while the ELISA competitive binding assay confirmed geraniin's interaction with rE-DIII with high affinity. Geraniin from the rind of Nephelium lappaceum has antiviral activity against DENV-2. It is postulated that the compound inhibits viral attachment by binding to the E-DIII protein and interferes with the initial cell-virus interaction. Our results demonstrate that geraniin has the potential to be developed into an effective antiviral treatment, particularly for early phase dengue viral infection.

Plasmodium falciparum Nucleosomes Exhibit Reduced Stability and Lost Sequence Dependent Nucleosome Positioning

PubMed Central

Silberhorn, Elisabeth; Schwartz, Uwe; Symelka, Anne; de Koning-Ward, Tania; Längst, Gernot

2016-01-01

The packaging and organization of genomic DNA into chromatin represents an additional regulatory layer of gene expression, with specific nucleosome positions that restrict the accessibility of regulatory DNA elements. The mechanisms that position nucleosomes in vivo are thought to depend on the biophysical properties of the histones, sequence patterns, like phased di-nucleotide repeats and the architecture of the histone octamer that folds DNA in 1.65 tight turns. Comparative studies of human and P. falciparum histones reveal that the latter have a strongly reduced ability to recognize internal sequence dependent nucleosome positioning signals. In contrast, the nucleosomes are positioned by AT-repeat sequences flanking nucleosomes in vivo and in vitro. Further, the strong sequence variations in the plasmodium histones, compared to other mammalian histones, do not present adaptations to its AT-rich genome. Human and parasite histones bind with higher affinity to GC-rich DNA and with lower affinity to AT-rich DNA. However, the plasmodium nucleosomes are overall less stable, with increased temperature induced mobility, decreased salt stability of the histones H2A and H2B and considerable reduced binding affinity to GC-rich DNA, as compared with the human nucleosomes. In addition, we show that plasmodium histone octamers form the shortest known nucleosome repeat length (155bp) in vitro and in vivo. Our data suggest that the biochemical properties of the parasite histones are distinct from the typical characteristics of other eukaryotic histones and these properties reflect the increased accessibility of the P. falciparum genome. PMID:28033404

Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae)

PubMed Central

Li, Guangwei; Chen, Xiulin; Li, Boliao; Zhang, Guohui; Li, Yiping; Wu, Junxiang

2016-01-01

Background The oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs) are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs) within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications. Methodology/Principal Finding Two antennae-specific general OBPs (GOBPs) of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2) for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC) experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1) exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition. Conclusion Two rGmolGOBPs exhibit different binding characteristics for tested ligands. rGmolGOBP1 has dual functions in recognition of host plant volatiles and sex pheromone components, while rGmolGOBP2 is mainly involved in minor sex pheromone component dodecanol perception. This study also provides empirical evidence for the predicted functions of key amino acids in recombinant protein ligand-binding characteristics. PMID:27152703

Persistent Graves' hyperthyroidism despite rapid negative conversion of thyroid-stimulating hormone-binding inhibitory immunoglobulin assay results: a case report.

PubMed

Ohara, Nobumasa; Kaneko, Masanori; Kitazawa, Masaru; Uemura, Yasuyuki; Minagawa, Shinichi; Miyakoshi, Masashi; Kaneko, Kenzo; Kamoi, Kyuzi

2017-02-06

Graves' disease is an autoimmune thyroid disorder characterized by hyperthyroidism, and patients exhibit thyroid-stimulating hormone receptor antibody. The major methods of measuring circulating thyroid-stimulating hormone receptor antibody include the thyroid-stimulating hormone-binding inhibitory immunoglobulin assays. Although the diagnostic accuracy of these assays has been improved, a minority of patients with Graves' disease test negative even on second-generation and third-generation thyroid-stimulating hormone-binding inhibitory immunoglobulins. We report a rare case of a thyroid-stimulating hormone-binding inhibitory immunoglobulin-positive patient with Graves' disease who showed rapid lowering of thyroid-stimulating hormone-binding inhibitory immunoglobulin levels following administration of the anti-thyroid drug thiamazole, but still experienced Graves' hyperthyroidism. A 45-year-old Japanese man presented with severe hyperthyroidism (serum free triiodothyronine >25.0 pg/mL; reference range 1.7 to 3.7 pg/mL) and tested weakly positive for thyroid-stimulating hormone-binding inhibitory immunoglobulins on second-generation tests (2.1 IU/L; reference range <1.0 IU/L). Within 9 months of treatment with oral thiamazole (30 mg/day), his thyroid-stimulating hormone-binding inhibitory immunoglobulin titers had normalized, but he experienced sustained hyperthyroidism for more than 8 years, requiring 15 mg/day of thiamazole to correct. During that period, he tested negative on all first-generation, second-generation, and third-generation thyroid-stimulating hormone-binding inhibitory immunoglobulin assays, but thyroid scintigraphy revealed diffuse and increased uptake, and thyroid ultrasound and color flow Doppler imaging showed typical findings of Graves' hyperthyroidism. The possible explanations for serial changes in the thyroid-stimulating hormone-binding inhibitory immunoglobulin results in our patient include the presence of thyroid-stimulating hormone receptor antibody, which is bioactive but less reactive on thyroid-stimulating hormone-binding inhibitory immunoglobulin assays, or the effect of reduced levels of circulating thyroid-stimulating hormone receptor antibody upon improvement of thyroid autoimmunity with thiamazole treatment. Physicians should keep in mind that patients with Graves' disease may show thyroid-stimulating hormone-binding inhibitory immunoglobulin assay results that do not reflect the severity of Graves' disease or indicate the outcome of the disease, and that active Graves' disease may persist even after negative results on thyroid-stimulating hormone-binding inhibitory immunoglobulin assays. Timely performance of thyroid function tests in combination with sensitive imaging tests, including thyroid ultrasound and scintigraphy, are necessary to evaluate the severity of Graves' disease and treatment efficacy.

Structure Determination of Ornithine-Linked Cisplatin by Infrared Multiple Photon Dissociation Action Spectroscopy

NASA Astrophysics Data System (ADS)

He, Chenchen; Kimutai, Bett; Hamlow, Lucas; Roy, Harrison; Nei, Y.-W.; Bao, Xun; Gao, Juehan; Martens, Jonathan K.; Berden, Giel; Oomens, Jos; Maitre, Philippe; Steinmetz, Vincent; McNary, Christopher P.; Armentrout, Peter B.; Chow, C. S.; Rodgers, M. T.

2016-06-01

Cisplatin [(NH_3)_2PtCl_2], the first FDA-approved platinum-based anticancer drug, has been widely used in cancer chemotherapy. Its pharmacological mechanism has been identified as its ability to coordinate to genomic DNA with guanine as its major target. Amino acid-linked cisplatin derivatives are being investigated as alternatives for cisplatin that may exhibit altered binding selectivity such as that found for ornithine-linked cisplatin (Ornplatin, [(Orn)PtCl_2]), which exhibits a preference for adenine over guanine in RNA. Infrared multiple photon dissociation (IRMPD) action spectroscopy experiments and complementary electronic structure calculations are performed on a series of Ornplatin complexes to elucidate the nature of binding of the Orn amino acid to the Pt center and how that binding is influenced by the local environment. The complexes examined in the work include: [(Orn-H)PtCl_2]-, [(Orn)PtCl]+, [(Orn)Pt(H_2O)Cl]+, and [(Orn)PtCl_2+Na]+. In contrast to that found previously for the glycine-linked cisplatin complex (Glyplatin), which binds via the backbone amino and carboxylate groups, binding of Orn in these complexes is found to involve both the backbone and sidechain amino groups. Extensive broadening of the IRMPD spectrum for the [(Orn)Pt(H_2O)Cl]+ complex suggests that either multiple structures are contributing to the measured spectrum or strong intra-molecular hydrogen-binding interactions are present. The results for Ornplatin lead to an interesting discussion about the differences in selectivity and reactivity versus cisplatin.

Antifreeze Protein Binds Irreversibly to Ice

NASA Astrophysics Data System (ADS)

Braslavsky, I.; Pertaya, N.; di Prinzio, C. L.; Wilen, L.; Thomson, E.; Wettlaufer, J. S.; Marshall, C. B.; Davies, P. L.

2006-03-01

Many organisms are protected from freezing by antifreeze proteins (AFPs), which bind to ice and prevent its growth by a mechanism not completely understood. Although it has been postulated that AFPs would have to bind irreversibly to arrest the growth of an ice crystal bathed in excess liquid water, the binding forces seem insufficient to support such a tight interaction. By putting a fluorescent tag on a fish AFP, we were able to visualize AFP binding to ice and demonstrate, by lack of recovery after photo-bleaching, that it is indeed irreversible. Because even the most avid protein/ligand interactions exhibit reversibility, this finding is key to understanding the mechanism of antifreeze proteins, which are becoming increasingly valuable in cryopreservation and improving the frost tolerance of crops.

Versatile function of the circadian protein CIPC as a regulator of Erk activation

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

Matsunaga, Ryota; Nishino, Tasuku; Yokoyama, Atsushi

2016-01-15

The CLOCK-interacting protein, Circadian (CIPC), has been identified as an additional negative-feedback regulator of the circadian clock. However, recent study on CIPC knockout mice has shown that CIPC is not critically required for basic circadian clock function, suggesting other unknown biological roles for CIPC. In this study, we focused on the cell cycle dependent nuclear-cytoplasmic shuttling function of CIPC and on identifying its binding proteins. Lys186 and 187 were identified as the essential amino acid residues within the nuclear localization signal (NLS) of CIPC. We identified CIPC-binding proteins such as the multifunctional enzyme CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase,more » and dihydroorotase), which is a key enzyme for de novo pyrimidine synthesis. Compared to control cells, HEK293 cells overexpressing wild-type CIPC showed suppressed cell proliferation and retardation of cell cycle. We also found that PMA-induced Erk activation was inhibited with expression of wild-type CIPC. In contrast, the NLS mutant of CIPC, which reduced the ability of CIPC to translocate into the nucleus, did not exhibit these biological effects. Since CAD and Erk have significant roles in cell proliferation and cell cycle, CIPC may work as a cell cycle regulator by interacting with these binding proteins. - Highlights: • CIPC is a cell cycle dependent nuclear-cytoplasmic shuttling protein. • K186 and 187are the essential amino acid residues within the NLS of CIPC. • CAD was identified as a novel CIPC-binding protein. • CIPC might regulate the activity and translocation of CAD in the cells.« less

Attenuation of cancer-initiating cells stemness properties by abrogating S100A4 calcium binding ability in head and neck cancers.

PubMed

Cheng, Li-Hao; Hung, Kai-Feng; Huang, Tung-Fu; Hsieh, Hsin-Pei; Wang, Shu-Ying; Huang, Chih-Yang; Lo, Jeng-Fan

2016-11-29

S100A4 is a calcium-binding protein capable of promoting epithelial-mesenchymal transition. Previously, we have demonstrated that S100A4 is required to sustain the head and neck cancer-initiating cells (HN-CICs) subpopulation. In this study, to further investigate the molecular mechanism, we established the head and neck squamous cell carcinoma (HNSCC) cell lines stably expressing mutant S100A4 proteins with defective calcium-binding sites on either N-terminal (NM) or C-terminal (CM), or a deletion of the last 15 amino-acid residues (CD). We showed that the NM, CM and CD harboring sphere cells that were enriched with HN-CICs population exhibited impaired stemness and malignant properties in vitro, as well as reduced tumor growth ability in vivo. Mechanistically, we demonstrated that mutant S100A4 proteins decreased the promoter activity of Nanog, likely through inhibition of p53. Moreover, the biophysical analyses of purified recombinant mutant S100A4 proteins suggest that both NM and CM mutant S100A4 were very similar to the WT S100A4 with subtle difference on the secondary structure, and that the CD mutant protein displayed the unexpected monomeric form in the solution phase.Taken together, our results suggest that both the calcium-binding ability and the C-terminal region of S100A4 are important for HN-CICs to sustain its stemness property and malignancy, and that the mechanism could be mediated by repressing p53 and subsequently activating the Nanog expression.

Intergenic Transcriptional Interference Is Blocked by RNA Polymerase III Transcription Factor TFIIIB in Saccharomyces cerevisiae

PubMed Central

Korde, Asawari; Rosselot, Jessica M.; Donze, David

2014-01-01

The major function of eukaryotic RNA polymerase III is to transcribe transfer RNA, 5S ribosomal RNA, and other small non-protein-coding RNA molecules. Assembly of the RNA polymerase III complex on chromosomal DNA requires the sequential binding of transcription factor complexes TFIIIC and TFIIIB. Recent evidence has suggested that in addition to producing RNA transcripts, chromatin-assembled RNA polymerase III complexes may mediate additional nuclear functions that include chromatin boundary, nucleosome phasing, and general genome organization activities. This study provides evidence of another such “extratranscriptional” activity of assembled RNA polymerase III complexes, which is the ability to block progression of intergenic RNA polymerase II transcription. We demonstrate that the RNA polymerase III complex bound to the tRNA gene upstream of the Saccharomyces cerevisiae ATG31 gene protects the ATG31 promoter against readthrough transcriptional interference from the upstream noncoding intergenic SUT467 transcription unit. This protection is predominately mediated by binding of the TFIIIB complex. When TFIIIB binding to this tRNA gene is weakened, an extended SUT467–ATG31 readthrough transcript is produced, resulting in compromised ATG31 translation. Since the ATG31 gene product is required for autophagy, strains expressing the readthrough transcript exhibit defective autophagy induction and reduced fitness under autophagy-inducing nitrogen starvation conditions. Given the recent discovery of widespread pervasive transcription in all forms of life, protection of neighboring genes from intergenic transcriptional interference may be a key extratranscriptional function of assembled RNA polymerase III complexes and possibly other DNA binding proteins. PMID:24336746

Occurrence of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus).

PubMed

Shiomi, K; Yamaguchi, S; Kikuchi, T; Yamamori, K; Matsui, T

1992-12-01

The shore crab (Hemigrapsus sanguineus) is highly resistant to tetrodotoxin (TTX) although it contains no detectable amount of TTX (less than 5 MU/g, where 1 MU is defined as the amount of TTX killing a 20 g mouse in 30 min). Its body fluid was examined for neutralizing effects against the lethal activity of TTX. When the mixture of the body fluid and TTX was injected i.p. into mice, the lethal activity of TTX was significantly reduced; 1 ml of the body fluid was evaluated to neutralize 3.6-4.0 MU of TTX. Higher neutralizing activity (7.2-12.5 MU/ml of the body fluid) was exhibited by i.v. administration of the body fluid into mice before or after i.p. challenge of TTX. The lethal effect of paralytic shellfish poisons was not counteracted by the body fluid. Analysis by gel filtration on Sepharose 6B revealed that the body fluid contained TTX-binding high mol. wt substances (> 2,000,000) responsible for the neutralizing activity of the body fluid against TTX, which accounts for the high resistibility of the crab to TTX. When the crude toxin extracted from the liver of puffer (Takifugu niphobles) was mixed with the body fluid and chromatographed on Sepharose 6B, almost pure TTX was obtained from the fractions containing the TTX-binding high mol. wt substances, suggesting that the TTX-binding high mol. wt substances could be useful in purification of TTX from biological samples.

Green reduction of graphene oxide via Lycium barbarum extract

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

Hou, Dandan, E-mail: houdandan114@163.com; Liu, Qinfu, E-mail: lqf@cumtb.edu.cn; Cheng, Hongfei, E-mail: h.cheng@cumtb.edu.cn

The synthesis of graphene from graphene oxide (GO) usually involves toxic reducing agents that are harmful to human health and the environment. Here, we report a facile approach for effective reduction of GO, for the first time, using Lycium barbarum extract as a green and natural reducing agent. The morphology and de-oxidation efficiency of the reduced graphene were characterized and results showed that Lycium barbarum extract can effectively reduce GO into few layered graphene with a high carbon to oxygen ratio (6.5), comparable to that of GO reduced by hydrazine hydrate (6.6). The possible reduction mechanism of GO may bemore » due to the active components existing in Lycium barbarum fruits, which have high binding affinity to the oxygen containing groups to form their corresponding oxides and other by-products. This method avoided the use of any nocuous chemicals, thus facilitating the mass production of graphene and graphene-based bio-materials. - Graphical abstract: Schematic illustration of the preparation of reduced graphene by Lycium barbarum extract. - Highlights: • The Lycium barbarum extract was used for the reduction of graphene oxide. • The obtained few layered graphene exhibited high carbon to oxygen ratio. • This approach can be applied in the preparation of graphene-based bio-materials.« less

Synthesis and structure of a heptanuclear nickel(II) complex uniquely exhibiting four distinct binding modes, two of which are novel, for a hydroxamate ligand.

PubMed

Gaynor, Declan; Starikova, Zoya A; Ostrovsky, Sergei; Haase, Wolfgang; Nolan, Kevin B

2002-03-07

The reaction of 2-(dimethylamino)phenylhydroxamic acid (2-dmAphaH) with NiSO(4).6H2O gives the complex [Ni7(2-dmAphaH-1)2(2-dmApha)8(H2O)2]SO(4).15H2O uniquely exhibiting four distinct hydroxamate binding modes, two of which are novel, and showing both antiferromagnetic and ferromagnetic interactions in contrast to [Cu5(2-dmAphaH-1)4(HSO4)2(MeOH)2].2MeOH, a strongly antiferromagnetic metallacrown formed with CuSO(4).5H2O.

A zwitterionic squaraine dye with a large Stokes shift for in vivo and site-selective protein sensing.

PubMed

Xu, Yongqian; Liu, Qin; Li, Xiaopeng; Wesdemiotis, Chrys; Pang, Yi

2012-11-28

A novel squaraine dye (SQ) exhibits improved fluorescence response toward protein detection by incorporation of a zwitterionic structure. With the aid of a dansylamide (DNSA) substituent, the new probe (DNSA-SQ) exhibits remarkable selectivity in binding to site I (a specific substructure in protein).

Dispersing Zwitterions into Comb Polymers for Nonviral Transfection: Experiments and Molecular Simulation.

PubMed

Ghobadi, Ahmadreza F; Letteri, Rachel; Parelkar, Sangram S; Zhao, Yue; Chan-Seng, Delphine; Emrick, Todd; Jayaraman, Arthi

2016-02-08

Polymer-based gene delivery vehicles benefit from the presence of hydrophilic groups that mitigate the inherent toxicity of polycations and that provide tunable polymer-DNA binding strength and stable complexes (polyplexes). However, hydrophilic groups screen charge, and as such can reduce cell uptake and transfection efficiency. We report the effect of embedding zwitterionic sulfobetaine (SB) groups in cationic comb polymers, using a combination of experiments and molecular simulations. Ring-opening metathesis polymerization (ROMP) produced comb polymers with tetralysine (K4) and SB pendent groups. Dynamic light scattering, zeta potential measurements, and fluorescence-based experiments, together with coarse-grained molecular dynamics simulations, described the effect of SB groups on the size, shape, surface charge, composition, and DNA binding strength of polyplexes formed using these comb polymers. Experiments and simulations showed that increasing SB composition in the comb polymers decreased polymer-DNA binding strength, while simulations indicated that the SB groups distributed throughout the polyplex. This allows polyplexes to maintain a positive surface charge and provide high levels of gene expression in live cells. Notably, comb polymers with nearly 50 mol % SB form polyplexes that exhibit positive surface charge similarly as polyplexes formed from purely cationic comb polymers, indicating the ability to introduce an appreciable amount of SB functionality without screening surface charge. This integrated simulation-experimental study demonstrates the effectiveness of incorporating zwitterions in polyplexes, while guiding the design of new and effective gene delivery vectors.

The in vitro kinetics of the interactions between PEG-ylated magnetic-fluid-loaded liposomes and macrophages.

PubMed

Martina, Marie-Sophie; Nicolas, Valerie; Wilhelm, Claire; Ménager, Christine; Barratt, Gillian; Lesieur, Sylviane

2007-10-01

Binding and uptake kinetics of magnetic-fluid-loaded liposomes (MFL) by endocytotic cells were investigated in vitro on the model cell-line J774. MFL consisted of unilamellar phosphatidylcholine vesicles (mean hydrodynamic diameter close to 200nm) encapsulating 8-nm nanocrystals of maghemite (gamma-Fe(2)O(3)) and sterically stabilized by introducing 5mol% of distearylphosphatidylcholine poly(ethylene glycol)(2,000) (DSPE-PEG(2,000)) in the vesicle bilayer. The association processes with living macrophages were followed at two levels. On one hand, the lipid vesicles were imaged by confocal fluorescence microscopy. For this purpose 1mol% of rhodamine-marked phosphatidylethanolamine was added to the liposome composition. On the other hand, the iron oxide particles associated with cells were independently quantified by magnetophoresis. All the experiments were similarly performed with PEG-ylated or conventional MFL to point out the role of polymer coating. The results showed cell association with both types of liposomes resulting from binding followed by endocytosis. Steric stabilization by PEG chains reduced binding efficiency limiting the amount of MFL internalized by the macrophages. In contrast, PEG coating did not change the kinetics of endocytosis which exhibited the same first-order rate constant for both conventional and PEG-ylated liposomes. Moreover, lipids and iron oxide particle uptakes were perfectly correlated, indicating that MFL vesicle structure and encapsulation rate were preserved upon cell penetration.

Incorporation of nisin into a meat binding system to inhibit bacteria on beef surfaces.

PubMed

Cutter, C N; Siragusa, G R

1998-07-01

In two separate experiments, the bacteriocin, nisin, was incorporated into a commercially available meat binding system (Fibrimex) and applied to meat surfaces as a way of inhibiting the meat spoilage organism, Brochothrix thermosphacta during extended refrigerated storage. In experiment 1, pre-rigor lean beef carcass tissue (BCT) was inoculated with B. thermosphacta, left untreated (U), treated with 10 micrograms ml-1 nisin (N), Fibrimex (F) or Fibrimex containing 10 micrograms ml-1 nisin (FN), held aerobically at 4 degrees C for up to 7 d, and populations of B. thermosphacta and nisin activity determined. Experiment 2 determined the effects of the same treatments but on post-rigor, frozen and thawed lean BCT that was inoculated, vacuum-packaged, and stored at 4 degrees C for up to 14 d. In both experiments, N- and FN-treated tissues exhibited significantly lower populations of B. thermosphacta compared to U- and F-treated tissues, for the duration of refrigerated storage. Nisin activity was detected up to 7 d in N- and FN-treated samples from experiment 1. However, activity was detected only to days 0 and 2 in FN- and N-treated samples, respectively, from experiment 2. These studies indicate that the addition of a bacteriocin to a meat binding system and application to meat surfaces may be useful in reducing undesirable bacteria in restructured meat products.

Structural determinants of Kvbeta1.3-induced channel inactivation: a hairpin modulated by PIP2.

PubMed

Decher, Niels; Gonzalez, Teresa; Streit, Anne Kathrin; Sachse, Frank B; Renigunta, Vijay; Soom, Malle; Heinemann, Stefan H; Daut, Jürgen; Sanguinetti, Michael C

2008-12-03

Inactivation of voltage-gated Kv1 channels can be altered by Kvbeta subunits, which block the ion-conducting pore to induce a rapid ('N-type') inactivation. Here, we investigate the mechanisms and structural basis of Kvbeta1.3 interaction with the pore domain of Kv1.5 channels. Inactivation induced by Kvbeta1.3 was antagonized by intracellular PIP(2). Mutations of R5 or T6 in Kvbeta1.3 enhanced Kv1.5 inactivation and markedly reduced the effects of PIP(2). R5C or T6C Kvbeta1.3 also exhibited diminished binding of PIP(2) compared with wild-type channels in an in vitro lipid-binding assay. Further, scanning mutagenesis of the N terminus of Kvbeta1.3 revealed that mutations of L2 and A3 eliminated N-type inactivation. Double-mutant cycle analysis indicates that R5 interacts with A501 and T480 of Kv1.5, residues located deep within the pore of the channel. These interactions indicate that Kvbeta1.3, in contrast to Kvbeta1.1, assumes a hairpin structure to inactivate Kv1 channels. Taken together, our findings indicate that inactivation of Kv1.5 is mediated by an equilibrium binding of the N terminus of Kvbeta1.3 between phosphoinositides (PIPs) and the inner pore region of the channel.

Physicochemical and biological characterization of single-walled and double-walled carbon nanotubes in biological media.

PubMed

Liu, Wen-Te; Bien, Mauo-Ying; Chuang, Kai-Jen; Chang, Ta-Yuan; Jones, Tim; BéruBé, Kelly; Lalev, Georgi; Tsai, Dai-Hua; Chuang, Hsiao-Chi; Cheng, Tsun-Jen

2014-09-15

To study the toxicity of nanoparticles under relevant conditions, it is important to reproducibly disperse nanoparticles in biological media in in vitro and in vivo studies. Here, single-walled nanotubes (SWNTs) and double-walled nanotubes (DWNTs) were physicochemically and biologically characterized when dispersed in phosphate-buffered saline (PBS) and bovine serum albumin (BSA). BSA-SWNT/DWNT interaction resulted in a reduction of aggregation and an increase in particle stabilization. Based on the protein sequence coverage and protein binding results, DWNTs exhibited higher protein binding than SWNTs. SWNT and DWNT suspensions in the presence of BSA increased interleukin-6 (IL-6) levels and reduced tumor necrosis factor-alpha (TNF-α) levels in A549 cells as compared to corresponding samples in the absence of BSA. We next determined the effects of SWNTs and DWNTs on pulmonary protein modification using bronchoalveolar lavage fluid (BALF) as a surrogate collected form BALB/c mice. The BALF proteins bound to SWNTs (13 proteins) and DWNTs (11 proteins), suggesting that these proteins were associated with blood coagulation pathways. Lastly, we demonstrated the importance of physicochemical and biological alterations of SWNTs and DWNTs when dispersed in biological media, since protein binding may result in the misinterpretation of in vitro results and the activation of protein-regulated biological responses. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

Targeting autocrine HB-EGF signaling with specific ADAM12 inhibition using recombinant ADAM12 prodomain

NASA Astrophysics Data System (ADS)

Miller, Miles A.; Moss, Marcia L.; Powell, Gary; Petrovich, Robert; Edwards, Lori; Meyer, Aaron S.; Griffith, Linda G.; Lauffenburger, Douglas A.

2015-10-01

Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, “decoy” antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling.

Signal transducer and activator of transcription 5B (STAT5B) modulates adipocyte differentiation via MOF.

PubMed

Gao, Peng; Zhang, Yuchao; Liu, Yuantao; Chen, Jicui; Zong, Chen; Yu, Cong; Cui, Shang; Gao, Weina; Qin, Dandan; Sun, Wenchuan; Li, Xia; Wang, Xiangdong

2015-12-01

The role and mechanism of signal transducer and activator of transcription 5B (STAT5B) in adipogenesis remain unclear. In this study, our data showed that Males absent on the first (MOF) protein expression was increased during 3 T3-L1 preadipocytes differentiation accompanied with STAT5B expression increasing. Over-expression STAT5B enhanced MOF promoter trans-activation in HeLa cells. Mutagenesis assay and ChIP analysis exhibited that STAT5B was able to bind MOF promoter. Knocking-down STAT5B in 3 T3-L1 preadipocytes led to decreased expression of MOF, but resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and fatty acid-binding protein 4 (Fabp4), which were important factors or enzymes for adipogenesis. We also found that knocking-down MOF in 3 T3-L1 preadipocytes resulted in increased expression of PPARγ, C/EBPα and Fabp4, which was in the same trend as STAT5B knocking-down. Over-expression MOF resulted in reduced promoter trans-activation activity of C/EBPα. These results suggest that STAT5B and MOF work as negative regulators in adipogenesis, and STAT5B modulates preadipocytes differentiation partially by regulating MOF expression. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Nanohole Arrays of Mixed Designs and Microwriting for Simultaneous and Multiple Protein Binding Studies

PubMed Central

Ji, Jin; Yang, Jiun-Chan; Larson, Dale N.

2009-01-01

We demonstrate using nanohole arrays of mixed designs and a microwriting process based on dip-pen nanolithography to monitor multiple, different protein binding events simultaneously in real time based on the intensity of Extraordinary Optical Transmission of nanohole arrays. The microwriting process and small footprint of the individual nanohole arrays enabled us to observe different binding events located only 16μm apart, achieving high spatial resolution. We also present a novel concept that incorporates nanohole arrays of different designs to improve confidence and accuracy of binding studies. For proof of concept, two types of nanohole arrays, designed to exhibit opposite responses to protein bindings, were fabricated on one transducer. Initial studies indicate that the mixed designs could help to screen out artifacts such as protein intrinsic signals, providing improved accuracy of binding interpretation. PMID:19297143

Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region

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

Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L.

Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or themore » doubly phosphorylated form of p38-alpha kinase.« less

Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

PubMed

Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

2016-04-01

Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. © 2016 Federation of European Biochemical Societies.

Interaction of herpes simplex virus glycoprotein gC with mammalian cell surface molecules.

PubMed Central

Tal-Singer, R; Peng, C; Ponce De Leon, M; Abrams, W R; Banfield, B W; Tufaro, F; Cohen, G H; Eisenberg, R J

1995-01-01

The entry of herpes simplex virus (HSV) into mammalian cells is a multistep process beginning with an attachment step involving glycoproteins gC and gB. A second step requires the interaction of glycoprotein gD with a cell surface molecule. We explored the interaction between gC and the cell surface by using purified proteins in the absence of detergent. Truncated forms of gC and gD, gC1(457t), gC2(426t), and gD1(306t), lacking the transmembrane and carboxyl regions were expressed in the baculovirus system. We studied the ability of these proteins to bind to mammalian cells, to bind to immobilized heparin, to block HSV type 1 (HSV-1) attachment to cells, and to inhibit plaque formation by HSV-1. Each of these gC proteins bound to conformation-dependent monoclonal antibodies and to human complement component C3b, indicating that they maintained the same conformation of gC proteins expressed in mammalian cells. Biotinylated gC1(457t) and gC2(426t) each bind to several cell lines. Binding was inhibited by an excess of unlabeled gC but not by gD, indicating specificity. The attachment of gC to cells involves primarily heparan sulfate proteoglycans, since heparitinase treatment of cells reduced gC binding by 50% but had no effect on gD binding. Moreover, binding of gC to two heparan sulfate-deficient L-cell lines, gro2C and sog9, both of which are mostly resistant to HSV infection, was markedly reduced. Purified gD1 (306t), however, bound equally well to the two mutant cell lines. In contrast, saturating amounts of gC1(457t) interfered with HSV-1 attachment to cells but failed to block plaque formation, suggesting a role for gC in attachment but not penetration. A mutant form of gC lacking residues 33 to 123, gC1(delta 33-123t), expressed in the baculovirus system, bound significantly less well to cells than did gC1(457t) and competed poorly with biotinylated gC1(457t) for binding. These results suggest that residues 33 to 123 are important for gC attachment to cells. In contrast, both the mutant and wild-type forms of gC bound to immobilized heparin, indicating that binding of these proteins to the cell surface involves more than a simple interaction with heparin. To determine that the contribution of the N-terminal region of gC is important for HSV attachment, we compared several properties of a mutant HSV-1 which contains gC lacking amino acids 33 to 123 to those of its parental virus, which contains full-length gC. The mutant bound less well to cells than the parental virus but exhibited normal growth properties.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7769707

Structure and Ligand Binding Properties of the Epoxidase Component of Styrene Monooxygenase

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

Ukaegbu, Uchechi E.; Kantz, Auric; Beaton, Michelle

2010-07-23

Styrene monooxygenase (SMO) is a two-component flavoprotein monooxygenase that transforms styrene to styrene oxide in the first step of the styrene catabolic and detoxification pathway of Pseudomonas putida S12. The crystal structure of the N-terminally histidine-tagged epoxidase component of this system, NSMOA, determined to 2.3 {angstrom} resolution, indicates the enzyme exists as a homodimer in which each monomer forms two distinct domains. The overall architecture is most similar to that of p-hydroxybenzoate hydroxylase (PHBH), although there are some significant differences in secondary structure. Structural comparisons suggest that a large cavity open to the surface forms the FAD binding site. Atmore » the base of this pocket is another cavity that likely represents the styrene binding site. Flavin binding and redox equilibria are tightly coupled such that reduced FAD binds apo NSMOA {approx}8000 times more tightly than the oxidized coenzyme. Equilibrium fluorescence and isothermal titration calorimetry data using benzene as a substrate analogue indicate that the oxidized flavin and substrate analogue binding equilibria of NSMOA are linked such that the binding affinity of each is increased by 60-fold when the enzyme is saturated with the other. A much weaker {approx}2-fold positive cooperative interaction is observed for the linked binding equilibria of benzene and reduced FAD. The low affinity of the substrate analogue for the reduced FAD complex of NSMOA is consistent with a preferred reaction order in which flavin reduction and reaction with oxygen precede the binding of styrene, identifying the apoenzyme structure as the key catalytic resting state of NSMOA poised to bind reduced FAD and initiate the oxygen reaction.« less

Cadherin Domains in the Polysaccharide-Degrading Marine Bacterium Saccharophagus degradans 2-40 Are Carbohydrate-Binding Modules▿

PubMed Central

Fraiberg, Milana; Borovok, Ilya; Bayer, Edward A.; Weiner, Ronald M.; Lamed, Raphael

2011-01-01

The complex polysaccharide-degrading marine bacterium Saccharophagus degradans strain 2-40 produces putative proteins that contain numerous cadherin and cadherin-like domains involved in intercellular contact interactions. The current study reveals that both domain types exhibit reversible calcium-dependent binding to different complex polysaccharides which serve as growth substrates for the bacterium. PMID:21036994

Structural insights into the anti-HIV activity of the Oscillatoria agardhii agglutinin homolog lectin family.

PubMed

Koharudin, Leonardus M I; Kollipara, Sireesha; Aiken, Christopher; Gronenborn, Angela M

2012-09-28

Oscillatoria agardhii agglutinin homolog (OAAH) proteins belong to a recently discovered lectin family. All members contain a sequence repeat of ~66 amino acids, with the number of repeats varying among different family members. Apart from data for the founding member OAA, neither three-dimensional structures, information about carbohydrate binding specificities, nor antiviral activity data have been available up to now for any other members of the OAAH family. To elucidate the structural basis for the antiviral mechanism of OAAHs, we determined the crystal structures of Pseudomonas fluorescens and Myxococcus xanthus lectins. Both proteins exhibit the same fold, resembling the founding family member, OAA, with minor differences in loop conformations. Carbohydrate binding studies by NMR and x-ray structures of glycan-lectin complexes reveal that the number of sugar binding sites corresponds to the number of sequence repeats in each protein. As for OAA, tight and specific binding to α3,α6-mannopentaose was observed. All the OAAH proteins described here exhibit potent anti-HIV activity at comparable levels. Altogether, our results provide structural details of the protein-carbohydrate interaction for this novel lectin family and insights into the molecular basis of their HIV inactivation properties.

Hyaluronic acid binding by human sperm indicates cellular maturity, viability, and unreacted acrosomal status.

PubMed

Huszar, Gabor; Ozenci, Ciler Celik; Cayli, Sevil; Zavaczki, Zoltan; Hansch, Eleonora; Vigue, Lynne

2003-06-01

To test, both in semen and washed-sperm fractions, whether hyaluronic acid (HA) binding is restricted to sperm that have completed cellular maturation. Comparisons of sperm in semen and in HA-bound sperm fractions. University-based diagnostic and research andrology laboratory. Semen samples originated in men being tested for infertility. The attributes of sperm maturity were tested by immunocytochemistry with creatine kinase and HspA2 antisera (highlights cytoplasmic retention in diminished-maturity sperm), aniline blue chromatin staining (detects persistent histones), pisum sativum lectin staining (reveals acrosomal integrity), and the FertiLight viability kit (highlights viable and nonviable sperm). All markers of sperm maturity and immaturity supported the hypothesis that HA-bound sperm are mature. Nonbinding sperm exhibited cytoplasmic and nuclear properties of diminished maturity. The acrosomal status of HA-bound sperm was either unreacted or slightly capacitated, but not acrosome reacted. Only viable sperm exhibited HA binding. Sperm that are able to bind to HA are mature and have completed the spermiogenetic processes of sperm plasma membrane remodeling, cytoplasmic extrusion, and nuclear histone-protamine replacement. Hyaluronic acid-bound sperm show unreacted acrosomes. These studies provide further insights into the relationship between spermiogenesis and sperm function.

Impaired β-arrestin recruitment and reduced desensitization by non-catechol agonists of the D1 dopamine receptor.

PubMed

Gray, David L; Allen, John A; Mente, Scot; O'Connor, Rebecca E; DeMarco, George J; Efremov, Ivan; Tierney, Patrick; Volfson, Dmitri; Davoren, Jennifer; Guilmette, Edward; Salafia, Michelle; Kozak, Rouba; Ehlers, Michael D

2018-02-14

Selective activation of dopamine D1 receptors (D1Rs) has been pursued for 40 years as a therapeutic strategy for neurologic and psychiatric diseases due to the fundamental role of D1Rs in motor function, reward processing, and cognition. All known D1R-selective agonists are catechols, which are rapidly metabolized and desensitize the D1R after prolonged exposure, reducing agonist response. As such, drug-like selective D1R agonists have remained elusive. Here we report a novel series of selective, potent non-catechol D1R agonists with promising in vivo pharmacokinetic properties. These ligands stimulate adenylyl cyclase signaling and are efficacious in a rodent model of Parkinson's disease after oral administration. They exhibit distinct binding to the D1R orthosteric site and a novel functional profile including minimal receptor desensitization, reduced recruitment of β-arrestin, and sustained in vivo efficacy. These results reveal a novel class of D1 agonists with favorable drug-like properties, and define the molecular basis for catechol-specific recruitment of β-arrestin to D1Rs.

Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. 1

PubMed Central

Meyer, Christiane; Feyerabend, Martin; Weiler, Elmar W.

1989-01-01

Using the novel radioligand, [3H]-9′-nor-fusicoccin-8′-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K+, Mg2+-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [3H]-9′-nor-fusicoccin-8′-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (KD), KD1 = 1.5 nanomolar and KD2 = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 ± 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin. PMID:16666603

Solution structure, backbone dynamics and chitin binding of the anti-fungal protein from Streptomyces tendae TU901.

PubMed

Campos-Olivas, R; Hörr, I; Bormann, C; Jung, G; Gronenborn, A M

2001-05-11

AFP1 is a recently discovered anti-fungal, chitin-binding protein from Streptomyces tendae Tü901. Mature AFP1 comprises 86 residues and exhibits limited sequence similarity to the cellulose-binding domains of bacterial cellulases and xylanases. No similarity to the Cys and Gly-rich domains of plant chitin-binding proteins (e.g. agglutinins, lectins, hevein) is observed. AFP1 is the first chitin-binding protein from a bacterium for which anti-fungal activity was shown. Here, we report the three-dimensional solution structure of AFP1, determined by nuclear magnetic resonance spectroscopy. The protein contains two antiparallel beta-sheets (five and four beta-strands each), that pack against each other in a parallel beta-sandwich. This type of architecture is conserved in the functionally related family II of cellulose-binding domains, albeit with different connectivity. A similar fold is also observed in other unrelated proteins (spore coat protein from Myxococcus xanthus, beta-B2 and gamma-B crystallins from Bos taurus, canavalin from Jack bean). AFP1 is therefore classified as a new member of the betagamma-crystallin superfamily. The dynamics of the protein was characterized by NMR using amide 15N relaxation and solvent exchange data. We demonstrate that the protein exhibits an axially symmetric (oblate-like) rotational diffusion tensor whose principal axis coincides to within 15 degrees with that of the inertial tensor. After completion of the present structure of AFP1, an identical fold was reported for a Streptomyces killer toxin-like protein. Based on sequence comparisons and clustering of conserved residues on the protein surface for different cellulose and chitin-binding proteins, we postulate a putative sugar-binding site for AFP1. The inability of the protein to bind short chitin fragments suggests that certain particular architectural features of the solid chitin surface are crucial for the interaction. Copyright 2001 Academic Press.

Surface properties of adipocyte lipid-binding protein: Response to lipid binding, and comparison with homologous proteins.

PubMed

LiCata, V J; Bernlohr, D A

1998-12-01

Adipocyte lipid-binding protein (ALBP) is one of a family of intracellular lipid-binding proteins (iLBPs) that bind fatty acids, retinoids, and other hydrophobic ligands. The different members of this family exhibit a highly conserved three-dimensional structure; and where structures have been determined both with (holo) and without (apo) bound lipid, observed conformational changes are extremely small (Banaszak, et al., 1994, Adv. Prot. Chem. 45, 89; Bernlohr, et al., 1997, Annu. Rev. Nutr. 17, 277). We have examined the electrostatic, hydrophobic, and water accessible surfaces of ALBP in the apo form and of holo forms with a variety of bound ligands. These calculations reveal a number of previously unrecognized changes between apo and holo ALBP, including: 1) an increase in the overall protein surface area when ligand binds, 2) expansion of the binding cavity when ligand is bound, 3) clustering of individual residue exposure increases in the area surrounding the proposed ligand entry portal, and 4) ligand-binding dependent variation in the topology of the electrostatic potential in the area surrounding the ligand entry portal. These focused analyses of the crystallographic structures thus reveal a number of subtle but consistent conformational and surface changes that might serve as markers for differential targeting of protein-lipid complexes within the cell. Most changes are consistent from ligand to ligand, however there are some ligand-specific changes. Comparable calculations with intestinal fatty-acid-binding protein and other vertebrate iLBPs show differences in the electrostatic topology, hydrophobic topology, and in localized changes in solvent exposure near the ligand entry portal. These results provide a basis toward understanding the functional and mechanistic differences among these highly structurally homologous proteins. Further, they suggest that iLBPs from different tissues exhibit one of two predominant end-state structural distributions of the ligand entry portal.

Modeling of the solution interaction properties of plastic materials used in pharmaceutical product container systems.

PubMed

Jenke, Dennis; Couch, Tom; Gillum, Amy; Sadain, Salma

2009-01-01

Material/water equilibrium binding constants (Eb) were determined for 14 organic solutes and 17 plastic raw materials that could be used in pharmaceutical product container systems. Correlations between the measured binding constants and the organic solute's octanol/water and hexane/water partition coefficients were obtained. In general, while the materials examined exhibited a wide range of binding characteristics, the tested materials by and large fell within two broad classes: (1) those that were octanol-like in their binding characteristics, and (2) those that were hexane-like. Materials of the same class (e.g., polypropylenes) generally had binding models that were very similar. Rank ordering of the materials in terms of their magnitude of drug binding (least binding to most binding) was as follows: polypropylene < polyethylene < polyamide < styrene-ethylene-butylene-styrene < copolyester ether elastomer approximately equal to amine-terminated poly fatty acid amide polymer. The utilization of the developed models to estimate drug loss via sorption by the container is discussed.

Molecular characterization and functional analysis of pheromone binding protein 1 from Cydia pomonella (L.).

PubMed

Tian, Z; Zhang, Y

2016-12-01

A full-length cDNA encoding Cydia pomonella pheromone binding protein 1 (CpomPBP1) was cloned and characterized. CpomPBP1, possessing the typical characteristics of lepidopteran odorant binding proteins, was detected to be specifically expressed in the antennae of male and female moths at the mRNA and protein level. Soluble recombinant CpomPBP1 was subjected to in vitro binding to analyse its binding properties and to search for potentially active semiochemicals. A competitive binding assay showed that three 12-carbon ligands, codlemone, 1-dodecanol and E,E-2,4-dodecadienal, were able to bind to CpomPBP1 in decreasing order of affinity. Moreover, unlike the wild-type CpomPBP1, the C-terminus truncated CpomPBP1 exhibited high affinity to ligands even in an acidic environment, suggesting that the C-terminus plays a role in preventing ligands from binding to CpomPBP1 in a lower pH environment. © 2016 The Royal Entomological Society.

Aza-Bambusurils En Route to Anion Transporters.

PubMed

Singh, Mandeep; Solel, Ephrath; Keinan, Ehud; Reany, Ofer

2016-06-20

Previous calculations of anion binding with various bambusuril analogs predicted that the replacement of oxygen by nitrogen atoms to produce semiaza-bambus[6]urils would award these new cavitands with multiple anion binding properties. This study validates the hypothesis by efficient synthesis, crystallography, thermogravimetric analysis and calorimetry. These unique host molecules are easily accessible from the corresponding semithio-bambusurils in a one-pot reaction, which converts a single anion receptor into a potential anion channel. Solid-state structures exhibit simultaneous accommodation of three anions, linearly positioned within the cavity along the main symmetry axis. The ability to hold anions at a short distance of about 4 Å is reminiscent of natural chloride channels in E. coli, which exhibit similar distances between their adjacent anion binding sites. The calculated transition-state energy for double-anion movement through the channel suggests that although these host-guest complexes are thermodynamically stable they enjoy high kinetic flexibility to render them efficient anion channels. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Design of a serotonin 4 receptor radiotracer with decreased lipophilicity for single photon emission computed tomography.

PubMed

Fresneau, Nathalie; Dumas, Noé; Tournier, Benjamin B; Fossey, Christine; Ballandonne, Céline; Lesnard, Aurélien; Millet, Philippe; Charnay, Yves; Cailly, Thomas; Bouillon, Jean-Philippe; Fabis, Frédéric

2015-04-13

With the aim to develop a suitable radiotracer for the brain imaging of the serotonin 4 receptor subtype (5-HT4R) using single photon emission computed tomography (SPECT), we synthesized and evaluated a library of di- and triazaphenanthridines with lipophilicity values which were in the range expected to favour brain penetration, and which demonstrated specific binding to the target of interest. Adding additional nitrogen atoms to previously described phenanthridine ligands exhibiting a high unspecific binding, we were able to design a radioiodinated compound [(125)I]14. This compound exhibited a binding affinity value of 0.094 nM toward human 5-HT4R and a high selectivity over other serotonin receptor subtypes (5-HTR). In vivo SPECT imaging studies and competition experiments demonstrated that the decreased lipophilicity (in comparison with our previously reported compounds 4 and 5) allowed a more specific labelling of the 5-HT4R brain-containing regions. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Discovery of potent cytotoxic ortho-aryl chalcones as new scaffold targeting tubulin and mitosis with affinity-based fluorescence.

PubMed

Zhu, Cuige; Zuo, Yinglin; Wang, Ruimin; Liang, Baoxia; Yue, Xin; Wen, Gesi; Shang, Nana; Huang, Lei; Chen, Yu; Du, Jun; Bu, Xianzhang

2014-08-14

A series of new ortho-aryl chalcones have been designed and synthesized. Many of these compounds were found to exhibit significant antiproliferation activity toward a panel of cancer cell lines. Selected compounds show potent cytotoxicity against several drug resistant cell lines including paclitaxel (Taxol) resistant human ovarian carcinoma cells, vincristine resistant human ileocecum carcinoma cells, and doxorubicin resistant human breast carcinoma cells. Further investigation revealed that active analogues could inhibit the microtubule polymerization by binding to colchicine site and thus induce multipolar mitosis, G2/M phase arrest, and apoptosis of cancer cells. Furthermore, affinity-based fluorescence enhancement was observed during the binding of active compounds with tubulin, which greatly facilitated the determination of tubulin binding site of the compounds. Finally, selected compound 26 was found to exhibit obvious in vivo antitumor activity in A549 tumor xenografts model. Our systematic studies implied a new scaffold targeting tubulin and mitosis for novel antitumor drug discovery.

Cold-sensitive mutants G680V and G691C of Dictyostelium myosin II confer dramatically different biochemical defects.

PubMed

Patterson, B; Ruppel, K M; Wu, Y; Spudich, J A

1997-10-31

Cold-sensitive myosin mutants represent powerful tools for dissecting discrete deficiencies in myosin function. Biochemical characterization of two such mutants, G680V and G691C, has allowed us to identify separate facets of myosin motor function perturbed by each alteration. Compared with wild type, the G680V myosin exhibits a substantially enhanced affinity for several nucleotides, decreased ATPase activity, and overoccupancy or creation of a novel strongly actin-binding state. The properties of the novel strong binding state are consistent with a partial arrest or pausing at the onset of the mechanical stroke. The G691C mutant, on the other hand, exhibits an elevated basal ATPase indicative of premature phosphate release. By releasing phosphate without a requirement for actin binding, the G691C can bypass the part of the cycle involving the mechanical stroke. The two mutants, despite having alterations in glycine residues separated by only 11 residues, have dramatically different consequences on the mechanochemical cycle.

Substrate binding site for nitrate reductase of Escherichia coli is on the inner aspect of the membrane.

PubMed Central

Kristjansson, J K; Hollocher, T C

1979-01-01

Escherichia coli grown anaerobically on nitrate exhibited the same transport barrier to reduction of chlorate, relative to nitrate, as that exhibited by Paracoccus denitrificans. This establishes that the nitrate binding site of nitrate reductase (EC 1.7.99.4) in E. coli must also lie on the cell side of the nitrate transporter which is associated with the plasma membrane. Because nitrate reductase is membrane bound, the nitrate binding site is thus located on the inner aspect of the membrane. Nitrate pulse studies on E. coli in the absence of valinomycin showed a small transient alkalinization (leads to H+/NO3- congruent to --0.07) which did not occur with oxygen pulses. By analogy with P. denitrificans, the alkaline transient is interpreted to arise from proton-linked nitrate uptake which is closely followed by nitrite efflux. The result is consistent with internal reduction of nitrate, whereas external reduction would be expected to give leads to H+/NO3-ratios approaching --2. PMID:374343

The mRNA-bound proteome of the early fly embryo

PubMed Central

Wessels, Hans-Hermann; Imami, Koshi; Baltz, Alexander G.; Kolinski, Marcin; Beldovskaya, Anastasia; Selbach, Matthias; Small, Stephen; Ohler, Uwe; Landthaler, Markus

2016-01-01

Early embryogenesis is characterized by the maternal to zygotic transition (MZT), in which maternally deposited messenger RNAs are degraded while zygotic transcription begins. Before the MZT, post-transcriptional gene regulation by RNA-binding proteins (RBPs) is the dominant force in embryo patterning. We used two mRNA interactome capture methods to identify RBPs bound to polyadenylated transcripts within the first 2 h of Drosophila melanogaster embryogenesis. We identified a high-confidence set of 476 putative RBPs and confirmed RNA-binding activities for most of 24 tested candidates. Most proteins in the interactome are known RBPs or harbor canonical RBP features, but 99 exhibited previously uncharacterized RNA-binding activity. mRNA-bound RBPs and TFs exhibit distinct expression dynamics, in which the newly identified RBPs dominate the first 2 h of embryonic development. Integrating our resource with in situ hybridization data from existing databases showed that mRNAs encoding RBPs are enriched in posterior regions of the early embryo, suggesting their general importance in posterior patterning and germ cell maturation. PMID:27197210

Identification of B. anthracis N(5)-carboxyaminoimidazole ribonucleotide mutase (PurE) active site binding compounds via fragment library screening.

PubMed

Lei, Hao; Jones, Christopher; Zhu, Tian; Patel, Kavankumar; Wolf, Nina M; Fung, Leslie W-M; Lee, Hyun; Johnson, Michael E

2016-02-15

The de novo purine biosynthesis pathway is an attractive target for antibacterial drug design, and PurE from this pathway has been identified to be crucial for Bacillus anthracis survival in serum. In this study we adopted a fragment-based hit discovery approach, using three screening methods-saturation transfer difference nucleus magnetic resonance (STD-NMR), water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR, and surface plasmon resonance (SPR), against B. anthracis PurE (BaPurE) to identify active site binding fragments by initially testing 352 compounds in a Zenobia fragment library. Competition STD NMR with the BaPurE product effectively eliminated non-active site binding hits from the primary hits, selecting active site binders only. Binding affinities (dissociation constant, KD) of these compounds varied between 234 and 301μM. Based on test results from the Zenobia compounds, we subsequently developed and applied a streamlined fragment screening strategy to screen a much larger library consisting of 3000 computationally pre-selected fragments. Thirteen final fragment hits were confirmed to exhibit binding affinities varying from 14μM to 700μM, which were categorized into five different basic scaffolds. All thirteen fragment hits have ligand efficiencies higher than 0.30. We demonstrated that at least two fragments from two different scaffolds exhibit inhibitory activity against the BaPurE enzyme. Published by Elsevier Ltd.

Lesions in two Escherichia coli type 1 pilus genes alter pilus number and length without affecting receptor binding.

PubMed Central

Russell, P W; Orndorff, P E

1992-01-01

We describe the characterization of two genes, fimF and fimG (also called pilD), that encode two minor components of type 1 pili in Escherichia coli. Defined, in-frame deletion mutations were generated in vitro in each of these two genes. A double mutation that had deletions identical to both single lesions was also constructed. Examination of minicell transcription and translation products of parental and mutant plasmids revealed that, as predicted from the nucleotide sequence and previous reports, the fimF gene product was a protein of ca. 16 kDa and that the fimG gene product was a protein of ca. 14 kDa. Each of the constructions was introduced, via homologous recombination, into the E. coli chromosome. All three of the resulting mutants produced type 1 pili and exhibited hemagglutination of guinea pig erythrocytes. The latter property was also exhibited by partially purified pili isolated from each of the mutants. Electron microscopic examination revealed that the fimF mutant had markedly reduced numbers of pili per cell, whereas the fimG mutant had very long pili. The double mutant displayed the characteristics of both single mutants. However, pili in the double mutant were even longer than those seen in the fimG mutant, and the numbers of pili were even fewer than those displayed by the fimF mutant. All three mutants could be complemented in trans with a single-copy-number plasmid bearing the appropriate parental gene or genes to give near-normal parental piliation. On the basis of the phenotypes exhibited by the single and double mutants, we believe that the fimF gene product may aid in initiating pilus assembly and that the fimG product may act as an inhibitor of pilus polymerization. In contrast to previous studies, we found that neither gene product was required for type 1 pilus receptor binding. Images PMID:1355769

The evolution of filamin – A protein domain repeat perspective

PubMed Central

Light, Sara; Sagit, Rauan; Ithychanda, Sujay S.; Qin, Jun; Elofsson, Arne

2013-01-01

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. PMID:22414427

The evolution of filamin-a protein domain repeat perspective.

PubMed

Light, Sara; Sagit, Rauan; Ithychanda, Sujay S; Qin, Jun; Elofsson, Arne

2012-09-01

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. Copyright © 2012 Elsevier Inc. All rights reserved.

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin.

PubMed

Treuheit, Nicholas A; Beach, Muneera A; Komives, Elizabeth A

2011-05-31

Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.

Tetrazepam: a benzodiazepine which dissociates sedation from other benzodiazepine activities. II. In vitro and in vivo interactions with benzodiazepine binding sites

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

Keane, P.E.; Bachy, A.; Morre, M.

1988-05-01

Tetrazepam is a 1,4-benzodiazepine (BZD) derivative which, in rodents, appears to have very little sedative and ataxic effects. In an attempt to identify the molecular mechanisms underlying this particular pharmacological profile we examined the interaction of tetrazepam with BZD binding sites. Tetrazepam interacted competitively with central and peripheral BZD binding sites and exhibited comparable affinities for both sites. Tetrazepam was approximately one-seventh as potent as diazepam at the central receptor and as potent as diazepam at the peripheral binding site. Tetrazepam did not distinguish type I from type II central BZD receptors, as evidenced by comparable affinities for the cerebellarmore » and hippocampal receptors. In vitro autoradiographic studies showed that tetrazepam displaced (3H)flunitrazepam from rat brain membranes without any clear regional specificity. Like all BZD receptor agonists, tetrazepam exhibited a gamma-aminobutyric acid shift, a photoaffinity shift and potentiated the binding of 35S-t-butyl-bicyclophosphorothionate to rat brain membranes. However, the latter effect was observed at relatively high concentrations of tetrazepam. In vivo, tetrazepam displaced specifically bound (3H)flunitrazepam from mouse brain (ID50, 37 mg/kg p.o. vs 3.5 mg/kg p.o. for diazepam) and from mouse kidney (ID50, 38 mg/kg p.o. vs. 21 mg/kg p.o. for diazepam). It is concluded that tetrazepam is a BZD receptor agonist; the molecular mechanisms which underly the low sedative potential of the drug cannot at present be explained by a particular interaction with either central or peripheral BZD binding sites, but may be related to the drug's relatively weak effect on 35S-t-butyl-bicyclophosphorothionate binding.« less

Conserved and divergent features of the structure and function of La and La-related proteins (LARPs)

PubMed Central

Bayfield, Mark A.; Yang, Ruiqing; Maraia, Richard J.

2010-01-01

Genuine La proteins contain two RNA binding motifs, a La motif (LAM) followed by a RNA recognition motif (RRM), arranged in a unique way to bind RNA. These proteins interact with an extensive variety of cellular RNAs and exhibit activities in two broad categories: i) to promote the metabolism of nascent pol III transcripts, including precursor-tRNAs, by binding to their common, UUU-3’OH containing ends, and ii) to modulate the translation of certain mRNAs involving an unknown binding mechanism. Characterization of several La-RNA crystal structures as well as biochemical studies reveal insight into their unique two-motif domain architecture and how the LAM recognizes UUU-3’OH while the RRM binds other parts of a pre-tRNA. Recent studies of members of distinct families of conserved La-related proteins (LARPs) indicate that some of these harbor activity related to genuine La proteins, suggesting that their UUU-3’OH binding mode has been appropriated for the assembly and regulation of a specific snRNP (e.g., 7SK snRNA assembly by hLARP7/PIP7S). Analyses of other LARP family members (i.e., hLARP4, hLARP6) suggest more diverged RNA binding modes and specialization for cytoplasmic mRNA-related functions. Thus it appears that while genuine La proteins exhibit broad general involvement in both snRNA-related and mRNA-related functions, different LARP families may have evolved specialized activities in either snRNA or mRNA related functions. In this review, we summarize recent progress that has led to greater understanding of the structure and function of La proteins and their roles in tRNA processing and RNP assembly dynamics, as well as progress on the different LARPs. PMID:20138158

Conserved and divergent features of the structure and function of La and La-related proteins (LARPs).

PubMed

Bayfield, Mark A; Yang, Ruiqing; Maraia, Richard J

2010-01-01

Genuine La proteins contain two RNA binding motifs, a La motif (LAM) followed by a RNA recognition motif (RRM), arranged in a unique way to bind RNA. These proteins interact with an extensive variety of cellular RNAs and exhibit activities in two broad categories: i) to promote the metabolism of nascent pol III transcripts, including precursor-tRNAs, by binding to their common, UUU-3'OH containing ends, and ii) to modulate the translation of certain mRNAs involving an unknown binding mechanism. Characterization of several La-RNA crystal structures as well as biochemical studies reveal insight into their unique two-motif domain architecture and how the LAM recognizes UUU-3'OH while the RRM binds other parts of a pre-tRNA. Recent studies of members of distinct families of conserved La-related proteins (LARPs) indicate that some of these harbor activity related to genuine La proteins, suggesting that their UUU-3'OH binding mode has been appropriated for the assembly and regulation of a specific snRNP (e.g., 7SK snRNP assembly by hLARP7/PIP7S). Analyses of other LARP family members suggest more diverged RNA binding modes and specialization for cytoplasmic mRNA-related functions. Thus it appears that while genuine La proteins exhibit broad general involvement in both snRNA-related and mRNA-related functions, different LARP families may have evolved specialized activities in either snRNA or mRNA-related functions. In this review, we summarize recent progress that has led to greater understanding of the structure and function of La proteins and their roles in tRNA processing and RNP assembly dynamics, as well as progress on the different LARPs.

Anethole potentiates dodecanol's fungicidal activity by reducing PDR5 expression in budding yeast.

PubMed

Fujita, Ken-Ichi; Ishikura, Takayuki; Jono, Yui; Yamaguchi, Yoshihiro; Ogita, Akira; Kubo, Isao; Tanaka, Toshio

2017-02-01

trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum and a weaker antimicrobial potency than other available antibiotics. When combined with polygodial, nagilactone E, and n-dodecanol, anethole has been shown to exhibit synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae, and a human opportunistic pathogenic yeast, Candida albicans. However, the mechanism underlying this synergistic effect of anethole has not been characterized. We studied this mechanism using dodecanol-treated S. cerevisiae cells and focusing on genes related to multidrug efflux. Although dodecanol transiently reduced the number of colony forming units, this recovered to levels similar to those of untreated cells with continued incubation beyond 24h. Reverse transcription polymerase chain reaction analysis revealed overexpression of an ATP-binding cassette (ABC) transporter gene, PDR5, in addition to a slight increase in PDR11, PDR12, and PDR15 transcriptions in dodecanol-treated cells. In the presence of anethole, these effects were attenuated and the fungicidal activity of dodecanol was extended. Dodecanol showed longer lasting fungicidal activity against a Δpdr5. In addition, Δpdr3 and Δlge1, lack transcription factors of PDR5 and PDR3, were partly and completely susceptible to dodecanol, respectively. Furthermore, combination of anethole with fluconazole was also found to exhibit synergy on C. albicans. These results indicated that although anethole reduced the transcription of several transporters, PDR5 expression was particularly relevant to dodecanol efflux. Anethole is expected to be a promising candidate drug for the inhibition of efflux by reducing the transcription of several ABC transporters. Copyright © 2016 Elsevier B.V. All rights reserved.

Programmed hyperphagia secondary to increased hypothalamic SIRT1.

PubMed

Desai, Mina; Li, Tie; Han, Guang; Ross, Michael G

2014-11-17

Small for gestational age (SGA) offspring exhibit reduced hypothalamic neural satiety pathways leading to programmed hyperphagia and adult obesity. Appetite regulatory site, the hypothalamic arcuate nucleus (ARC) contains appetite (NPY/AgRP) and satiety (POMC) neurons. Using in vitro culture of hypothalamic neuroprogenitor cells (NPC) which form the ARC, we demonstrated that SGA offspring exhibit reduced NPC proliferation and neuronal differentiation. bHLH protein Hes1 promotes NPC self-renewal and inhibits differentiation by repressing neuronal differentiation genes (Mash1, neurogenin3). We hypothesized that Hes1/Mash1 and ultimately ARC neuronal differentiation and expression of NPY/POMC neurons are influenced by SIRT1 which is a nutrient sensor and a histone deacetylase. Control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to 21 (SGA). In vivo studies showed that SGA newborns and adult offspring had increased protein expression of hypothalamic/ARC SIRT1 and AgRP with decreased POMC. Additionally, SGA newborns had decreased expression of hypothalamic neurogenic factors with reduced in vivo NPC proliferation. In vitro culture of hypothalamic NPCs showed similar changes with elevated SIRT1 binding to Hes1 in SGA newborn. Silencing SIRT1 increased NPC proliferation and Hes1 and Tuj1expression in both Control and SGA NPCs. Although SGA NPC proliferation remained below that of Controls, it was higher than Control NPCs in the absence of SIRT1 siRNA. The direct impact of SIRT1 on NPC proliferation and differentiation were further confirmed with pharmacologic SIRT1 inhibitor and activator. Thus, in SGA newborns elevated SIRT1 induces premature differentiation of NPCs, reducing the NPC pool and cell proliferation. Copyright © 2014 Elsevier B.V. All rights reserved.

Programmed Hyperphagia secondary to Increased Hypothalamic SIRT1

PubMed Central

Desai, Mina; Li, Tie; Han, Guang; Ross, Michael G.

2014-01-01

Small for gestational age (SGA) offspring exhibit reduced hypothalamic neural satiety pathways leading to programmed hyperphagia and adult obesity. Appetite regulatory site, the hypothalamic arcuate nucleus (ARC) contains appetite (NPY/AgRP) and satiety (POMC) neurons. Using in vitro culture of hypothalamic neuroprogenitor cells (NPC) which form the ARC, we demonstrated that SGA offspring exhibit reduced NPC proliferation and neuronal differentiation. bHLH protein Hes1 promotes NPC self-renewal and inhibits differentiation by repressing neuronal differentiation genes (Mash1, neurogenin3). We hypothesized that Hes1/Mash1 and ultimately ARC neuronal differentiation and expression of NPY/POMC neurons are influenced by SIRT1 which is a nutrient sensor and a histone deacetylase. Control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to 21 (SGA). In vivo studies showed that SGA newborns and adult offspring had increased protein expression of hypothalamic/ARC SIRT1 and AgRP with decreased POMC. Additionally, SGA newborns had decreased expression of hypothalamic neurogenic factors with reduced in vivo NPC proliferation. In vitro culture of hypothalamic NPCs showed similar changes with elevated SIRT1 binding to Hes1 in SGA newborn. Silencing SIRT1 increased NPC proliferation and Hes1 and Tuj1expression in both Control and SGA NPCs. Although SGA NPC proliferation remained below that of Controls, it was higher than Control NPCs in the absence of SIRT1 siRNA. The direct impact of SIRT1 on NPC proliferation and differentiation were further confirmed with pharmacologic SIRT1 inhibitor and activator. Thus, in SGA newborns elevated SIRT1 induces premature differentiation of NPCs, reducing the NPC pool and cell proliferation. PMID:25245521

Distinct mechanisms for N-acetylcysteine inhibition of cytokine-induced E-selectin and VCAM-1 expression.

PubMed

Faruqi, R M; Poptic, E J; Faruqi, T R; De La Motte, C; DiCorleto, P E

1997-08-01

We have examined the effects of N-acetyl-L-cysteine (NAC), a well-characterized, thiol-containing antioxidant, on agonist-induced monocytic cell adhesion to endothelial cells (EC). NAC inhibited interleukin-1 (IL-1 beta)-induced, but not basal, adhesion with 50% inhibition at approximately 20 mM. Monocytic cell adhesion to EC in response to tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), alpha-thrombin, or phorbol 12-myristate 13-acetate (PMA) was similarly inhibited by NAC. Unlike published studies with pyrrolidinedithiocarbamate, which specifically inhibited vascular cell adhesion molecule 1 (VCAM-1), NAC inhibited IL-1 beta-induced mRNA and cell surface expression of both E-selectin and VCAM-1. NAC had no effect on the half-life of E-selectin or VCAM-1 mRNA. Although NAC reduced nuclear factor-kappa B (NF-kappa B) activation in EC as measured by gel-shift assays using an oligonucleotide probe corresponding to the consensus NF-kappa B binding sites of the VCAM-1 gene (VCAM-NF-kappa B), the antioxidant had no appreciable effect when an oligomer corresponding to the consensus NF-kappa B binding site of the E-selectin gene (E-selectin-NF-kappa B) was used. Because NF-kappa B has been reported to be redox sensitive, we studied the effects of NAC on the EC redox environment. NAC caused an expected dramatic increase in the reduced glutathione (GSH) levels in EC. In vitro studies demonstrated that whereas the binding affinity of NF-kappa B to the VCAM-NF-kappa B oligomer peaked at a GSH-to-oxidized glutathione (GSSG) ratio of approximately 200 and decreased at higher ratios, the binding to the E-selectin-NF-kappa B oligomer appeared relatively unaffected even at ratios > 400, i.e., those achieved in EC treated with 40 mM NAC. These results suggest that NF-kappa B binding to its consensus sequences in the VCAM-1 and E-selectin gene exhibits marked differences in redox sensitivity, allowing for differential gene expression regulated by the same transcription factor. Our data also demonstrate that NAC increases the GSH-to-GSSG ratio within the EC suggesting one possible mechanism through which this antioxidant inhibits agonist-induced monocyte adhesion to EC.

Compositional tuning of epoxide-polyetheramine "click" reaction toward cytocompatible, cationic hydrogel particles with antimicrobial and DNA binding activities.

PubMed

Tang, Shuangcheng; Huang, Lu; Daniels-Mulholland, Robert J; Dlugosz, Elizabeth; Morin, Emily A; Lenaghan, Scott; He, Wei

2016-10-01

The "click" characteristics of nucleophilic opening of epoxide have recently been exploited for the development of a functional hydrogel particle system based on commercially available bisepoxide and triamine polyetheramine monomers. Key features of these particles include high cationic charges and responsiveness to temperature, pH, and oxidation. Despite these advantages, the cytocompatibility of these particles must be considered prior to use in biomedical applications. Here we demonstrate that, by introducing a diamine polyetheramine as a comonomer in the "click" reaction, and tuning its molar ratio with the triamine monomer, cationic nanoparticles with improved cytocompatibility can be prepared. The reduced cytotoxicity is primarily due to the hydrophilic backbone of the diamine comonomer, which has polyethylene glycol as a primary component. The resulting nanoparticles formed from the diamine comonomer exhibited a lower surface charge, while maintaining a comparable size. In addition, the responsiveness of the nanoparticles to temperature, pH, and oxidation was conserved, while achieving greater colloidal stability at basic pH. Results from this study further demonstrated that the nanoparticles were able to encapsulate Nile red, a model for hydrophobic drug molecules, were effective against the bacteria Staphylococcus aureus, and were capable of binding DNA through ionic complexation. Based on the results from this work, the use of diamine comonomers significantly reduces the cytotoxicity of similarly developed hydrogel nanoparticles, allowing for numerous biomedical applications, including nanocarriers for therapeutic agents with poor water solubility, treatment of bacterial infection, and non-viral vectors for gene therapy. In recent years significant attention has been placed on the development of nanocarriers for numerous biomedical applications. Of particular interest are cationic polymers, which contain high positive surface charges that allow binding of numerous therapeutic agents. Unfortunately, the advantages of cationic polymers for binding, are often negated by the tendency of these polymers to be cytotoxic. Previous studies have developed highly responsive cationic hydrogel nanoparticles, which meet several of the criteria for biomedical applications, but were acutely cytotoxic. In this work, cationic hydrogel nanoparticles, with significantly improved cytocompatibility, were synthesized using simple, green epoxy chemistry. In addition, the ability of these nanoparticles to maintain a small size (<500nm), bind DNA, encapsulate hydrophobic drugs, and kill bacteria was maintained. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Interactions between neutral endopeptidase (EC 3.4.24.11) and the substance P (NK1) receptor expressed in mammalian cells.

PubMed

Okamoto, A; Lovett, M; Payan, D G; Bunnett, N W

1994-05-01

Interactions between neutral endopeptidase-24.11 (NEP) and the substance P receptor (SPR; NK1) were investigated by examining substance P (SP) degradation, SP binding and SP-induced Ca2+ mobilization in epithelial cells transfected with cDNA encoding the rat SPR and rat NEP. Expression of NEP accelerated the degradation of SP by intact epithelial cells and by membrane preparations, and degradation was reduced by the NEP inhibitor thiorphan. In cells expressing SPR alone, specific 125I-SP binding after 20 min incubation at 37 degrees C was 92.2 +/- 3.1% of maximal binding and was unaffected by thiorphan. Coexpression of NEP in the same cells as the SPR markedly reduced SP binding to 13.9 +/- 0.5% of maximal, and binding was increased to 82.7 +/- 2.4% of maximal with thiorphan. Coexpression of NEP in the same cells as the SPR also reduced to undetectable the increase in intracellular Ca2+ in response to low concentrations of SP (0.3 and 0.5 nM), and significantly reduced the response to higher concentrations (1 and 3 nM). The Ca2+ response was restored to control values by inhibition of NEP with thiorphan. In contrast, SP binding and SP-induced Ca2+ mobilization were only slightly reduced when cells expressing SPR alone were mixed with a 3- to 24-fold excess of cells expressing NEP alone. Therefore, in this system, NEP markedly down-regulates SP binding and SP-induced Ca2+ mobilization only when coexpressed in the same cells as the SPR.

Timing of Galectin-1 Exposure Differentially Modulates Nipah Virus Entry and Syncytium Formation in Endothelial Cells

PubMed Central

Garner, Omai B.; Yun, Tatyana; Pernet, Olivier; Aguilar, Hector C.; Park, Arnold; Bowden, Thomas A.; Freiberg, Alexander N.

2014-01-01

ABSTRACT Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate-binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 postinfection results in reduced production of progeny virus and syncytium formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome. IMPORTANCE Nipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed endothelial cells, can both promote Nipah virus infection of endothelial cells by “bridging” the virus to the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects of a host lectin for a virus in one type of host cell. PMID:25505064

Affinities of penicillins and cephalosporins for the penicillin-binding proteins of Escherichia coli K-12 and their antibacterial activity.

PubMed Central

Curtis, N A; Orr, D; Ross, G W; Boulton, M G

1979-01-01

The affinities of a range of penicillins and cephalosporins for ther penicillin-binding proteins of Escherichia coli K-12 have been studied, and the results were compared with the antibacterial activity of the compounds against E. coli K-12 and an isogenic permeability mutant. Different penicillins and cephalosporins exhibited different affinities for the "essential" penicillin-binding proteins of E. coli K-12, in a manner which directly correlated with their observed effects upon bacterial morphology. Furthermore, the affinities of the compounds for their "primary" lethal penicillin-binding protein targets showed close agreement with their antibacterial activities against the permeability mutant. Images PMID:393164

Dual binding in cohesin-dockerin complexes: the energy landscape and the role of short, terminal segments of the dockerin module.

PubMed

Wojciechowski, Michał; Różycki, Bartosz; Huy, Pham Dinh Quoc; Li, Mai Suan; Bayer, Edward A; Cieplak, Marek

2018-03-22

The assembly of the polysaccharide degradating cellulosome machinery is mediated by tight binding between cohesin and dockerin domains. We have used an empirical model known as FoldX as well as molecular mechanics methods to determine the free energy of binding between a cohesin and a dockerin from Clostridium thermocellum in two possible modes that differ by an approximately 180° rotation. Our studies suggest that the full-length wild-type complex exhibits dual binding at room temperature, i.e., the two modes of binding have comparable probabilities at equilibrium. The ability to bind in the two modes persists at elevated temperatures. However, single-point mutations or truncations of terminal segments in the dockerin result in shifting the equilibrium towards one of the binding modes. Our molecular dynamics simulations of mechanical stretching of the full-length wild-type cohesin-dockerin complex indicate that each mode of binding leads to two kinds of stretching pathways, which may be mistakenly taken as evidence of dual binding.

System controls on the aqueous distribution of mercury in the northern Florida Everglades

USGS Publications Warehouse

Hurley, J.P.; Krabbenhoft, D.P.; Cleckner, L.B.; Olson, M.L.; Aiken, G.R.; Rawlik, P.S.

1998-01-01

The forms and partitioning of aqueous mercury species in the canals and marshes of the Northern Florida Everglades exhibit strong spatial and temporal variability. In canals feeding Water Conservation Area (WCA) 2A, unfiltered total Hg (HgT(U)) is less than 3 ng L-1 and relatively constant. In contrast, methyl mercury (MeHg) exhibited a strong seasonal pattern, with highest levels entering WCA-2A marshes during July. Stagnation and reduced flows also lead to particle enrichment of MeHg. In the marshes of WCA-2A, 2B and 3A, HgT(U) is usually <5 ng L-1 with no consistent north-south patterns. However, for individual dates, aqueous unfiltered MeHg (MeHg(U)) levels increase from north to south with generally lowest levels in the eutrophied regions of northern WCA-2A. A strong relationship between filtered Hg species and dissolved organic carbon (DOC), evident for rivers draining wetlands in Wisconsin, was not apparent in the Everglades, suggesting either differences in the binding sites of DOC between the two regions, or non-organic Hg complexation in the Everglades.

Perturbed desmosomal cadherin expression in grainy head-like 1-null mice.

PubMed

Wilanowski, Tomasz; Caddy, Jacinta; Ting, Stephen B; Hislop, Nikki R; Cerruti, Loretta; Auden, Alana; Zhao, Lin-Lin; Asquith, Stephen; Ellis, Sarah; Sinclair, Rodney; Cunningham, John M; Jane, Stephen M

2008-03-19

In Drosophila, the grainy head (grh) gene plays a range of key developmental roles through the regulation of members of the cadherin gene family. We now report that mice lacking the grh homologue grainy head-like 1 (Grhl1) exhibit hair and skin phenotypes consistent with a reduction in expression of the genes encoding the desmosomal cadherin, desmoglein 1 (Dsg1). Grhl1-null mice show an initial delay in coat growth, and older mice exhibit hair loss as a result of poor anchoring of the hair shaft in the follicle. The mice also develop palmoplantar keratoderma, analogous to humans with DSG1 mutations. Sequence analysis, DNA binding, and chromatin immunoprecipitation experiments demonstrate that the human and mouse Dsg1 promoters are direct targets of GRHL1. Ultrastructural analysis reveals reduced numbers of abnormal desmosomes in the interfollicular epidermis. These findings establish GRHL1 as an important regulator of the Dsg1 genes in the context of hair anchorage and epidermal differentiation, and suggest that cadherin family genes are key targets of the grainy head-like genes across 700 million years of evolution.

Perturbed desmosomal cadherin expression in grainy head-like 1-null mice

PubMed Central

Wilanowski, Tomasz; Caddy, Jacinta; Ting, Stephen B; Hislop, Nikki R; Cerruti, Loretta; Auden, Alana; Zhao, Lin-Lin; Asquith, Stephen; Ellis, Sarah; Sinclair, Rodney; Cunningham, John M; Jane, Stephen M

2008-01-01

In Drosophila, the grainy head (grh) gene plays a range of key developmental roles through the regulation of members of the cadherin gene family. We now report that mice lacking the grh homologue grainy head-like 1 (Grhl1) exhibit hair and skin phenotypes consistent with a reduction in expression of the genes encoding the desmosomal cadherin, desmoglein 1 (Dsg1). Grhl1-null mice show an initial delay in coat growth, and older mice exhibit hair loss as a result of poor anchoring of the hair shaft in the follicle. The mice also develop palmoplantar keratoderma, analogous to humans with DSG1 mutations. Sequence analysis, DNA binding, and chromatin immunoprecipitation experiments demonstrate that the human and mouse Dsg1 promoters are direct targets of GRHL1. Ultrastructural analysis reveals reduced numbers of abnormal desmosomes in the interfollicular epidermis. These findings establish GRHL1 as an important regulator of the Dsg1 genes in the context of hair anchorage and epidermal differentiation, and suggest that cadherin family genes are key targets of the grainy head-like genes across 700 million years of evolution. PMID:18288204

Design and synthesis of novel and highly-active pan-histone deacetylase (pan-HDAC) inhibitors.

PubMed

Tashima, Toshihiko; Murata, Hiroaki; Kodama, Hidehiko

2014-07-15

Histone deacetylase (HDAC) inhibitions are known to elicit anticancer effects. We designed and synthesized several HDAC inhibitors. Among these compounds, compound 40 exhibited a more than 10-fold stronger inhibitory activity compared with that of suberoylanilide hydroxamic acid (SAHA) against each human HDAC isozyme in vitro (IC50 values of 40: HDAC1, 0.0038μM; HDAC2, 0.0082μM; HDAC3, 0.015μM; HDAC8, 0.0060μM; HDAC4, 0.058μM; HDAC9, 0.0052μM; HDAC6, 0.058μM). The dose of the administered HDAC inhibitors that contain hydroxamic acid as the zinc-binding group may be reduced by 40. Because the carbostyril subunit is a time-tested structural component of drugs and biologically active compounds, 40 most likely exhibits good absorption, distribution, metabolism, excretion, and toxicity (ADMET). Thus, compound 40 is expected to be a promising therapeutic agent or chemical tool for the investigation of life process. Copyright © 2014 Elsevier Ltd. All rights reserved.

Probing C-O bond activation on gas-phase transition metal clusters: Infrared multiple photon dissociation spectroscopy of Fe, Ru, Re, and W cluster CO complexes

NASA Astrophysics Data System (ADS)

Lyon, Jonathan T.; Gruene, Philipp; Fielicke, André; Meijer, Gerard; Rayner, David M.

2009-11-01

The binding of carbon monoxide to iron, ruthenium, rhenium, and tungsten clusters is studied by means of infrared multiple photon dissociation spectroscopy. The CO stretching mode is used to probe the interaction of the CO molecule with the metal clusters and thereby the activation of the C-O bond. CO is found to adsorb molecularly to atop positions on iron clusters. On ruthenium and rhenium clusters it also binds molecularly. In the case of ruthenium, binding is predominantly to atop sites, however higher coordinated CO binding is also observed for both metals and becomes prevalent for rhenium clusters containing more than nine atoms. Tungsten clusters exhibit a clear size dependence for molecular versus dissociative CO binding. This behavior denotes the crossover to the purely dissociative CO binding on the earlier transition metals such as tantalum.

Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region.

PubMed

Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L; Hood, Molly M; Lord, John W; Lu, Wei-Ping; Miller, David F; Patt, William C; Smith, Bryan D; Vogeti, Lakshminarayana; Kaufman, Michael D; Petillo, Peter A; Wise, Scott C; Abendroth, Jan; Chun, Lawrence; Clark, Robin; Feese, Michael; Kim, Hidong; Stewart, Lance; Flynn, Daniel L

2010-10-01

Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase. Copyright © 2010 Elsevier Ltd. All rights reserved.

Binding-Induced Fluorescence of Serotonin Transporter Ligands: A Spectroscopic and Structural Study of 4-(4-(Dimethylamino)phenyl)-1-methylpyridinium (APP+) and APP+ Analogues

PubMed Central

2014-01-01

The binding-induced fluorescence of 4-(4-(dimethylamino)-phenyl)-1-methylpyridinium (APP+) and two new serotonin transporter (SERT)-binding fluorescent analogues, 1-butyl-4-[4-(1-dimethylamino)phenyl]-pyridinium bromide (BPP+) and 1-methyl-4-[4-(1-piperidinyl)phenyl]-pyridinium (PPP+), has been investigated. Optical spectroscopy reveals that these probes are highly sensitive to their chemical microenvironment, responding to variations in polarity with changes in transition energies and responding to changes in viscosity or rotational freedom with emission enhancements. Molecular docking calculations reveal that the probes are able to access the nonpolar and conformationally restrictive binding pocket of SERT. As a result, the probes exhibit previously not identified binding-induced turn-on emission that is spectroscopically distinct from dyes that have accumulated intracellularly. Thus, binding and transport dynamics of SERT ligands can be resolved both spatially and spectroscopically. PMID:24460204

Non-intercalative, deoxyribose binding of boric acid to calf thymus DNA.

PubMed

Ozdemir, Ayse; Gursaclı, Refiye Tekiner; Tekinay, Turgay

2014-05-01

The present study characterizes the effects of the boric acid binding on calf thymus DNA (ct-DNA) by spectroscopic and calorimetric methods. UV-Vis absorbance spectroscopy, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), isothermal titration calorimetry (ITC), and Fourier transform infrared (FT-IR) spectroscopy were employed to characterize binding properties. Changes in the secondary structure of ct-DNA were determined by CD spectroscopy. Sizes and morphologies of boric acid-DNA complexes were determined by transmission electron microscopy (TEM). The kinetics of boric acid binding to calf thymus DNA (ct-DNA) was investigated by isothermal titration calorimetry (ITC). ITC results revealed that boric acid exhibits a moderate affinity to ct-DNA with a binding constant (K a) of 9.54 × 10(4) M(-1). FT-IR results revealed that boric acid binds to the deoxyribose sugar of DNA without disrupting the B-conformation at tested concentrations.

Interaction studies of resistomycin from Streptomyces aurantiacus AAA5 with calf thymus DNA and bovine serum albumin

NASA Astrophysics Data System (ADS)

Vijayabharathi, R.; Sathyadevi, P.; Krishnamoorthy, P.; Senthilraja, D.; Brunthadevi, P.; Sathyabama, S.; Priyadarisini, V. Brindha

2012-04-01

Resistomycin, a secondary metabolite produced by Streptomyces aurantiacus AAA5. The binding interaction of resistomycin with calf thymus DNA (CT DNA) and bovine serum albumin (BSA) was investigated by spectrophotometry, spectrofluorimetry, circular dichroism (CD) and synchronous fluorescence techniques under physiological conditions in vitro. Absorption spectral studies along with the fluorescence competition with ethidium bromide measurements and circular dichroism clearly suggest that the resistomycin bind with CT DNA relatively strong via groove binding. BSA interaction results revealed that the drug was found to quench the fluorescence intensity of the protein through a static quenching mechanism. The number of binding sites 'n' and apparent binding constant 'K' calculated according to the Scatchard equation exhibit a good binding property to bovine serum albumin protein. In addition, the results observed from synchronous fluorescence measurements clearly demonstrate the occurrence of conformational changes of BSA upon addition of the test compound.

Newly identified invertebrate-type lysozyme (Splys-i) in mud crab (Scylla paramamosain) exhibiting muramidase-deficient antimicrobial activity.

PubMed

Zhou, Jian; Zhao, Shu; Fang, Wen-Hong; Zhou, Jun-Fang; Zhang, Jing-Xiao; Ma, Hongyu; Lan, Jiang-Feng; Li, Xin-Cang

2017-09-01

Lysozymes are widely distributed immune effectors exerting muramidase activity against the peptidoglycan of the bacterial cell wall to trigger cell lysis. However, some invertebrate-type (i-type) lysozymes deficient of muramidase activity still exhibit antimicrobial activity. To date, the mechanism underlying the antimicrobial effect of muramidase-deficient i-type lysozymes remains unclear. Accordingly, this study characterized a novel i-type lysozyme, Splys-i, in the mud crab Scylla paramamosain. Splys-i shared the highest identity with the Litopenaeus vannamei i-type lysozyme (Lvlys-i2, 54% identity) at the amino acid level. Alignment analysis and 3D structure comparison show that Splys-i may be a muramidase-deficient i-type lysozyme because it lacks the two conserved catalytic residues (Glu and Asp) that are necessary for muramidase activity. Splys-i is mainly distributed in the intestine, stomach, gills, hepatopancreas, and hemocytes, and it is upregulated by Vibrio harveyi or Staphylococcus aureus challenge. Recombinant Splys-i protein (rSplys-i) can inhibit the growth of Gram-negative bacteria (V. harveyi, Vibrio alginolyticus, Vibrio parahemolyticus, and Escherichia coli), Gram-positive bacteria (S. aureus, Bacillus subtilis, and Bacillus megaterium), and the fungus Candida albicans to varying degrees. In this study, two binding assays and a bacterial agglutination assay were conducted to elucidate the potential antimicrobial mechanisms of Splys-i. Results demonstrated that rSplys-i could bind to all nine aforementioned microorganisms. It also exhibited a strong binding activity to lipopolysaccharide from E. coli and lipoteichoic acid and peptidoglycan (PGN) from S. aureus but a weak binding activity to PGN from B. subtilis and β-glucan from fungi. Moreover, rSplys-i could agglutinate these nine types of microorganisms in the presence of Ca 2+ at different protein concentrations. These results suggest that the binding activity and its triggered agglutinating activity might be two major mechanisms of action to realize the muramidase-deficient antibacterial activity. In addition, rSplys-i can hydrolyze the peptidoglycan of some Gram-positive bacteria because it exhibits weak isopeptidase activities in salt and protein concentration-dependent manner. This result indicates that such an isopeptidase activity may contribute to the muramidase-deficient antimicrobial activity to a certain degree. In conclusion, Splys-i is upregulated by pathogenic bacteria, and it inhibits bacterial growth by binding and agglutination activities as well as isopeptidase activity, suggesting that Splys-i is involved in immune defense against bacteria through several different mechanisms of action. Copyright © 2017 Elsevier Ltd. All rights reserved.

Atomic interactions of neonicotinoid agonists with AChBP: Molecular recognition of the distinctive electronegative pharmacophore

PubMed Central

Talley, Todd T.; Harel, Michal; Hibbs, Ryan E.; Radić, Zoran; Tomizawa, Motohiro; Casida, John E.; Taylor, Palmer

2008-01-01

Acetylcholine-binding proteins (AChBPs) from mollusks are suitable structural and functional surrogates of the nicotinic acetylcholine receptors when combined with transmembrane spans of the nicotinic receptor. These proteins assemble as a pentamer with identical ACh binding sites at the subunit interfaces and show ligand specificities resembling those of the nicotinic receptor for agonists and antagonists. A subset of ligands, termed the neonicotinoids, exhibit specificity for insect nicotinic receptors and selective toxicity as insecticides. AChBPs are of neither mammalian nor insect origin and exhibit a distinctive pattern of selectivity for the neonicotinoid ligands. We define here the binding orientation and determinants of differential molecular recognition for the neonicotinoids and classical nicotinoids by estimates of kinetic and equilibrium binding parameters and crystallographic analysis. Neonicotinoid complex formation is rapid and accompanied by quenching of the AChBP tryptophan fluorescence. Comparisons of the neonicotinoids imidacloprid and thiacloprid in the binding site from Aplysia californica AChBP at 2.48 and 1.94 Å in resolution reveal a single conformation of the bound ligands with four of the five sites occupied in the pentameric crystal structure. The neonicotinoid electronegative pharmacophore is nestled in an inverted direction compared with the nicotinoid cationic functionality at the subunit interfacial binding pocket. Characteristic of several agonists, loop C largely envelops the ligand, positioning aromatic side chains to interact optimally with conjugated and hydrophobic regions of the neonicotinoid. This template defines the association of interacting amino acids and their energetic contributions to the distinctive interactions of neonicotinoids. PMID:18477694